ADITI DILIP SATPUTE UNIVERSITY OF FLORIDA

Transcription

1 DIFFERENTIAL EFFECT OF IMPROVED CITRUS ROOTSTOCKS AND NUTRITION ON THE GENE EXPRESSION IN Candidatus Liberibacter asiaticus (CaLas) - INFECTED VALENCIA SWEET ORANGE TREES By ADITI DILIP SATPUTE A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2017

2 2017 Aditi Dilip Satpute

3 To the Florida citrus growers and researchers

4 ACKNOWLEDGMENTS I would like to express my thanks to my outstanding advisor Dr. Jude Grosser for giving me the opportunity to work on my doctoral degree and his guidance throughout my studies. Dr. Grosser encouraged my research and allowed me to grow as a research scientist. His dedication and perseverance towards the betterment of the citrus industry inspire me to support the agricultural community. I would like to extend my thanks to my committee members Drs. Christine Chase, Fred Gmitter and Matias Kirst for their suggestions, brilliant comments, and input to achieve the goal of my Ph.D. Project. Thanks to the Kirst lab members Christopher Dervinis and Isabela Sant Anna for their help in sequencing preparation and data analysis. Thanks to Quibin Yu in the Gmitter lab for introducing me to different software which I used for functional analysis of the genes. would like to recognize Dr. Manjul Dutt for his guidance in my greenhouse study. Thanks to my laboratory colleagues and greenhouse team who helped me in conducting my laboratory and greenhouse work smoothly and efficiently. I would like to acknowledge the Horticultural Sciences Department chair Dr. Kevin Folta, the Citrus Research and Development Foundation, and the New Varieties Development and Management Corporation for their financial support of this project. My spiritual sustenance thrives through a trust and faith in the Almighty. I would not be able to come to the USA and pursue a doctoral degree without the support of my family and friends back in India. Thanks to my parents and sister for supporting me financially and emotionally in my endeavors. I am grateful to my dear friends, Jian Wu, Utpal Handique, Kiran Dixit, Flavia Zambon, and Mathew Mattia for their unconditional support, brainstorming discussions, and making my life more enjoyable throughout 4

5 hardships of my Ph.D. program. There are also many people who contributed pleasant memories throughout my Ph.D. program. Thanks to all of them. 5

6 TABLE OF CONTENTS page ACKNOWLEDGMENTS... 4 LIST OF TABLES LIST OF FIGURES ABSTRACT CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW Introduction Field Study Greenhouse Study Literature Review Citrus Genomics Breeding for citrus improvement Effect of citrus rootstock on scion in scion/rootstock combinations Citrus Bacterial Disease: Huanglongbing (HLB) Geographic distribution of HLB HLB complex, pathogen-host-vector interaction Status of HLB in Florida and recent developments in HLB research Effect of HLB infection on scion performance; nutritional deficiencies and low quality fruit production Interaction of citrus roots with HLB Breeding of citrus scion and rootstock for HLB tolerance and resistance.. 38 Use of Sequencing Technologies in Plants Sequencing technologies in non-model plants Transcriptome analysis using RNA sequencing Citrus-HLB interaction: omics studies Transcriptomic analysis of CaLas-infected leaves of rough lemon and sweet orange Transcriptomic analysis between CaLas-infected and healthy roots and stem sampled from Valencia /Swingle combination Transcriptomic analysis of DEGs in CaLas-infected leaves between non-grafted US-897 and Cleopatra mandarin rootstocks Genes associated with HLB susceptibility Genes related to HLB tolerance Comprehensive transcriptome and gene expression network analysis of HLB-citrus interactions

7 Transcriptomic and proteomic analysis of CaLas-infected and noninfected roots of Sanhu red tangerine Proteomic analysis between healthy and CaLas-infected Madam Vinous sweet orange Comparative proteomic analysis of symptomatic and pre-symptomatic CaLas-infected grapefruit leaves Research Overview COMPARATIVE ANALYSIS OF TRANSCRIPTOME AND PLANT PHENOTYPES OF CaLas-INFECTED SCION/ROOTSTOCK COMBINATIONS.. 54 Introduction Materials and Methods Plant Materials Sampling Fruit Juice Quality Analysis CaLas and Citrus Tristeza Virus (CTV) Detection RNA Extraction and Quantification RNA Library Preparation and RNA Sequencing Raw Data Processing Functional Analysis and Gene Ontology of DEGs Validation of the RNA-seq Data Results Fruit Juice Quality Analysis PCR Detection of HLB and ELISA Detection of CTV RNA Extraction, RNA Library Preparation, and RNA Sequencing Raw Data Processing Functional Analysis of Differentially Expressed Genes Validation of Differentially Expressed Genes using qrt-pcr Discussion DIFFERENTIAL EXPRESSION ANALYSIS OF HORMONAL METABOLISM- ASSOCIATED GENES IN LEAVES AND ROOTS OF TWO SCION/ROOTSTOCK COMBINATIONS AT ASYMPTOMATIC AND SYMPTOMATIC STAGES OF HLB DISEASE Introduction Materials and Methods Plant Material Sampling, RNA extraction, and RNA sequencing Results HLB Detection and RNA Sequencing Output Differential Expression of Hormonal Regulation-Associated Genes in Leaves and Roots of Asymptomatic VAL/CAN and VAL/SW Combinations. 96 Leaf samples Root samples

8 Differential Expression of Hormonal Regulation-Associated Genes in Leaves and Roots of Symptomatic VAL/CAN and VAL/SW Combinations Leaf samples Root samples Discussion DIFFERENTIAL EXPRESSION OF PLANT IMMUNITY AND DEFENSE- ASSOCIATED GENES IN LEAVES AND ROOTS OF TWO SCION/ROOTSTOCK COMBINATIONS AT ASYMPTOMATIC AND SYMPTOMATIC STAGES OF HLB DISEASE Introduction Materials and Methods Plant Material Sampling, RNA extraction, and RNA sequencing Results HLB Detection and RNA Sequencing Output Differential Expressed of Defense-Associated Genes in Leaves and Roots of Asymptomatic VAL/CAN and VAL/SW combinations Leaf samples Root samples Differentially Expression of Defense-Associated Genes in Leaves and Roots of Symptomatic VAL/CAN and VAL/SW combinations Leaf Samples Root Samples Discussion DIFFERENTIAL EXPRESSION OF PLANT GROWTH AND DEVELOPMENT- ASSOCIATED GENES IN LEAVES AND ROOTS OF TWO SCION/ROOTSTOCK COMBINATIONS AT ASYMPTOMATIC AND SYMPTOMATIC STAGES OF HLB DISEASE Introduction Materials and Methods Plant Material Sampling, RNA Extraction, and RNA Sequencing Results HLB Detection and RNA Sequencing Output Differentially Expressed Plant Growth and Development-Associated Genes in Leaves and Roots of Asymptomatic VAL/CAN and VAL/SW combinations Leaf samples Root samples Differentially Expressed Plant Growth and Development-Associated Genes in Leaves and Roots of Symptomatic VAL/CAN and VAL/SW Combinations Leaf samples

9 Root samples Discussion EFFECTS OF IMPROVED CITRUS ROOTSTOCK AND ENHANCED NUTRIENT FORMULATION ON HLB-DISEASE SEVERITY IN VALENCIA SWEET ORANGE SCION Introduction Materials and Methods Plant Material and Nutrition Treatment CaLas-Inoculation and Detection Sampling RNA Extraction and Gene Expression Quantification Plant Phenotype Analysis Results CaLas-Detection DGE Analysis of The Defense and Transporter Genes in Leaves Differential expression analysis of the NPR1 gene Differential expression analysis of the NPR3 gene Differential expression analysis of the PP2B gene Differential expression analysis of the ZRT2 gene Differential expression analysis of the NRAMP2 gene Differential expression analysis of the NIP6 gene DGE Analysis of Defense and Transporter Genes in Roots Plant Phenotype Analysis Discussion SUMMARY AND CONCLUSIONS APPENDIX A SUPPLEMENTAL DATA FOR CHAPTER B SUPPLEMENTAL DATA FOR CHAPTER LIST OF REFERENCES BIOGRAPHICAL SKETCH

10 LIST OF TABLES Table page 2-1 Experimental treatments and combinations Comparison pairs used for differential gene expression analysis in leaves Analysis of HLB and CTV detection in the experimental samples Reads obtained from the sequencing run Functional analysis of significant DEGs in leaves of the asymptomatic treatment between VAL/CAN and Functional analysis of significant DEGs in leaves of the symptomatic treatment between Functional analysis of significant DEGs in roots of the symptomatic treatment between VAL/CAN and VAL/SW Genes used for validation of RNA-sequencing results Experimental treatments and scion/rootstock combinations Comparison pairs used for DGE analysis in leaves and roots of the... experimental scion/rootstock combinations Differentially expressed hormonal metabolism-associated genes significantly upregulated in leaves of Differentially expressed hormonal metabolism-associated genes significantly upregulated in leaves of Differentially expressed hormonal metabolism-associated genes significantly upregulated in leaves of Differentially expressed hormonal metabolism-associated genes significantly upregulated in leaves of the Differentially expressed hormonal metabolism-associated genes significantly upregulated in roots of the Differentially expressed hormonal metabolism-associated genes significantly upregulated in roots of the Experimental treatments and combinations

11 4-2 Comparison pairs used for differential gene expression analysis in leaves... and roots of the experimental scion/rootstock combinations Differentially expressed immunity and defense-associated genes significantly upregulated in leaves of Differentially expressed immunity and defense-associated genes significantly upregulated in leaves of Differentially expressed immunity and defense-associated genes significantly upregulated in leaves of Differentially expressed immunity and defense-associated genes significantly upregulated in leaves of the Differentially expressed immunity and defense-associated genes significantly upregulated in roots of the Differentially expressed hormonal metabolism-associated genes significantly upregulated in roots of the Experimental treatments and scion/rootstock combinations Comparison pairs used for differential gene expression analysis in leaves and roots of the experimental scion/rootstock combinations Differentially expressed growth and development -associated genes significantly upregulated in leaves of Differentially expressed growth and development-associated genes significantly upregulated in leaves of Differentially expressed growth and development -associated genes significantly upregulated in leaves of Differentially expressed growth and development-associated genes significantly upregulated in leaves of the Differentially expressed growth and development -associated genes significantly upregulated in roots of the Differentially expressed growth and development -associated genes significantly upregulated in roots of the Rootstock treatments Controlled release fertilizer formulations

12 6-3 Primer sequences Gene expression analyses in the roots Phenotypic measurements including tree diameters below and above the graft union, and no.of A-1 Identification of samples for RNA-seq run A-2 Individual RNA-seq library pooling calculations for the sequencing A-3 Shell Script used to identify DGE between the comparisons mentioned in B-1 Differentially expressed and significantly upregulated growth associated genes in leaves of the B-2 Differentially expressed and significantly upregulated growth associated genes in the leaves of B-3 Differentially expressed and significantly upregulated growth associated genes in leaves of the symptomatic B-4 Differentially expressed and significantly upregulated growth associated genes in the leaves of the B-5 Differentially expressed and significantly upregulated growth associated genes in the roots of B-6 Differentially expressed and significantly upregulated growth associated genes in roots of the

13 LIST OF FIGURES Figure page 2-1 VAL/CAN combination field trees VAL/SW combination field trees Juice qualityt analysis. A) Year 2015, B) Year RNA quality and quantity analysis. A) Leaf RNA B) Root RNA Read mapping statistics Display of Volcano plot of using CummeRbund Blast2GO functional analysis of DEGs in leaves (A and B) and roots (C and D) of the asymptomatic VAL/CAN and VAL/SW combinations Blast2GO functional analysis of DEGs in leaves (A and B) and roots (C and D) of the symptomatic VAL/CAN and VAL/SW combintions qrt-pcr based DEGs validation Graphical presentation of Hormonal regulation in plants Graphical presentation of DEGs involved in hormonal metabolism Graphical presentation of DEGs involved in environmental biotic and Graphical presentation of DEGs involved in biotic and abiotic stress Display of HLB-induced biotic stress responses in the leaves of the HLB... asymptomatic VAL/CAN and VAL/SW leaves Display of HLB-induced biotic stress responses in leaves of the HLB-... symptomatic VAL/CAN and VAL/SW leaves Display of HLB-induced biotic stress responses in roots of the HLB-... symptomatic VAL/CAN and VAL/SW leaves Graphical presentation of secondary metabolite biosynthesis pathway Graphical presentation of cell wall modification-associated pathways Graphical presentation of nutrient transportation-associated genes found in DGE analysis of asymptomatic and symptomatic VAL/CAN

14 5-4 Graphical presentation of Nitrogen metabolism-associated... genes found DGE analysis of asymptomatic and symptomatic Graphical presentation of carbohydrate metabolism-associate genes Graphical presentation of genes encoding transcription regulators... in plant growth and development, and found in DGE analysis of CaLas-infected Valencia sweet orange (VAL) stick grafts CaLas detection from leaves and roots of different combinations Gene expression analysis of the selected genes in leaves. A) NPR1, B) Phenotypic differences in rootstock-nutrient formulation combinations Plant phenotype at 37 WAB. A) VAL grafted onto Sw rootstock. B) VAL Graphic presenting summary of CaLas-infected VAL/SW combination Graphic presenting summary of CaLas-infected VAL/CAN combination A-1 Tuxedo pipeline components. (Adapted from Trapnell et al., 2012)

15 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy DIFFERENTIAL EFFECT OF IMPROVED CITRUS ROOTSTOCKS AND NUTRITION ON THE GENE EXPRESSION IN Candidatus Liberibacter asiaticus (CaLas) - INFECTED VALENCIA SWEET ORANGE TREES Chair: Jude Grosser Major: Horticultural Sciences By Aditi Dilip Satpute August 2017 Rootstocks are a key component of commercial citrus production. Therefore, rootstock improvement is a major breeding objective of citrus breeding programs. Improved citrus rootstocks are a potential solution to combat Huanglongbing (HLB), a bacterial disease which is caused by putative causal agent CaLas. The citrus breeding program at the University of Florida, Citrus Research and Education Center (UF-CREC) has developed many putative HLB-tolerant rootstocks that can enhance Valencia sweet orange scion sustainability and fruit quality under endemic HLB condition. Differential transcriptomic analysis of HLB -asymptomatic and -symptomatic Valencia (VAL) scion grafted onto UF-CREC improved candidate (CAN) rootstock (a putatively HLB tolerant rootstock, hybrid of Hirado Buntan pummelo and Cleopatra mandarin) and commercially used Swingle (SW) rootstock, showed significant differential expression regulation of transcripts involved in the jasmonic acid (JA), ethylene (ET), abscisic acid (ABA), auxin (AU) and brassinosteroid (BR) hormonal metabolism. In asymptomatic (leaves) and symptomatic (leaves and roots) VAL/SW leaves showed significant 15

16 upregulation of genes encoding in ABA response regulation, AU biosynthesis, and ET biosynthesis and receptors as compared to the respective treatments of VAL/CAN indicating possible activation of response to abiotic stress, and strong involvement of AU and ET mediated responses in CaLas-infected VAL/SW. In VAL/CAN, significant upregulation of AU response factors and BR response genes suggests that the enhanced plant sustainability might be the outcome of AU-BR interactions. VAL/SW also showed upregulation of different JA biosynthesis genes suggesting a defense activation, possibly against the psyllid phloem feeding. The transcriptome comparison results also showed a greater number of defense-associated genes upregulated in leaves and roots of VAL/SW combination which seem to exhibit a high energy requirement condition that compromises plant growth. Therefore, strong upregulation of defense genes in VAL/SW seems to be a reason for poor plant health in the advanced stage of CaLas-infection. Whereas significant upregulation of nutrient transporters, cell wall modification genes, phloem regeneration associated genes, growth factors and AU- BR interactions suggest a better energy distribution balance between defense and growth in VAL/CAN plants. In a greenhouse study, VAL grafted onto a UF-CREC created improved complex tetraploid (4x) rootstock and SW showed significant differences in the plant phenotype and nutrient transporter genes expression. CaLasinfected-VAL/4x plants had a superior phenotype and lower HLB bacterial titer as compared to VAL/SW under traditional and enhanced controlled release fertilizer (ECRF). Also, CaLas infected -VAL/SW phenotype improved under ECRF. Our findings in the field and greenhouse experiments support the hypothesis that rootstock can differentially reprogram CaLas-infected scion to improve plant performance. Moreover, 16

17 there appears to be a significant rootstock-nutrition interaction that plays a role in the defense response. 17

18 CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW Introduction Orange juice is a popular beverage consumed all around the world. Florida ranks as the 2 nd largest producer of the orange juice in the world and the leading supplier of orange juice in the USA. However, the endemic spread of Huanglongbing (HLB) or citrus greening disease started a rapid decline of the Florida citrus industry. The major concerns associated with HLB to Florida citrus industry are decreasing fruit and juice production. HLB is caused by a putative Candidatus species, Candidatus Liberibacter asiaticus (CaLas) in the USA. CaLas is a gram-negative (Garnier et al., 1984), biotrophic and phloem residing bacteria belonging to the alpha subdivision of the proteobacteria (Jagoueix et al., 1994). The dissemination of CaLas is possible because of its vector the Asian citrus psyllid (Diaphrina citri Kuwayama) which was found first in 1998 in Florida (Halbert and Manjunath, 2004). Currently, there is no definite solution to control HLB. HLB control practices in Florida mostly relied on vector suppression and nutrition management that increases production costs. HLB preventive practices are not cost-effective, rather in some cases, they are a great financial burden to the citrus farmers. In Florida, an increasing number of HLB-affected citrus trees in the past few years have been leading to many abandoned citrus orchards. Declining fruit production is also decreasing the juice production and, also, has caused business losses to farmers. Long term-effective and affordable solutions are required to achieve sustainable citrus production that will make farmers less dependent on chemical control strategies. Citrus breeding has the potential to offer a solution to HLB through the creation of tolerant or resistant cultivars that can mitigate HLB symptoms or even 18

19 challenge the CaLas survival. Hence, breeding of HLB tolerant cultivars is important to lessen the disease severity and improve sustainable high quality fruit and juice production. In Florida, declining juice quality of HLB affected fruit is a matter of grave concern, as domestic and international juice markets are highly competitive. Most commercial citrus rootstock and scion cultivars that are used in Florida are highly susceptible to HLB. It has also been reported that once a plant is CaLas-infected, bacteria can move quickly into the root system, and lead to significant loss of feeder roots, especially in the Swingle citrumelo (Citrus paradisi [Macf.] x Poncirus trifoliata [L.] Raf) rootstock (Graham et al., 2013). Thus, feeder root loss leads to overall poor tree health and significantly reduced productivity. Also, HLB susceptible scion/rootstock combinations showed callose and starch deposition, and vascular system blockage which are the primary reasons for CaLas-infected plant health deterioration (Etxeberria et al., 2009; Achor et al., 2010). Improved HLB-tolerant citrus cultivars are badly needed with mechanisms that reduce HLB-affected plant phloem damage and root loss. Scion and rootstock are equally important for plant performance. Woody fruit-tree rootstocks impart many beneficial traits such as disease resistance, soil ph tolerance, better fruit quality, and improved root systems (Martines-Ballista et al., 2012; Car, 1973; Webster, 1995). Citrus rootstocks are also crucial to improve plant horticultural performance (Ribeiro et al., 2014). The citrus breeding program at the University of Florida- Citrus Research and Education Center, Lake Alfred, FL (UF-CREC) has a major citrus rootstock improvement program. The rootstock improvement program includes conventional breeding at the diploid level, somatic hybridization via protoplast fusion to produce allotetraploid rootstock hybrids (Grosser and Gmitter, 2011), and more recently 19

20 conventional breeding at the tetraploid level (Grosser et al., 2015). The program also is heavily involved in the evaluation of UF-CREC created rootstock germplasm and material obtained from rootstock breeding programs around the world. Growers are actively involved in the evaluation of newly developed citrus varieties in field trials for release in a recently employed fast-track release program. One of the priorities of the CREC-rootstock breeding program is to develop HLB-tolerant rootstocks for high quality fruit and juice production. Putative HLB-tolerant candidate rootstocks (CANs) in greenhouse pre-testing and field trials showed that CaLas-infected sweet orange (Citrus sinensis [L.] Osbeck) and grapefruit (Citrus paradisi [Macf.]) scion varieties grafted onto CAN rootstocks had better plant performance as compared to commercial rootstocks; this was attributed to regulatory mechanisms that are altered due to differential rootstock genetics (Castle et al., 2015). Therefore, differential regulatory process analysis should shed light on factors contributing to the improved plant phenotype in Valencia/CAN combinations. Improved plant performances and increased sustainability of CaLas-infected Valencia/CANs combinations as compared to Valencia/Swingle combination led to our hypothesis that CAN rootstocks can alter the scion gene expression by triggering changes in the plant defenses, hormonal signaling pathways, plant growth and development regulations, and nutrient transportation processes, when the plant is CaLas-infected. Considering the promising results of UF-CREC developed CAN rootstock in reducing HLB disease severity in the grafted scion, the aim of this project is to gain a better understanding of the molecular mechanisms that are responsible for the tolerance of the Valencia/CAN combinations using a transcriptomic approach. The 20

21 comparative differential gene expression (DGE) analysis between Valencia/Swingle and Valencia/CAN combinations will also be helpful to study the HLB-citrus interactions at asymptomatic and symptomatic stages of CaLas-infection. Field Study Comparative DGE analysis of leaves and roots of CaLas-infected and the asymptomatic Valencia/CAN and Valencia/Swingle combinations Comparative DGE analysis of leaves and roots of CaLas-infected and the symptomatic Valencia/CAN and Valencia/Swingle combinations Greenhouse Study Study of phenotypic differences in CaLas-infected plants response to interaction between citrus rootstock and nutrient formulations combinations DGE analysis of transporter and defense associated genes in CaLas-infected plants in response to citrus rootstocks and nutrient formulations combination Citrus Genomics Breeding for citrus improvement Literature Review Citrus fruits; sweet orange, mandarin (Citrus reticulata Blanco.), lemon (Citrus limon L. [Burm.] f.), grapefruit varieties are among the highly consumed horticultural crops globally (Burke, 1967). The origin of citrus is claimed by some to be in the Indian subcontinent (Scora, 1975). It is also reported by others that Yunnan province in China a region where citrus might have originated (Gmitter and Hu, 1990) Tropical and subtropical climatic conditions favor citrus production (Burke, 1967). The commercially produced citrus cultivars are mostly hybrids that originated from four citrus progenitors, namely mandarin, pummelo (Citrus maxima Merr.), citron (Citrus medica L.) and papeda species (Scora, 1975; Li et al., 2010). Citrus species are primarily diploid (2n= 2x) and constituted of 18 chromosomes (x=9). A few naturally occurring citrus cultivars 21

22 are triploid (3x) and occasionally tetraploid (4x). Genomic studies of progenitor citrus species showed that the smallest and largest haploid genome size of citrus species, respectively, are 360 MB in mandarins and 390 MB in citron (Ollitrault et al., 1994). The haploid genome size of secondary species of citrus such as sweet oranges, sour oranges (Citrus aurantium L.), grapefruit, and lemons is between 360 and 390 MB/haploid(Gmitter et al., 2012). The joint venture of international citrus genetics researchers and sequencing centers worldwide initiated the International Citrus Genome Consortium (ICGC). ICGC is a platform for citrus genetics and breeding researchers worldwide to share and exchange the knowledge of contemporary citrus genetics research (ICGC, 2004). ICGS facilitates free citrus genome-based databases and tools through a portal called Phytozome (Michael and Jackson, 2013). Citrus genome databases are widely used to study citrus population genetics and domestication events. The citrus genome databases also used to study targeted genome editing of sweet orange using CRISPR technology (Jia and Wang, 2014), genetic transformation (Dutt et al., 2015), gene predictions that are responsible for plant phenotypes (Dornelas et al., 2007), and a genome wide association study (Minamikawa et al., 2017). The study of diverse mandarins, pummelos, and orange genome sequencing showed that cultivated mandarins are the results of admixture of pummelo into the mandarin, and also, found that a Chinese wild mandarin is significantly diverse from ancestral mandarin species Citrus reticulata (Wu et al., 2014). Information available in the sequenced citrus genomes databases has opened many opportunities for citrus breeders and molecular biologists to perform advanced citrus research. 22

23 Major human edible or non-edible crops in the world evolved through adaptation, domestication and manual breeding processes. Human civilization stimulated intervention in the natural breeding process to develop cultivars that are better suited for climatic, social, and economic conditions of human society at a given space and time. Hence, breeding is an inseparable part of any agricultural and horticulture based industry, and so it is with citrus cultivar development. Citrus species origine is proposed to be in the Asia continent, in parts of China and India (Scora, 1975; Gmitter and Hu, 1990). Over the years, trade between the different geographical locations led to the transport of citrus species from eastern countries to the rest of the world. Also, citrus hybrids are being developed in all major citrus breeding programs through conventional and modern breeding techniques. The conventional citrus breeding method includes hybridization followed by a selection of superior performing individuals. Marker-assisted selection (MAS) enables efficient screening of superior individuals. Somatic hybridization via protoplast fusion is also the most popular and widely used biotechnique in citrus breeding (Gmitter, 1990; Grosser and Gmitter, 2011). Protoplast fusion techniques have overcome some limitations of conventional breeding, bypassing the barriers including sexual incompatibles between certain breeding parents, and male or female sterility issues. Protoplast fusion allows ploidy manipulation, inter- or intraspecies organelle exchange and gene transfer to create somatic hybrids and cybrids (Grosser et al., 1992; Guo et al., 2013). Citrus improvement programs also use natural or induced mutations and somaclonal variation for variety development. An Individual with a desired mutation or somaclonal variation can be used as a potential parent in the hybridization/selection program. Omics-assisted, molecular marker-assisted, and 23

24 genetic-engineering techniques are gaining popularity in modern plant improvement research. The Agrobacterium-based transformation has also been successfully used in citrus breeding to transfer target genes (Dutt and Grosser, 2011). However, controversies over genetically modified organisms (GMO) and environmental regulations limit their commercial use. Newly emerging non-gmo genome editing CRISPR Cas9 technique has been gaining the attention of citrus breeders for potential to achieve certain citrus breeding objectives (Jia and Wang, 2014) Commercial citrus plants are grown by grafting a scion onto a rootstock. Unlike non-grafted plants, both the scion and rootstock are equally important for plant performance and survival. Therefore, scion and rootstock genetic improvements are equally important objectives of citrus breeding programs. One primary objective of citrus scion breeding is focused on manipulating ploidy level to create seedless triploids and high-quality fruits. Somatic hybridization is key to creating allotetraploid breeding parents that can be used in interploid crosses to generate seedless triploids (Grosser and Gmitter 2011). In citrus improvement, embryo rescue technique is used for efficient triploid embryo recovery (Viloria and Grosser, 2005; Shen et al., 2011). Polyembryony is associated with nucellar embryony which produces multiple embryos in a single one seed, all clones of the mother. Polyembryonic selections are difficult to use as a female parent in conventional crosses due to a lack of zygotic embryo production. Therefore, in scion breeding, efficient production of zygotic embryos (monoembryony) is preferred to the nucellar embryony (polyembryony) in diverse female parents (Grosser and Gmitter, 2011). Other objectives of scion breeding programs are developing cultivars that combine cold hardiss, easy peeling, disease resistance, improved juice flavor and color, 24

25 and expanded periods of harvesting. Among disease resistant cultivars, Lakeland limequat is a well known cultivar that is a canker resistant hybrid of kumquat (Fortunella [Swing.]) and Key lime (Citrus aurantifolia Swing.) (Viloria and Grosser, 2005). LB8-9 SugarBelle has proven to be the most HLB-tolerant commercially available scion (Stover et al., 2016), and it is being used heavily in the UF-CREC breeding program. Allotetraploid and autotetraploid zipperskin mandarins can be used as elite parents to create easy peel and triploid seedless fruits (Grosser et al., 2010). UF-CREC scion breeding program has, also, developed various mandarin, orange and grapefruit cultivars which are accepted for commercial citrus production (Grosser et al., 2015). Protoplast fusion also generates cybrids that allow the exchange of parental organelles (mitochondria and/or chloroplast DNA) with or without mixing of the nuclear genomes. Cybridization also has good potential to improve certain traits. Diploid cybrids generated from the fusion of embryogenic callus of Dancy mandarin with Ruby red grapefruit showed increased brix of the juice and extended fruit harvesting period (Satpute et al., 2015). The mechanism for this change has not been determined. Many commercially used citrus scions are developed at United States Department of Agriculture (USDA)- citrus breeding program. Some of the imporatnt commercially accepted are Orlando and Minneola Tangelos (Citrus paradisi x Citrus reticulata), Osceola, Lee, Nova and Robinson which are hybrids of Clementine mandarion with Orlando tangelo, Page a hybrid of Minneola Tangelos with Clementine, Sunburst ( Robinson x Osceola ) (McCollum, 2007). Another important component of citrus improvement programs is rootstock breeding. Rootstock breeding history goes back to approximately 1900 AC and can be 25

26 defined as early-, and modern-era based on international trade of citrus fruits, development of budwood protection programs, and commercialization of the citrus industry (Castle, 2010). Citrus rootstock characteristics evaluations are different from those of scion breeding. Therefore, sources of genetic variation and selection methods are also different than for scion breeding. Sexual and somatic hybridization are primary approaches for rootstock development. Genetic engineering may also play a role in citrus rootstock improvement programs. In a citrus rootstock development program, plant material from any source or developed by any technique can be entered into the system at any time point, that can then be screened based on the potential of the plant material to improve a desirable trait or multiple traits (Gmitter et al., 2007). Today, different citrus rootstocks such as sour orange (Citrus aurantium L.), Cleopatra mandarin, rough lemon (Citrus jambhiri Lush.), Volkamer lemon (Citrus volkmeriana Ten. & pesq.), citranges (P. trifoliata x C. sinensis), and citrumelos (C. Paradisi x P. trifoliata) are used in commercial citrus production. Citrus rootstock breeding is primarily aimed to develop cultivars with improved disease resistance and tree size-controlling attributes. In addition, there are many other rootstock-related citrus tree attributes that can be achieved through citrus rootstock improvement (Castle, 2010). These attributes include yield, precocity of bearing, fruit quality, adaptation to high soil ph, scion compatibility, and ease of propagation. Louzada et al. (1992) reported the creation of potential disease resistant 4x somatic hybrids created via protoplast fusion. Somatic hybrids created between Caipira sweet orange embryogenic parent fused with Volkamer lemon, Rangpur lime (Citrus limonia L. osbeck), and sour orange as the non-embryogenic parents showed potential resistance to citrus blight and 26

27 citrus tristeza virus CTV (Mendes et al., 2001). The soil borne disease complex Diaprepes/Phytophthora is a serious problem for citrus production in Florida. The rootstock program at UF-CREC has developed several improved rootstock cultivars. Among these, a somatic hybrid combining Nova mandarin + Hirado Buntan Pink HBP pummelo and its progeny exhibited tolerance to the Diaprepes/Phytophthora complex (Grosser and Gmitter, 2011). Somatic hybrids created at the UF-CREC rootstock breeding program have shown many improved horticultural traits, including dwarf plant stature and higher fruit production in field trials with commercial scions. Somatic hybrids created between sour orange + Rangpur lime and sour orange + Palestine sweet lime yielded 20,000 kg fruit per acre with 3-4 m tree height (Grosser and Gmitter, 2011). Traditional rootstock propagation in nurseries requires polyembryonic rootstock seed as necessary to generate true-to-type uniform seedlings. Therefore, embryo genetics is highly important in rootstock breeding. The citrus breeding program at the UF-CREC has also pioneered the creation of superior 4x allotetraploid tetrazyg cultivars. Tetrazyg rootstocks are a combination of three or four cultivars that bring desirable traits from different citrus germplasm into one cultivar via conventional breeding using somatic hybrids or other tetrazygs as parents. Some of these tetrazygs were derived from the following crosses: [ Nova mandarin + HBP pummelo x Cleopatra mandarin + argentine trifoliate orange], [ Nova mandarin + HBP pummelo x Succari + argentine trifoliate orange], [ Nova mandarin + HBP pummelo x Cleopatra mandarin + sour orange], [Nova + HBP pummelo x sour orange + Palestine sweet lime] (Grosser and Gmitter, 2011). In addition to the improved horticultural traits, UF-CREC developed 2X and 4x rootstocks have also shown a lower incidence of HLB as compared to the 27

28 commercially used rootstocks in the grafted scions after 5 years in the field under heavy HLB pressure (Grosser, 2013; Castle et al., 2016). Many USDA developed rootstocks are also commercially used to grow citrus. Some of USDA-developed rootstocks: US- 812 US-802, US-897, and US-942, have potential to combat against CTV, Diaprepes/Phytophthora complex, and also, act as candidate rootstock for HLB tolerance (Bowman et al., 2016). In Florida, the epidemic of HLB has brough many challenges to the use of traditional scion and rootstock cultivars. The newly developed scions and rootstocks are the potential cultivars that may establish in the citrus industry because of their their ability to fight against HLB. Effect of citrus rootstock on scion in scion/rootstock combinations A majority of commercially important horticultural crops are grown using scion/rootstock combinations. In horticultural crops, fruits are commercially important plant parts. In citrus, fruit and juice are equally important commodities. Citrus rootstocks have a great impact on regulating fruit and juice quality (Castle, 1995; Ghnaim et al., 2006). Rootstocks have been used for crop production for over 2000 years ( Webster, 1995). Rootstocks significantly contribute to increasing or decreasing tree vigor, higher plant productivity, increasing precocity, increasing plant immunity, changes in plant biomass, plant growth pattern, and the biochemical and physiological status of the plant. The roles of rootstocks in fruit crops like mango, apple, citrus, and stone fruits have been thoroughly studied. In apple, the Malling (M) series of clonal rootstocks is well known for dwarfing and use in high density plantings. The Polish (P) series is used for collar rot resistance. The Budagovasky (Bud) series is used worldwide for winterhardiness. In pear, the famous Quince rootstock is widely used to obtain higher quality fruits from the grafted scion (Webster, 1995). In citrus: rough lemon, sour orange, 28

29 Rangpur lime, Cleopatra mandarin, Swingle citrumelo, and Carrizo citrange (Poncirus trifoliata X Washington sweet orange) rootstocks are popular (Castle, 2010). Although rootstocks are critical in regulating scion performance, the mechanisms responsible for changes in scion performance are still unclear. It is hypothesized that the rootstockscion union leads to internal changes in the quantity and ratio of endogenous hormones, movement of plant assimilates such as sugar, amino acids, mineral nutrients, and the amount of water uptake (Webster, 1995). Biotechnological research tools have facilitated the understanding of molecular mechanisms involved in rootstockscion interactions. A new discovery in this direction is the mrna, small RNA, peptides, or amino acids long distance transport through phloem in grafted vegetables and woody plants. A heterografting experiment between tomato rootstock and potato scion showed the physical transfer of mrna between rootstock and scion that led to changes in scion leaf morphology (Kudo and Harada, 2007). In woody plants, long distance transport of mrna between rootstock and scion was reported in apple (Stoeckli et al., 2011). Also, gibberellic acid-insensitive (GAI) mrna transcript (Zhang et al., 2012), and transcript encoding NAC domain protein exchanges were found across graft unions in pear scion and rootstock (Zhang et al., 2013). Considering the importance of the rootstock in regulating scion performance, improved citrus rootstocks can be a potential antidote to reduce the detrimental effects of HLB in CaLas-infected commercial citrus trees. Citrus Bacterial Disease: Huanglongbing (HLB) Huanglongbing is the mandarin language-originated word meaning yellow dragon disease. Worldwide, HLB is known by different names such as greening in South Africa, mottle leaf in Philippines, and dieback in India. In South Africa, greeningaffected sweet oranges were found to have disintegrated and necrotic phloem present 29

30 in their vascular system. In Indonesia, HLB is recognized as Vein phloem degeneration.' The different names of HLB in the disease affected areas were commonly derived from typical symptoms of HLB on the host plant. HLB affected areas were first found to be reported in India. In 1919, Reinking reported yellow shoot of citrus in the study of diseases of economic plants in southern China and later it was known as HLB. In the 21 st century, HLB has become a highly destructive epidemic citrus disease (Bové, 2006; Bové, 2014). Geographic distribution of HLB The origin of HLB is found to be in the Asian continent. In the 18 th century, the die back symptoms were first observed in citrus orchards in India (Capoor, 1963), and yellow shoot symptoms were found in southern China citrus orchards in the late 1800 s (Zhao, 1981). This suggests the origin of HLB to be in the Asian continent, either in India or China. In Africa, HLB was first observed in the late 1920s (da Graca et al., 2016). In 2004, HLB was first confirmed in the western world in the state of Sao Paulo in Brazil, and later, in 2005, it was confirmed in the state of Florida in the USA (Halbert, 2005). In the USA, HLB is also detected in other citrus producing states; Texas, Arizona, California, South Carolina, Georgia and Louisiana (da Graca et al., 2016). The expanding HLB infection is becoming a threat to other citrus producing countries too. These countries include Australia, Mediterranean basin countries, Middle Eastern countries: Iran, Turkey, and other Caribbean countries: Cuba, Belize, and Mexico. HLB complex, pathogen-host-vector interaction The presumed causal agent of HLB is Candidatus Liberibacter spp. bacteria. In 1970, the causal organism of HLB was confused with a virus or Mycoplasma like organism (MLO) because of its presence in the plant's phloem and yellow shoot 30

31 symptoms (Doi et al., 1967). However, in 1970, Laflache and Bove proved that the HLB associated pathogen is a gram-negative bacterium that has a triple layer of outer peptidoglycan membrane, and it belongs to the alpha-proteobacteria in the family of Rhizobiaceae. Presumably, three species of the HLB causing Candidatus Liberibacter genus have been identified: americanus, asiaticus, and africanus. The most destructive species of putative HLB causing bacteria is Candidatus Liberibacter asiaticus which is prominently found in the American and Asian continents. The other two species, namely africanus, and americanus have been found in African countries and in Brazil, respectively (Bové, 2006). The current research shows that the putative HLB causing bacteria is pleomorphic during growth, and is not limited to the shoots. Rather, bacteria colonize the roots even before the appearance of visible symptoms on leaves, and cause stunted root growth (Graham et al., 2013). CaLas bacteria are thus far nonculturable which continues to be a major constraint in understanding the host-pathogen interactions (Jagoueix et al., 1997). In earlier days, CaLas diagnosis was conducted using biological index or antibodies against HLB (Garnier et al., 1991). However, in recent years, the HLB diagnosis has been routinely performed using polymerase chain reaction (PCR) technique (Li et al., 2006; Ananthakrishnan et al., 2013). The complete genome sequencing of CaLas obtained from an infected psyllid revealed that the bacterium has a small genome size of 1.23 MB, and lacks type III and IV secretion systems. Gene annotation showed that CaLas genome contains a high number of genes, particularly for low a small genome, involved in cell motility (4.5%) and transport activity (8%) (Duan et al., 2009). Li et al. (2012) showed several ATI-binding cassette 31

32 (ABC) transporters are also present in the CaLas that are involved in importing metabolites, co-enzymes, and heavy metals from host plants. Another important component of the HLB disease complex is the insect vector; namely Asian citrus psyllid (ACP) Diaphorina citri Kuwayama and Trioza erytreae that cause transmission of CaLas. ACP is a vector of CaLas, whereas, T. erytreae is a vector of Candidatus Liberibacter africanus. The interaction of scion-rootstock grafts and its effect on the HLB complex is crucial since CaLas is a phloem-limited- bacteria and can also be transmitted by grafting. The third key component of the HLB disease complex is the host. Most Citrus species are HLB-susceptible (Folimonova et al., 2009). Murraya paniculata belongs to the family Rutaceae and is also highly susceptible to HLB (Damsteegt et al., 2010). Whereas, trifoliate orange and rough lemon have shown moderate tolerance to HLB (Albrecht and Bowman, 2012a; Fan et al., 2012). Interactions of HLB and citrus cultivars are discussed in the following sections. HLB symptom development depends on the geographic location, temperature, and citrus types. CaLas is observed to be heat-tolerant. Therefore, CaLas pathogen can survive and develop symptoms in the temperatures above 30 degrees in hot tropical and subtropical regions of the world, whereas africanus species is heat-sensitive and cannot survive hot temperatures. The African psyllid vector T. erytreae can survive in cool temperatures. Hence, Candidatus Liberibacter africanus-caused HLB is predominantly found in African countries or in cool and dry places. HLB affected plants are found to have non-systemic or localized presence of CaLas in different plant organs such as leaves, roots, and fruit. Hilf (2011) reported that CaLas DNA could not be detected from seedlings grown from CaLas-infected Sanguenelli and Conner sweet 32

33 orange fruit. A similar study investigating potential seed mediated CaLas transmission from sweet orange fruits also demonstrated that seedlings grown from infected fruits were not HLB positive over a period of up three years. The authors did not detect any of the typical HLB symptoms such as blotchy mottle or chlorosis from the regenerated seedlings and these plants remained qpcr negative for the duration of the study (Hartung et al., 2010). HLB-affected trees exhibit leaves with mottled, blotchy appearance and corky mid-veins, as well as stunted root growth and significant loss of lateral roots biomass (Graham et al., 2013). HLB-affected fruits are lopsided with small sizes (Vashisth et al., 2016). The internal fruit quality of HLB-affected fruit is highly compromised. Juice extracted from HLB-affected fruits possesses off flavor and low total soluble content (Baldwin et al., 2010; Dagulo et al., 2010; Plotto et al., 2010). An HLB-affected tree also shows the presence of small pointed and erect leaves known as rabbit ears, twin dieback, fruit drop, and off-season flowering. Status of HLB in Florida and recent developments in HLB research Florida is the 2 nd largest orange juice supplier in the world. Valencia and Hamlin sweet oranges are the most popular juice producing scion varieties in Florida, and they are highly susceptible to HLB (Castle, 2013). In Florida, HLB was first found in Miami Dade county, in 2005 (Bové, 2006). Since then, in the last 13 years, the HLB infection rate in Florida has approached almost 80% (Singerman and Useche, 2016). The endemic spread of HLB is causing severe economic losses to the citrus growers and the citrus industry. However, the combination of UF-CREC and USDA citrus research team efforts, financial support from the government, and growers determination may help to achieve sustainable citrus production under the pressure of HLB. 33

34 Prevalent HLB, in Florida, has not only affected the citrus production but also affected the livelihood of people depending on the citrus industry. Statistical analysis of economic impacts of citrus greening (HLB) in Florida reported that during through , the state of Florida had severe income losses because of reduction in in citrus industry generated revenues, and about 48% total jobs were lost in the citrus industry associated employment. Each year, Florida orange production is declining in comparison to the previous year. Efforts are being taken to improve the production of HLB affected trees. The citrus research teams at the UF-CREC and USDA are involved in developing improved HLB tolerant and resistant citrus cultivars, efficient nutrition and HLB management practices, control of psyllids, and pathological identification of early disease detection, etc. The citrus breeding program at the UF-CREC has developed new improved HLB tolerant scion and rootstock selections using conventional and modern biotechnological approaches. These selections are being tested under controlled greenhouse conditions and at different trial locations to analyze their response under HLB. Nutrition is a key factor in HLB disease development. Macro- and micro-nutrient deficiencies such as zinc (Zn), phosphorous (P), boron (B), calcium (Ca) and magnesium (Mg) are prominent in the HLB-affected trees. Advanced foliar nutrition programs are found to be expensive and less effective (Gottwald et al., 2012). The recent studies of soil application of controlled release fertilizers supplemented with enhanced micro-nutrient concentrations have shown improved health of HLB-affected commercial trees (Spyke et al., 2017). Psyllid control is crucial to minimize the spread of HLB in the citrus orchard. Insecticidal foliar sprays and scouting are being implemented to identify the presence of 34

35 the vector and control its population in HLB-affected orchards. Neonicotinoid-containing pesticides are systemic and have long-lasting residual effects which are used in controlling psyllids (Boina and Bloomquist, 2015). In addition, engineering-inspired automated drone surveys and aerial imaging techniques have been used to identify the spacial patterns of HLB infection in the citrus orchards (Garcia-Ruiz et al., 2013). Thermotherapy, chemotherapy, and antibiotics have also shown promising but mixed results. However, these may not be curable solutions but a temporary resort for HLBaffected plants. The successful development of a viable HLB solution also depends on the awareness of growers about the dynamics of the disease, information on available options to manage infected orchards, and sharing HLB success stories among growers. The citrus extension specialists are the mediators that reach out to the growers and can effectively convey the research from the laboratory to the field. Programs like the Citrus Health Management program (CHMA) are being implemented to bring citrus growers together to combat HLB and become involved in the area-wide HLB management programs. Also, the UF-implemented fast-track release program provides growers the opportunity to test newly developed putative HLB-tolerant rootstocks or scions to accelerate their commercialization. Effect of HLB infection on scion performance; nutritional deficiencies and low quality fruit production Mineral nutrients are invaluable for plant growth, development and defense. Plants encounter environmental and biotic stresses which may cause nutrient deficiencies in the plants. The nutritional deficiencies in the plants are the biomarkers of the plant stress and are mostly seen as symptoms on the plants (McCauley et al., 2011). Balanced nutrition is a key for healthy and productive life for all living beings on 35

36 the earth, and plants are not an exception. Plant physiology and defense are highly interrelated to the nutrition level of plants (Dordas, 2008). Imbalance in the plant nutritional status affects plant growth and thus, reduces immunity to fight against pest and diseases, resulting in declining yields. HLB-affected plants exhibit Zn deficiency like-blotchy mottled symptoms on the leaves. In addition, B deficiency is prominent in the HLB-affected plants, which show vein-corking symptoms on the leaves. HLB-affected plants are found to be deficient in Mn, Fe, Ca, Mg and P, whereas, potassium (K) level is found to be increased (Spann and Schuman, 2009). Therefore, HLB-affected plants seem to be severely depleted in the pool of essential secondary and micronutrients. Nutrient uptake depends on the plant s demand for nutrition, the activity of nutrient transporters, and assimilation and movement of the nutrients to the sink tissue. Uptake and movement of nutrients in the plants may be inhibited by many constraints such as inadequate nutrient supply, dysfunctional transporters, or impaired transporting passages. Of these reasons, reduced nutrient supply and impaired vascular (phloem) system are responsible for nutrient deficiencies and source-sink imbalance in the HLB affected and the symptomatic citrus plants. Callose depositions, starch accumulation, and phloem degeneration also hinder translocation of nutrients in HLB-affected plants, causing premature fruit-drop and low-quality fruits. Small and lopsided fruits produced from HLB-affected plants are non-marketable in the fresh fruit market and produce mediocre quality juice (Bessanezi et al., 2009). Fruits harvested from HLB- symptomatic and asymptomatic trees have been found to contain higher concentrations of limonin and nominin, and undesirable juice flavor (Baldwin et al., 2010). Consistently declining 36

37 fruit yields and fruit juice quality, and escalating expenditures on nutrition programs due to HLB, affect the grower s interest in investing in citrus orchard management or in new citrus plantings. Interaction of citrus roots with HLB The citrus rootstock is invaluable for the citrus tree health, enhanced immunity, fruit quality, production, and tree hardiness (Ribeiro et al., 2014). The performances of citrus scions grafted on commercial rootstocks such as Swingle, Cleopatra, Sour orange have been observed to be highly compromised under HLB pressure in Florida. Fibrous roots are important for nutrient and water uptake. HLB-affected plants have been observed to show a decline of 30%-50% capacity of water and nutrient uptake due to the stunted root growth and loss of fibrous root density (Johnson and Graham, 2015). Also, presymptomatic CaLas-infected root systems have been found to lose about 30% root density (Graham et al., 2013). Johnson et al. (2014), reported presymptomatic colonization of CaLas in infected plants. A greenhouse study on the movement of CaLas in HLB-affected plants showed that CaLas colonizes roots first, uses root-starch to multiply, and then moves to the scion. HLB-affected plants exhibit substantial dieback and low starch content (Etxeberria et al., 2009). A similar observation was reported by Li et al. (2003) showing a rapid decline in the carbohydrate reserve in the roots of phloem disrupted and girdled plants. The decrease in the CaLas-infected roots starch suggests that CaLas multiplies at the cost of root-starch and thus, reduces the supply of carbohydrates for root growth. Root starvation leads to stunted root growth and depletion of nutrients and water uptake (Johnson et al., 2014). Increasing awareness about root damage in HLB-citrus interactions is drawing the attention of growers to the importance of rootstocks in HLB disease control and maintaining root health. 37

38 Breeding of citrus scion and rootstock for HLB tolerance and resistance HLB is prevalent in Florida and now affects more than 80% of citrus trees (Singerman and Useche, 2016), and has reduced Florida citrus fruit and juice marketability. Commercial sweet orange and grapefruit varieties account for 95% of citrus fruit production in Florida, all which are highly susceptible to HLB. The popular rootstocks: Swingle citrumelo, Cleopatra mandarin, and Carrizo citrange, and scions: Valencia,' and Hamlin sweet oranges, and Ruby Red and Marsh grapefruits are intolerant to HLB. New rootstock varieties in Florida have been developed for Diprepes/Phytophthora complex resistance, citrus tristeza virus (CTV) resistance, cold hardiness, higher vigor, etc. Florida-grown sweet orange and grapefruit juice quality is favored in the domestic and international market. However, the epidemic of HLB in citrus orchards has been decreasing tree survival and sustainable fruit production. Understanding the urgent need for HLB tolerant or resistant cultivars, the citrus breeders at UF-CREC and USDA have been developing improved cultivars that can survive and produce sustainable fruit yield under the high impact of HLB. In the case of HLB, sometimes CaLas-infected plants show biological tolerance. However, it is short lived. There are no truly HLB-resistant cultivars available. Realistic expectations and knowledge of scion/rootstock combination field performance are required to produce biologically HLB-tolerant and economically profitable citrus rootstock or scion cultivars (Castle, 2016). In addition, the knowledge of CaLas strains, vector and nutrition dynamics are important in finding true tolerance to HLB. Field evaluations of UF-CREC developed rootstocks at St. Helena, Dundee, FL, showed fruits harvested from seven-year old CaLas-infected Valquarius and Vernia scions grafted onto different tetraploid somatic hybrid and tetrazyg rootstocks had 38

39 higher amounts of soluble solids/per box than commercial rootstocks (Grosser et al., 2013). Scions grafted onto UF-CREC developed tetraploid and diploid rootstock showed a lower percentage of CaLas-infection as compared to the scions grafted onto commercial rootstocks after 5 years (Grosser 2013). The UF-CREC developed UFR series rootstock hybrids have shown tolerance and lower incidence of HLB with increased yield/tree (Castle et al., 2016). Also, UF-CREC developed scions LB8-9 (Sugar Belle ), and Orie Lee Late (OLL) sweet orange varieties have shown promising tolerance to HLB when grafted onto conventional rootstocks (Grosser et al.; Stover et al., 2016). The USDA citrus breeding program has also developed improved HLBtolerant rootstock cultivars, of which mandarin and Poncirus trifoliata hybrid rootstocks are showing promising HLB tolerance, such as US-942 (Vicky, 2014). Conventional scion/rootstock combinations LB8-9 (Sugar Belle ) /sour orange and Tango/Kurhakse have been reported to show high growth rate and increased production in the presence of HLB (Stover et al., 2016) Use of Sequencing Technologies in Plants Sequencing technologies in non-model plants Model plant species have many biological and physiological characteristics that make them an appropriate choice for genomic research. The complete genome sequence of Arabidopsis has opened the door for genomic studies of other plants with more complex genomes. Plant genomic information is useful for breeding and understanding the evolutionary development of the species. However, not all plant species genomes are amenable to sequencing using available technologies. The four major constraints in building high quality genome assembly in non-model plants are discussed by Hirsch and Buell (2013). These constraints are genome duplication, ploidy 39

40 level, heterozygosity, and repetitive sequences. In non-model plants, sequencing guided genomic studies may produce inadequate information. Hence, model plants such as Arabidopsis, rice, and maize genomes are commonly used as a reference genome for alignment and annotation of newly studied plant species. In recent years, the next generation sequencing (NGS) platforms have enabled fast and reliable extraction of genomic information from non-model species (Strickler et al., 2012; Unamba et al., 2015). Today, many plant genomes have been sequenced including Citrus species such as Clementine mandarin (Citrus clementine) and Citrus sinensis (Wu et al., 2014). NGS platforms are less time-consuming, and comparatively inexpensive. NGS-high throughput sequencing (HTS) platforms are dominated by companies such as Illumina (sequencing by synthesis), and ABI/SOLiD (bridge amplification), and new innovative technologies providers are emerging as competitors. Bioinformatics are important to analyze the sequencing information to identify genes and their functional characterization (Thimm et al., 2004; Conesa et al., 2005; Magi et al., 2010). The sequencing technologies have facilitated genome sequencing, transcriptome profiling, development of single nucleotide polymorphism (SNP) and microsatellite markers, genome wide association (GWAS) of markers with traits of interest, identification of copy number variation, co-expression analysis of genes, and methylation pattern profiling (Lister et al., 2009; Voelkerding et al., 2009). In horticultural crops, NGS platforms have been used to sequence genomes de novo and to study regulatory mechanisms (Abdurakhmonov, 2016). NGS platforms facilitate analysis of regulatory genes, metabolic pathways, protein networking, and identifying molecular 40

41 markers associated with horticultural traits. The information obtained from NGS platforms can be used to improve the marketable traits of the plant produce through breeding. Transcriptome analysis using RNA sequencing RNA sequencing (RNA-seq) technology is a revolutionary tool of NGS that can perform transcriptome profiling, gene expression analysis, and re-sequencing. According to the central dogma of molecular biology, sequential conversion of DNA to proteins goes through two steps. The first one is a transcription of biopolymer DNA into RNA, and the second is a translation of RNA into proteins. The aim of RNA-seq technology is to catalogue the transcriptome of an organism. Plants contain messenger RNA (mrna), ribosomal RNA (rrna), and transfer RNA (trna). In addition, small RNAs, micro RNAs, non-coding RNAs have been found to play a key role in plant responses (Chu and Rana, 2006; Wolfswinkel and Ketting, 2010). Transcriptomic studies use different approaches to profile the quantitative or qualitative differential RNA changes in the given condition of an organism (Wang et al., 2009). One of the approaches in studying transcriptomics is the use of microarray analysis that allows identifying gene expression changes using a hybridization technique. However, its application is limited to quantify known genes of the species (Wang et al., 2009). In contrast to the microarray method, RNA-seq is a sequence-based approach, and more reliable because of short reads and deep sequencing (Sims et al., 2014). RNA-seq produces raw data in the form of short strings of nucleotides that are called reads. The reads can be reproducible for any biological and technical replicates. However, technical difficulties may arise in laboratory preparations of cdna that may need to be optimized for different species. Analysis of RNA-seq generated data requires a highly 41

42 precise computational method for mapping and quantifying the genes or transcripts. The computational techniques perform reads cleaning, reads alignment to the reference genome, expression quantification, normalization of gene expression counts, differential gene/isoform expression, and in some cases transcriptome reconstruction (Garber et al., 2011). Citrus-HLB interaction: omics studies Citrus cultivars have shown a various range of reactions to HLB in infected plants. Among these, most of commercially used mandarin, sweet orange and grapefruit varieties are sensitive to HLB, showed strong symptoms of HLB. Whereas, Volkameriana and Eureka lemons, Mexican,' Persian and Palestine Sweet limes, Meiwa Kumquat, Citron, Citrus micrantha, Poncirus trifoliata, and Severnia buxifolia are moderately tolerant or tolerant to HLB. Pummelo varieties; HBP,' Siamese Sweet, Ling Ping Yau, and other citrus such as C. amblycarpa, C. indica, and Cleopatra mandarin showed variable interaction with HLB, exhibiting mild HLB symptoms and less reduction in growth as compared to the sensitive cultivars (Folimonova et al., 2009). The UF-CREC developed the UFR series rootstocks and LB8-9 SugarBelle scion, and the USDA created the US series rootstocks and some scion showing improved tolerance to HLB. The interactions between citrus and HLB are also studied using transcriptomic and proteomic analyses which showed that HLB infection could significantly alter the expression of the genes that are involved in plant defense, hormonal regulations, sugar metabolism, nutrient metabolism, and vascular tissue development. The gene expression and protein profile changes differ with the stage of symptoms, plant tissue, and the cultivars. Some of these interactions are discussed in the following sections. 42

43 Transcriptomic analysis of CaLas-infected leaves of rough lemon and sweet orange Time-course dependent comparative differential gene expression analysis was conducted between CaLas-infected and non-grafted leaves sampled from Rough lemon and Madam Vinous sweet orange using microarray technology (Fan et al., 2012). The results of microarray analysis showed that rough lemon upregulated callose hydrolyzing 1 3 β glucanase transcripts at the late stage (27 weeks after infection) of the infection. Rough lemon also overexpressed group of genes encoding cell wall modifying proteins xyloglucan endotransglycosylases (XET) at the late stage of infection compared to the sweet orange. In addition, CaLas-infected rough lemon leaves showed activation of genes encoding defense associated WRKY transcription factors and signaling kinases at the early stage of infection (5 weeks after infection). In contrast, CaLas-infected sweet orange leaves upregulated defense related genes at the late stage of infection. The results of the comparative transcriptome analysis between CaLas-infected leaves of rough lemon and Madam Vinous sweet orange showed the variety dependent response of citrus to HLB. This study showed that the delayed defense in the sweet orange leaves might contribute to the disease development. Whereas, early stage defense activation and cell wall modifying enzymes in rough lemon might inhibit the further spread of CaLas, and create a barrier to the spread of HLB in the non-infected plant parts. Transcriptomic analysis between CaLas-infected and healthy roots and stem sampled from Valencia /Swingle combination Microarray based comparative transcriptome analysis between HLB-affected and healthy Valencia /Swingle combinations showed that a total of 885 genes in stems and 111 genes in roots were differentially expressed (Aritua et al., 2013). The 43

44 downregulated genes in the HLB-affected plant tissue belonged to carbohydrate metabolism, cell wall biogenesis, biotic and abiotic stress response, transportation, cell wall organization, hormone signaling, metal binding and redox functional categories. In the same study, microscopy analysis of HLB-affected roots was also conducted. Microscopy of the HLB affected stem tissue showed the collapse and thickening of the cell wall, and also showed the depletion of starch in the roots of CaLas-infected plants as compared to the healthy plants. Transcriptomic analysis of DEGs in CaLas-infected leaves between non-grafted US-897 and Cleopatra mandarin rootstocks Microarray assisted comparative transcriptomic analysis between CaLas-infected USDA developed US-897, and commercial Cleopatra mandarin rootstocks (both nongrafted) showed 3412 genes were significant differently expressed in response to CaLas infection (Albrecht and Bowman, 2012b). A total of 326 genes were considerably overexpressed in Cleopatra compared to only 17 genes in the US-897 rootstock. Genes belonging to different biological functional categories were upregulated in CaLasinfected Cleopatra leaves. These include genes encoding expansin proteins (6 fold), pathogenesis related (PR) proteins, enzymes of carbohydrate metabolism, proteins involved in oxidation-reduction processes and other unknown proteins. Genes involved in plant defense were also upregulated (10-40 fold) in Cleopatra leaves. In CaLasinfected US-897 leaves, genes encoding for biotic stress related protein CONSTITUTIVE DISEASE RESISTANCE (CDR1), COPPER/ZINC SUPEROXIDE DISMUTASE 1 (CDS1), and VITAMIN C DEFECTIVE 2 (VTC 2) were upregulated. CDR1 is associated with salicylic acid mediated plant defense. CDS1 is a reactive oxygen species (ROS) scavenger that downregulates the hormful effects of ROS. The 44

45 results of this study suggested that upregulation of antioxidants such as CSD1 and defense related gene CDR1 in US-897 might be a possible mechanisms that counters the attack the HLB pathogen. Overall, the DGE analysis in leaves of CaLas-infected tolerant US-897 and susceptible Cleopatra mandarin suggested that susceptibility or tolerance to HLB is correlated to the magnitude and speed of the plant defense mechanism to counter attack the pathogen infection. Genes associated with HLB susceptibility Symptoms are the physical manifestation of the disease. The disease symptoms are the results of stress generated in the plant because of unfavorable pathogen colonization and/or plant defense response at the different stages of disease development. Comparative DGE analysis of CaLas-infected leaves of susceptible Cleopatra mandarin and tolerant US-897 showed upregulation of genes that were overexpressed only in Cleopatra but not in US-897 (Albrecht and Bowman, 2012b). Among these, expression of the gene encoding Myb-like HTH transcriptional regulator family protein was found to be increased by 200-fold in Cleopatra but not in US-897. A Myb-like transcriptional regulator was also expressed in Bos Noir phytoplasma infection of grapevine and related to the resistance to the phytoplasma infection (Albertazzi et al., 2009). In the case of HLB, higher expression of the gene encoding for Myb-like HTH transcriptional regulator was found in susceptible Cleopatra cultivar. Therefore, this study suggested that the Myb-like HTH transcription regulator, unlike in grapevine disease, is associated with HLB susceptibility (Albrecht and Bowman, 2012b). HLBaffected plants exhibit symptoms similar to that of Zn deficiency. Therefore, susceptible CaLas-infected Cleopatra showed upregulation of ZINC TRANSPORTER 5 PRECURSOR (ZIP5) to compensate for disease-induced Zn deficiency. Upregulation of 45

46 ZIP5 was also observed in HLB-affected Valencia fruit (Martinelli et al., 2013). CaLasinfected plants show excessive accumulation of starch (Etxeberria et al., 2009). CaLasinfected Cleopatra mandarin and other sweet oranges such as Valencia and Madam Vinous showed the significantly elevated level of genes encoding enzymes involved in the starch biosynthesis (Fan et al., 2012; Albrecht and Bowman, 2012b). The higher accumulation of starch in HLB-affected plants is supported by a phenomenon called microbial volatile-induced starch accumulation process (MIVOISAP) which is a microbial strategy for the survival inside the host tissue where pathogen-induced volatiles affect the carbohydrate metabolism and accumulation of starch in the plant organs (Ezquer et al., 2010). Also, downregulation of plastidial thioredoxin level is associated with plant pathogen activated volatiles which cause starch accumulation (Ezquer et al., 2010). Thioredoxin family protein level was also downregulated in CaLas-infected Cleopatra suggesting its connection to the starch accumulation and therefore, susceptibility to HLB. Transcripts encoding PHLOEM PROTEIN 2-B15 (PP2-B15) was strongly upregulated in Cleopatra compared to the US-897. At the initial stage of HLB development, upregulation of transcripts encoding PP2 is beneficial in being involved in differentiation of vascular tissue and plugging of sieve plates. In the advanced stage of HLB, upregulated expression of transcripts encoding PP2 results in a barrier for the translocation of nutrients through phloem which ultimately creates local or systemic nutrient deficiencies. A homologue of GAST1 (GASA1) was found to be highly upregulated (>15 fold change) in the CaLas-infected Madam Vinous sweet orange leaves compared to the CaLas-infected rough lemon leaves, suggesting its potential role as an HLB-susceptibility associated gene (Fan et al., 2012). 46

47 Genes related to HLB tolerance In the absence of HLB resistance, long-lasting HLB tolerance is vital to keep citrus production sustainable. Citrus types such as certain pummelos, rough lemon, Poncirus trifoliata, and some other citrus hybrids have been found to exhibit tolerance to HLB. However, the endurance and production capacity of the HLB-tolerant cultivars are not verified. Transcriptome comparison of non-grafted tolerant US-897, and commercial and HLB-susceptible Cleopatra rootstock was reported (Albrecht and Bowman, 2012b). The genes that were upregulated in CaLas-infected US-897 and healthy Cleopatra identify HLB-tolerance candidate genes. Among these, oxido-reductase family 2- OXOGLUTARATE (2OG) AND FE (II) DEPENDENT OXYGENASE, VEIN PATTERNING 1 (VEP1), GLUCOSE TRANSPORTER 1 (GLT1), that are involved in plant defense, herbivore defense, and in export of photoassimilates from chloroplasts respectively, were upregulated over 30-fold (Albrecht and Bowman, 2012b). UDPglycosyl transferase (UGT) superfamily proteins are crucial to impart tolerance to the virus infection in tobacco. Transcripts encoding UGT superfamily proteins were expressed in abundance in non-infected US-897. Transcripts encoding PR proteins; OSMOTIN LIKE PROTEINS (OSM 34) and PLANT DEFENSIN (PDF2.2) were also overexpressed in CaLas-infected US-897 (Albrecht and Bowman, 2012b). Proteomic analysis conducted by Martinelli et al., (2016) reported the significant role of detoxification enzyme; S-glutathione transferases in the moderate HLB tolerant Volkamer lemon leaves. Transcriptomic comparison between putative tolerant Jackson grapefruit and susceptible Marsh grapefruit showed the upregulated expression of auxin-negative regulators SAUR-like genes and upregulated basal defense genes in the Jackson grapefruit (Wang et al., 2016). UF-CREC developed 47

48 UFR series rootstocks and SugarBelle scion have also shown sustainable tolerance to HLB in different scion/rootstock combinations (Stover et al., 2016). However, genetic regulators that contribute the potential tolerance in the UFR rootstocks and LB8-9 SugarBelle scion need to be analyzed. Comprehensive transcriptome and gene expression network analysis of HLBcitrus interactions A comprehensive view of HLB response networks was presented using different HLB-citrus transcriptomic studies (Zheng and Zhao, 2013). The results of the previously studied HLB-citrus interaction transcriptome datasets were used to build a system view of the HLB response network using Pearson correlation coefficient-based gene coexpression analysis. The gene coexpression networks were presented by the core network, subnetworks, and hubs that showed the interrelation of the genes with each other. Among these, carbohydrate metabolism, transport, and hormone response networks had large hubs that were then further subdivided into subnetworks. The hormonal network showed that plant hormones such as ethylene (ET), abscisic acid (ABA), salicylic acid (SA), jasmonic acid (JA), and gibberellin (GA) response network were overrepresented in the HLB-citrus interaction. Subnetwork analysis of SA showed overrepresentation of protein degradation component UBIQUTINATION 10-LIKE (UBQ10), transcriptional regulator WRKY40, ASYMMETRIC LEAVES 1 (AS1), MYB, and carbohydrate metabolism enzyme GSTU7. At the early stage of CaLas infection, it is difficult to find any visible HLB symptoms, but transcriptome analysis of early stage HLB infection showed that the plant is already responding to the HLB infection by overexpressing associated defense genes. In the early stage, the HLB response subnetwork showed upregulation of ENHANCED DISEASE SUSCEPTIBILITY (EDS-1) 48

49 like gene, transcripts encoding Tetratricopeptide repeat (TPR)- like superfamily protein and NAC domain containing NAC096 transcription factor. Further subnetwork analysis revealed that transport is a key process that overexpressed in the HLB response. Overexpression of the PP2-LIKE gene was reported in resistance to phloem feeding aphids in Arabidopsis (Zhang et al., 2011a). Upregulation of closest homolog of Arabidopsis PP2 in citrus at the late and very late stage of infection suggests its role in defense response to CaLas infection. The subnetwork of PP2 showed the presence of genes encoding Zn transporter proteins. The presence of Zn family transporter transcripts in the PP2 subnetwork suggests that Zn binding proteins are important in arresting the development of disease at an early stage of infection. The results of this work also showed the overlapping and interconnection of different biological processes that were activated or suppressed in response to HLB and will be helpful in understanding the key genes and mechanisms that are potentially involved in regulation of CaLas-citrus interaction. Citrus co-expression networks built from microarray data also showed significant presence of HLB network (Du et al., 2015). The results of this study showed 28 nodes, 75 edges, and density of the HLB network using random matrix theory. The highest rank gene ontology (GO) terms represented in HLB module was programmed cell death (PCD). The PCD network showed upregulation of transcripts encoding POLYUBIQUITIN 10, SPHINGOID BASE HYDROXYLASE, Glutaredoxin family proteins, RING finger E3 ubiquitin ligases, and BCL-2-ASSOCIATED ANTHOGENE. 49

50 Transcriptomic and proteomic analysis of CaLas-infected and non-infected roots of Sanhu red tangerine Comparative analysis of transcriptome and proteome of CaLas-infected and noninfected roots of Sanhu red tangerine showed a total of 3956 genes, and 70 proteins were differentially regulated (Zhong et al., 2015). CaLas-infected Sanhu tangerine roots showed downregulation of genes involved in ubiquitin dependent protein degradation pathway, secondary metabolism, cytochrome P450s, UDP-glucosyl transferase, and pentatricopeptide repeat containing proteins. Upregulation of SPERM SPECIFIC PROTEIN 411, COPPER ION BINDING protein, GERMIN-LIKE proteins, SUBTILIN-LIKE proteins and SERINE CARBOXYPEPTIDASE-LIKE 40 protein were observed in the proteomic comparisons, and the transcriptomic study showed that the transcripts encoding same genes were also uregulated in the same comparison suggesting CaLas has evolved a counter attack to the host through proteolysis. CaLasinfected Sanhu roots showed upregulation of a gene encoding PP2, and downregulation of GLS7, a callose synthase gene. A gene encoding Beta-glucosidase, a callose hydrolyzing enzyme, was upregulated in the CaLas-infected roots as compared to the non-infected roots in Sanhu.' Based on these results, authors of this study concluded that the roots of Sanhu tangerine are trying to achieve a balance between callose synthesis and degradation enzymes as a part of the defense against HLB. Proteomic analysis between healthy and CaLas-infected Madam Vinous sweet orange itraq based proteomic analysis between healthy and CaLas-infected sweet orange leaves showed differential proteomic changes in the asymptomatic and symptomatic leaves (Fan et al., 2011). A total of 686 proteins were expressed uniquely 50

51 in the differential comparison between asymptomatic and symptomatic leaves. Among these, MIRACULIN LIKE proteins, CU/ZN SUPEROXIDE DISMUTASE, and CHITINASE were significantly accumulated in the CaLas-infected asymptomatic leaves. Comparative proteomic analysis of symptomatic and pre-symptomatic CaLasinfected grapefruit leaves Duncan grapefruit leaves were analyzed for comparative protein profiles changes in response to the CaLas infection using 2D protein gels (Nwugo et al., 2013b). This study identified 191 significantly altered protein spots in response to CaLas infection. Of these, 56 proteins were downregulated in CaLas-infected and symptomatic Duncan leaves as compared to healthy leaves. These proteins were associated with photosynthesis, protein synthesis, and metabolism. Whereas, an OXYGEN-EVOLVING ENHANCER (OEE) and RNA polymerase B TRANSCRIPTIONAL FACTOR 3 (BTF3) proteins were downregulated in pre-symptomatic and symptomatic Duncan leaves. This study also found CU/ZN SUPEROXIDE DISMUTASE, CHITINASE, lectin-related proteins, MIRACULIN-LIKE proteins, and peroxiredoxin proteins were upregulated in CaLas infected Duncan leaves. The significance of this study was to establish the correlation between protein and nutritional profiles in CaLas-infected and symptomatic Duncan leaves, where increased concentrations of potassium in the CaLas-infected Duncan leaves were correlated to the upregulated granule-bound starch synthase proteins. Research Overview Since the first incidence of HLB in Florida, there are a considerable number of studies conducted that have generated a lot of information about the HLB disease complex. The lack of enough information about CaLas biology limits the research 51

52 opportunities to find solutions for HLB control. HLB is a high impact disease that is causing vast economic losses to the Florida citrus industry. The contemporary HLB research is focused on developing CaLas and psyllid control strategies including thermotherapy, antibiotics, and chemical control. However, to control the endemic spread of HLB, economically viable solutions are required. The vulnerability of existing commercial citrus cultivars to HLB has been studied under both laboratory and field conditions. The improved citrus scions or rootstocks may bring a practical solution that can reverse the negative impact of HLB in CaLas-infected plants. Therefore, economically enduring-hlb tolerant citrus cultivars can be a long-term solution to combat the existing sustainability issues associated with HLB-affected plants. There are a few rootstocks and scion cultivars showing some enhanced level of HLB tolerance that have been released for commercial citrus production, and several additional promising selections in the breeding pipeline. The overall goal of this study is to identify the molecular mechanisms that potentially contribute to the sustainability of Valencia sweet orange scion grafted onto two selected putative HLB-tolerant CAN rootstocks currently in the breeding pipeline: 42x ( HBP x Cleopatra mandarin) and 4x hybrid ( Amblycarpa mandarin + Volkamer lemon cybrid) x [ Nova + HBP pummelo x Argentine trifoliate orange]. In addition, the effect of root applied enhanced controlledrelease micronutrient formulations was also analyzed to test the performance of the HLB susceptible Valencia/Swingle as well as the latter Valencia/CAN combination above. The results of this study will be a useful contribution in developing HLB tolerant cultivars by providing insights into the mechanisms responsible for the tolerance 52

53 response. Finally, a better understanding of rootstock genetics/nutrition interactions should lead to improved nutrition practices for HLB management. 53

54 CHAPTER 2 COMPARATIVE ANALYSIS OF TRANSCRIPTOME AND PLANT PHENOTYPES OF CaLas-INFECTED SCION/ROOTSTOCK COMBINATIONS Introduction In Florida, commercial citrus production is focused on growing sweet orange (Citrus sinensis [L.] Osbeck) and grapefruit (Citrus paradisi [Macf.]) varieties. Sweet orange cultivars are grown for homeland and premium export quality juice production. However, HLB-affected citrus trees make it more difficult to fulfill the export quality juice standards because of off-flavors and color, and lowered Brix-acid ratio. The endemic spread of HLB has resulted in the consistently declining net productivity of HLB-affected citrus orchards. In , Florida commercial citrus production was the lowest recorded citrus production with increased gross loss as compared to previous years since 2009 (USDA, 2014). Increasing economic losses caused by HLB in Florida, and the lack of true resistance to HLB in the citrus germplasm demand economically enduring HLB-tolerant cultivars that have potential to reduce HLB severity and increase sustainability of HLB-affected plants. Field- and greenhouse-based studies of the interaction between various Citrus species and closely related genera with HLB have been conducted. The interactions between citrus cultivars and HLB are classified as sensitive (chlorosis, vein corking and reduced growth, death), tolerant (little or no HLB symptoms), moderately tolerant (mild HLB symptoms on older leaves or scattered chlorosis on groups of leaves), or inconsistent presence or absence of bacteria based on symptomatology and Candidatus Liberibacter asiaticus (CaLas) bacteria titer in the plants (Folimonova et al., 2009). Of these responses, tolerant or moderately tolerant responses were found in some closely related species and genera of citrus. Trifoliate orange (Poncirus trifoliata [L.] Raf), rough lemon (Citrus jambhiri Lush.), pummelos 54

55 (Citrus maxima Merr.), lemons (Citrus limon L. [Burm.] f.), and their hybrids have shown tolerance to HLB (Fan et al., 2012; Albrecht and Bowman, 2012b; Aritua et al., 2013; Fan et al., 2013; Martinelli et al., 2016). However, the tolerance of these citrus species and their hybrids is not consistent. The performance consistency of HLB-tolerant cultivars is highly dependent on the psyllid control, interaction with other diseases, environmental challenges, nutritional program, and farm management practices (Gottwald et al., 2012; Haapalainen, 2014; Castle, 2015). Against all the odds of HLB, citrus breeders have engaged in developing improved citrus cultivars for HLB tolerance, and perhaps even resistance using different breeding approaches. The University of Florida, Citrus Research and Education Center (UF-CREC) at Lake Alfred is the world renowned citrus research facility. UF-CREC research teams have contributed to the Florida citrus industry over the past 100 years through their research, extension, and teaching programs ( The breeding program at UF-CREC has always been engaged in developing the improved citrus cultivars that can fulfill the demand of growers and citrus-associated food industries (Grosser et al., 2015). In the UF-CREC citrus breeding program, putative HLB-tolerant rootstock and scion hybrids have been developed by crossing non-commercial HLBtolerant and commercially accepted citrus cultivars. There is no citrus cultivar available to defend against HLB and improved plant performance when the plant is CaLasinfected. However, HLB tolerance can be improved by enhancing regeneration ability of the phloem, regrowth of the affected root system, and reducing CaLas titer. Differential gene expression (DGE) analysis of CaLas-infected rough lemon and sweet orange showed the higher phloem regeneration ability of rough lemon (Fan et al., 2012). Rough 55

56 lemon tolerance to HLB is also reported in India (Cheema et al., 1982). Some of the pummelo cultivars and trifoliate orange are found to be less affected by HLB (Shokrollah et al., 2011). However, these cultivars cannot produce commercially accepted fruit and juice as compared to the commercially used rootstock/scion combinations. In Florida, commercially used rootstock cultivars: Swingle citrumelo (Citrus paradisi x Poncirus trifoliata), Cleopatra mandarin (Citrus reticulata Blanco), Carrizo citrange (Poncirus trifoliata X Washington sweet orange), and scions Valencia and Hamlin sweet oranges, and grapefruit cultivars are HLB susceptible. Therefore, to create HLB-tolerant and commercially viable cultivars, hybrids between HLB-tolerant Citrus species and high quality fruit producing cultivars are required. The UF-CREC citrus breeding program has developed scion and rootstock hybrids which exhibit HLB-tolerance with improved plant performance. The newly developed putative-hlb tolerant rootstocks have been showing promising results in mitigating HLB severity in the CaLas-infected plants. Some of these putative HLBtolerant rootstocks, which have been commercially available since 2009, yield higher fruit production and increased juice quality in grafted CaLas-infected scion (Castle et al., 2016). Also, some of the scion hybrids have shown reduced HLB symptoms. However, there is no published data available on commercial scion tolerance except LB8-9 SugarBelle variety (Stover et al., 2016). The tolerance of the newly developed cultivars might not be economically viable because of possible scion-rootstock compatibility issues, the impact of rootstock on the scion, or undesirable changes in the fruit quality. Field evaluations of the CaLas-infected scion/newly developed rootstock combinations are important to test the economic viability of the new rootstock hybrids. A field trial of 56

57 one of these newly developed and putative HLB-tolerant candidate rootstocks (CAN) exhibited superior plant phenotype of CaLas-infected Valencia scion grafted onto it as compared to the Valencia/Swingle control combination after 7 years in the field under heavy HLB pressure. The performances of these combinations were measured in terms of visual inspection of HLB symptoms on leaves and fruit quality parameters (Brix, acid, Brix-acid ratio, and total sugars.). The consistency of HLB-tolerance and plant sustainability of CaLas-infected Valencia /CAN suggested a more enduring performance of the rootstock when the plant is CaLas-infected. Therefore, Valencia /CAN trees were selected to analyze transcriptomic differences in the leaves and roots of trees identified in the field exhibiting asymptomatic and symptomatic stages as compared to the commercial standard Valencia /Swingle combination. In the contemporary biological sciences, omics-assisted methods of research are very popular and widely used to reveal the regulatory mechanisms in the different living organisms. The omics studies require precision instrumentation and high throughput computing technology to give quicker and highly accurate results. The demand and competition between technological developments are out competing the old techniques of omics-assisted research, and transcriptome studies are no exception. The next generation sequencing-based RNA sequencing (RNA-seq) techniques are rapidly evolving. RNA-seq not only identifies the known transcripts/genes but also discloses the unidentified genes, identifies alternate splicing events, quantifies the isoforms, measures differential allelic expression, and detects non-coding RNAs. High throughput, improved resolution, and less background noise have increased the popularity of NGS techniques in the global gene expression studies of plants, animals, microbes, and 57

58 humans. The International Citrus Genomics Consortium (ICGC) is a collaboration of a worldwide group of scientists, students, and industry leaders associated with citrus. The goal of ICGC is to understand the underlying genetic structure of citrus. Many diverse pummelo, mandarin and sweet orange citrus genomes have sequenced (Xu et al., 2013; Wu et al., 2014). The Clementine mandarin (Citrus clementina) and sweet orange genomes have also sequenced, and data are available on the Phytozome portal. Phytozome is the plant comparative genomics portal of the Department of Energy s Joint Genome Institute (Goodstein et al., 2012). The NGS or microarray technologies have been used to perform DGE analysis in many citrus studies. These studies are physiological development (Terol et al., 2016), nutrient deficiency (Licciardello et al., 2013), disease and pest interactions (Gandía et al., 2007; Boava et al., 2011; Wang et al., 2016), genotypic differences, and gene expression network (Du et al., 2015). HLBcitrus interactions are also studied using global gene expression transcriptomic approach (Albrecht and Bowman, 2008; Fan et al., 2011; Febres et al., 2012; Martinelli et al., 2012). RNA-seq technology has been used successfully in understanding the molecular mechanisms that cause phenotypic changes. The aim of this study is to determine potential molecular mechanisms that enhance sustainability in Valencia grafted onto the CAN rootstock with RNA-seq. The results of this study are important to the understanding of possible molecular mechanisms which are involved in reducing HLB damage in citrus and developing HLB-tolerant or resistant citrus cultivars using conventional breeding as well as advanced biotechnological approaches such as gene transfer and genome editing. 58

59 Materials and Methods Plant Materials Two scion/rootstock combinations were used in this experiment. Field grown seven year-old experimental plants were identified in the Lee Family's Alligator Grove, east of St. Cloud, Florida. The first combination of trees was Valencia' (VAL) sweet orange grafted onto a putative HLB-tolerant candidate (CAN) rootstock. The CAN rootstock (46x ) is a hybrid of Hirado Buntan Pink' (HBP) pummelo and Cleopatra mandarin. The 2 nd combination was Valencia' (VAL) scion grafted onto Swingle citrumelo (SW), a standard commercial rootstock. Swingle is a hybrid of grapefruit and trifoliate orange. In each combination of VAL/CAN and VAL/SW, plants were divided into two treatments based on the visible presence of HLB-like symptoms (Table 2-1). Highly infected and symptomatic trees in each combination grouped into the symptomatic treatment (Figure 2-1A and B; Figure 2-2A and B). Whereas, trees with very few or no visible symptoms were grouped into the asymptomatic treatment (Figure 2-1C and D; Figure 2-2C and D). All biological replicates in each treatment and combination were tested using quantitative PCR (qpcr) based CaLas detection, which validated that all trees in the study were indeed infected. Enzyme-linked immunosorbent assay (ELISA) was performed to detect Citrus Tristeza Virus (CTV) in the samples. Sampling Sampling of different plant organs (leaves, roots, and fruits) was conducted to study DGE analysis and fruit juice quality of the plants. In each treatment of both scion/rootstock combinations, leaves and fibrous roots from all quadrants of the tree were collected for CaLas detection, DGE analysis and real time PCR based gene expression validation studies, in April Leaves (December 2014, April 2015, 59

60 December 2015) and fruits (April 2015 and April 2016) were sampled in two consecutive years to analyze changes in HLB status and fruit juice quality. Three separate biological replicates of asymptomatic and symptomatic treatments were selected in each VAL/CAN and VAL/SW combination for DGE analysis and juice quality testing. Leaf and root tissues collected for the RNA sequencing, were saved at -80º Celsius until RNA extraction. Fruit Juice Quality Analysis Juice quality of fruit collected from each biological replicate in each treatment was analyzed. In each biological replicate, approximately fruit were collected to analyze the juice quality. The commercial juice quality is judged by its Brix, acid percentage, and Brix-acid ratio. The same analyses were conducted for fruit harvested in years 2015 and 2016 from the both treatments of VAL/SW and VAL/CAN combinations. Fruit Juice analyses were conducted at the Tropicana facility located at Bradenton, FL. CaLas and Citrus Tristeza Virus (CTV) Detection Approximately 10 fully expanded leaves and enough root tips were collected from both the treatments in VAL/CAN and VAL/SW combinations to extract DNA for CaLasdetection. GenElute Plant genomic DNA Miniprep kit was used to extract DNA from the petiole and leaf midrib (Sigma Aldrich, Woodlands, TX). Root DNA was isolated using PowerMax Soil DNA Isolation kit (MOBIO Laboratories, Inc., Carlsbad, CA). The extracted DNA was then quantified in a NanoDrop ND-100 spectrophotometer (Thermo Scientific, Wilmington, DE). The qpcr assays were performed using CaLas specific probe and primer pairs which are developed by Ananthakrishnan et al. (2013). The cytochrome c oxidase (cox) subunit Vc gene forward and reverse primer pair, and 60

61 corresponding probe were used as internal control. Amplification was performed for 40 cycles using ABI7500 real-time PCR machine (Applied Biosystems, Foster City, CA) and the TaqMan Gene Expression Master Mix (Applied Biosystems). All reactions were carried out in a 25 µl reaction volume containing 50 ng DNA and 0.3 µm probe and primer pair concentrations. The CTV detection was performed using the ELISA plates that are pre-coated with CTV specific antibody (Agdia Co, Elkhart, IN). A total of 0.25 gram of leaf midrib was used to diagnose the CTV from each leaf sample. The procedure for CTV detection was performed according to the manufacturer s protocol (SRA78900, Agdia). CTV-36 and healthy plant tissue were used as the positive and negative controls, respectively. RNA Extraction and Quantification PureLink Plant RNA Reagent-small scale procedure was used to extract total RNA from 0.1 gram samples of leaves or roots (ThermoFisher Scientific, Waltham, MA). PureLink Plant RNA reagent contains Trizol reagent which uses the guanidium isothiocyanate RNase inhibitor and phenol-chloroform protein extraction method. It is a non-column based RNA extraction method that may cause DNA contamination in the eluted product. Hence, total extracted RNA was treated with DNase. Approximately 100 µl of eluted total RNA was treated with DNA-free kit (ThermoFisher Scientific). For each biological replicate of leaf and root samples for both treatments, RNA was isolated twice and then pooled together as one sample for downstream processes. RNA extracted from leaves was tagged A and B followed by the experiment ID, and the roots RNA samples named as C and D followed by the experiment ID (Table A-1). The quality and quantity of DNase treated total RNA were determined using the 0.8% agarose gel and Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA). RNA integrity (RIN) 61

62 number generated by Bioanalyzer was used to determine the quality of RNA and its acceptability for RNA-seq library preparations. RNA Library Preparation and RNA Sequencing DNase treated total RNA with acceptable RIN value was used to prepare RNA libraries. Poly-A mrna magnetic isolation kit (NEW ENGLAND Biolabs Inc, Ipswich, MA) and NEBNext Ultra Directional RNA Library Kit for Illumina (NEW ENGLAND Biolabs Inc) were used to prepare an RNA library for each sample. NEBNext Ultra Directional RNA Library Kit for Illumina contains Illumina-specific adapters and multiplexing index that can be used in Illumina sequencing platform. The NEBNextdeveloped protocol is optimized for 200 base pair (bp) RNA insert. For RNA library preparation, one microgram RNA was used as the starting amount. The manufacturer s protocol was followed to prepare RNA libraries of each sample. The protocol contains steps for preparation of first strand reaction buffer and random primer mix, mrna isolation, fragmentation and priming starting with total RNA, first strand cdna synthesis, second strand cdna synthesis, purification of the double-stranded cdna using 1.8X Agencourt AMPure XP Beads (Beckman Coulter Inc., Indianapolis, IN), end preparation of cdna library, adaptor ligation, purification of the ligation reaction using AMPure XP Beads, PCR enrichment of adaptor-ligated DNA, and purification of the PCR reaction using AMPure XP Beads. A total 24 RNA libraries was prepared and sent to the UF Interdisciplinary Center for Biotechnology Research (ICBR) gene expression core for sequencing. All RNA libraries were diluted and then pooled together to test the quality parameters suitable for sequencing (Table A-2). Two sets of pooled libraries were used for RNA sequencing. Paired-end sequencing, 2 x 150, was carried on Illumina Nextseq500 high throughput platform using the sequencing by synthesis (SBS) method. 62

63 Raw Data Processing Nextseq500-generated raw sequencing data were processed using the Tuxedo bioinformatics pipeline to get the comparative DGE results (Trapnell et al., 2012) (Figure A-1). The UF High Performance Computing (HPC) developed HiPerGator.1 server was used to compute and store data processing results. Illumina Nextseq500 platform generates raw data in the form of fastq files. Raw data processing was executed using a Unix-based shell script command line (Table A-3). The raw data obtained from each biological replicate were organized into directories to perform the pairwise comparison between the treatments, tissues, and the scion/rootstock combinations (Table 2-2). The Trimmomatic program was used to remove the low-quality reads and trim the Illumina adapters by assigning leading (20), trailing (20), slidedown (4:20), minlen (36), and phredscore (33) parameters. Trimmomatic algorithm-generated output files contained the data which were ready to use for the alignment. The Bowtie 2 program was used to index the Trimmomatic generated clean sequencing reads and C. clementina, a reference genome (Phytozome.org.net). The indexed RNA seq reads were then mapped to the indexed C. clementine genomic data using a TopHat2 algorithm (Trapnell et al., 2012). Cufflinks and Cuffmerge algorithms were used to quantify the abundance of the transcripts in each biological replicate and then consolidated into a master transcriptome file (Trapnell et al., 2012). The differential gene expression changes between the treatments (Table 2-2) were analyzed using the Cuffdiff program. Cuffdiff normalizes the expression level of differentially expressed genes (DEGs) based on fragments per kilobase per million (FPKM) as explained by Trapnell et al., The DGE changes were expressed in the log2 fold change (log2fc). CummeRbund, an R 63

64 statistical package based program, was used to overview and visualize the results of Cufffdiff output (Goff et al., 2012). Functional Analysis and Gene Ontology of DEGs DEGs were chosen based on significance determined by a Cuffdiff algorithm, and used for gene ontology (GO) and functional analysis studies. DEGs obtained in the comparative analysis between VAL/CAN and VAL/SW treatments were annotated to the Arabidopsis thaliana genes, which are available in the Phytozome database. Functional analysis of the differentially expressed and A. thaliana annotated genes was performed using MapMan tool (Usadel et al., 2009).The signaling pathways and regulatory components of the significant differentially expressed genes were analyzed using Pathway Studio (Nikitin et al., 2003) and MapMan. DEGs were assigned into gene ontology (GO) biological processes using a Blast2GO algorithm (Conesa et al., 2005). The biological importance of the significant differentially expressed genes was determined by combining the results from MapMan, Blast2GO and Pathway Studio. Validation of the RNA-seq Data Quantitative real-time reverse transcriptase (qrt-rt) PCR technique was used to validate the genes that were differentially expressed in the RNA-seq analysis. Seven genes were selected to validate gene expression from RNA-seq results (Figure 2-9). These genes were selected based on their significant changes in the expression of levels in the leaves and roots of symptomatic and asymptomatic treatments between VAL/CAN and VAL/SW combinations. These genes encode BON ASSOCIATED PROTEIN 2 (BAP2), BR (Brassinosteroid) LEUCINE RICH RECEPTOR BRI1, NPR1- like 3 (NPR3), MYO-INOSITOL PHOSPHATE SYNTHASE 2 (MIPS2), Zim domain containing JAZ8, Pectin methyl esterase inhibitor (PMEI) and LIPID TRANSFER 64

65 PROTEIN 1 (LTP1). The gene encoding BRI1 (Ciclev ) is a BR receptor kinase and found to be significantly upregulated in the asymptomatic VAL/CAN leaves as compared to the asymptomatic VAL/SW leaves. BAP2 is identified as a negative regulator of programmed cell death (PCD) in Arabidopsis (Yang et al., 2007). The loss of BAP2 function was found to induce a hypersensitive response in Arabidopsis. BAP2 was significantly upregulated in the symptomatic leaves and roots, and downregulated in the asymptomatic leaves as compared to respective treatments and organs in the VAL/SW, Therefore, BAP2 was selected to validate the (Ciclev ) expression. NPR3 is a SA signaling receptor, however, it is a negative regulator of defense response through its interaction with NPR1 (Shi et al., 2013). Significant downregulation of NPR3 (Ciclev ) in the asymptomatic and symptomatic VAL/CAN leaves indicates the possibility of upregulating NPR1 expression and, therefore NPR1- induced defense in the VAL/CAN leaves as compared to VAL/SW. Considering the significant role of NPR3 in regulating NPR1 expression, NPR3 was selected to validate its expression pattern. MIPS is involved in a rate limiting step in the synthesis of myoinositol which is a critical component in the developmental stages (Loewus, 1970; Donahue et al., 2010). The gene encoding (Ciclev ). was upregulated in the asymptomatic and symptomatic VAL/CAN leaves as compared to the respective VAL/SW leaves. Therefore, MIPS2 was selected to validate its expression pattern. JAZ8 acts as JA repressor (Wasternack and Hause, 2013). Significant changes in the expression of JAZ8 may be a clue to understand a JA-SA antagonism in CaLas-infected plants. Therefore JAZ8 (Ciclev ) was selected to validate its expression pattern. PMEIs are involved in plant development and defense. The overexpression PMEI in 65

66 Arabidopsis is found to influence the Botrytis cinerea induced susceptibility, and thereby, confers resistance to Botrytis cinereal (Lionetti et al., 2007). Therefore, PMEI (Ciclev ) was selected to validate RNA-seq expression results. Lipid transfer proteins (LTPs) is a small class of proteinsthat is involved in various plant functions such as cutin formation, embryogenesis, defense against phytopathogens, and plant adaptations to various environmental conditions (Finkina et al., 2016). LTP1 (Ciclev ) expression was significantly downregulated in asymptomatic and symptomatic VAL/CAN leaves as compared to respective VAL/SW leaves. Considering the critical role of LTP in plant growth and defense, and significant downregulation in VAL/CAN leaves, LTP1 was also selected for validation. Forward and reverse primers were designed for the selected genes using Integrated DNA Technologies (IDT) technologies Primer Quest Tool (IDT, Coralville, IA), and then amplified using a SYBR green based gene expression protocol. For normalization, the GLYCERADEHYDE-3-PHOSPHATE DEHYDROGENASE C2 (GAPC) gene was used as the reference gene control. Power SYBR green RNA-to- Ct 1-Step RT-PCR (Applied Biosystems) master mix was used to convert total RNA into cdna, and then cdna fragments were amplified with the primer pair. A total of 50 ng DNase free RNA and 500 nm of each primer concentration were used to amplify and quantify the qrt-rt gene expression. Amplification was performed over 40 cycles on a StepOnePlus real-time PCR machine (Applied Biosystems). Relative gene expression quantification was calculated using the ΔΔCt method (Livak and Schmittgen, 2001). For each sample, the Ct value of GAPC was subtracted from the Ct value for the gene of interest (ΔCt). ΔΔCt was calculated by subtracting ΔCt 66

67 of VAL/SW from ΔCt of VAL/CAN, and the results are presented as log2fc scale. The visual presentation of the data was created using Microsoft Office Excel10. Results Fruit Juice Quality Analysis In 2015, the Brix values of fruit collected from all the treatments and combinations were not significantly different. The average Brix value of fruit collected from the asymptomatic VAL/SW was higher followed by asymptomatic VAL/CAN, symptomatic VAL/SW and symptomatic VAL/CAN (Figure 2-3A). In 2015, although the Brix value remained nonsignificantly different in fruit collected from all scion/rootstock combinations, the acidity percentage was significantly higher in fruit collected from the symptomatic VAL/SW plants (Figure 2-3A). In 2016, fruit collected from the symptomatic VAL/CAN combination followed the similar pattern of Brix, acidity percentage, and BAR ratio as the symptomatic and asymptomatic VAL/SW combination (Figure 2-3B). PCR Detection of HLB and ELISA Detection of CTV All 24 samples were tested to identify the CaLas presence in leaf and root tissues collected from the symptomatic and asymptomatic treatments of VAL/CAN and VAL/SW combinations (Table 2-3). In April 2015, leaves collected from asymptomatic VAL/SW biological replicates were CaLas negative; whereas roots collected from asymptomatic VAL/SW were CaLas positive (Table 2-3). All biological replicates in symptomatic and asymptomatic VAL/SW combination were CTV positive except one biological replicate (sample #21) in the asymptomatic treatment. In the same year, symptomatic leaves and roots collected in VAL/SW combination were all CaLas positive. In asymptomatic VAL/CAN combination, leaves collected from sample #22 was CaLas and CTV negative, and sample #23 leaves were CaLas and CTV positive. In 67

68 asymptomatic VAL/CAN combination, sample #24 was CTV-infected with the absence of CaLas in the leaves, but roots were CaLas-infected. All biological replicates in the symptomatic VAL/CAN combination were CTV negative; however, VAL leaves were CaLas positive (Table 2-3). Roots sampled from symptomatic VAL/CAN combination were either negative (sample #5) or found to have higher Ct value in the range of In December 2015, all biological replicates in symptomatic and asymptomatic treatments of VAL/CAN and VAL/SW combinations were PCR CaLas positive (Table 2-3). RNA Extraction, RNA Library Preparation, and RNA Sequencing RNA extracted from leaves showed a 260/280 ratio of 2 and clear separation of 18s and 26s ribosomal bands on the 0.8% agarose gel (Figure 2-4A). Whereas, it was difficult to get high quality RNA from root tissue (Figure 2-4B). Multiple RNA extractions were conducted to get high quality RNA from the roots. The average size of libraries prepared for the sequencing was 390 bp as quantified by qpcr results at the ICBR facility (Table A-2). The leaf samples tested in this study obtained about million reads/sample, and the root samples obtained million reads/sample (Table 2-4). Raw Data Processing Trimmomatic trimming, Bowtie indexing, and Tophat alignment generated 60% of mapped and paired reads of total raw reads in leaf samples of both the treatments. except two leaf samples in the asymptomatic VAL/CAN combination that generated a lower percentage of alignment to the reference genome. In root samples of both the treatments, a total 56 to 97% of the raw reads were mapped to the C. clementine genome (Figure 2-5). The leaf and root samples that generated a lower percentage of alignment to the reference genome were re-sequenced to increase the reliability of the 68

69 DEG analysis. The Cuffdiff significance test showed that comparative analysis between the asymptomatic treatment of both scion/rootstock combinations had 1163 genes that were significant differentially expressed in leaves with a false discovery rate (FDR) q 0.04 (Figure 2-6A). Whereas roots of the asymptomatic treatment showed very few, only 49 genes were significant (q 0.04) differentially regulated. Comparative transcriptomic analysis of leaves of the symptomatic VAL/CAN and VAL/SW combination showed 1437 significant (q 0.04) differentially expressed genes (Figure 2-6B). Whereas, roots in the same treatment showed 2025 significantly regulated (q 0.04) genes (Figure 2-6C). Functional Analysis of Differentially Expressed Genes Functional analysis software: MapMan, Blast2Go, and Pathway Studio were used to find functional annotation of the differentially regulated genes. Comparative gene expression analysis between leaves of the asymptomatic VAL grafted onto CAN and SW rootstock showed differentially regulated genes belonged to signaling, transport, biotic stress, hormone metabolism, jasmonic acid (JA) and ethylene (ET) stimulus, salicylic acid (SA) signaling pathway, response to chitin and wounding, intracellular signal transduction, starch and sucrose metabolism, response to water deprivation functional, and transport categories (Table 2-5, and Figure 2-7A and B). However, the comparative DGE analysis in the roots of the asymptomatic treatment of VAL/SW and VAL/CAN did not show the involvement of functional categories except photosynthesis processes, glutamine metabolism, response to light, and microtubule polymerization (Figure 2-7C and D). In the symptomatic treatment, DGE analysis between VAL grafted onto the CAN, and SW rootstocks showed significant differences in the genes belonging to following 69

70 functional categories: flavonoid metabolism, cell wall pectin esterases metabolism, lipid metabolism, response to chitin and wounding, lignin biosynthesis, water deprivation, defense response to bacterium, respiratory burst involved in the defense, response to JA stimulus, and response to water deprivation (Table 2-6, and Figure 2-8A and B). Whereas, comparative transcriptomic analysis between CAN and SW roots in the symptomatic VAL/CAN, and VAL/SW combinations showed that genes belonging to RNA regulation of transcription factors (TF), wounding response, response to water deprivation, flavonoid biosynthesis, nitrate transport, response to auxin (AU) stimulus, JA-mediated signaling pathway, ET stimulus, starch and sucrose metabolism, brassinosteroid (BR) metabolism, phenylpropanoid biosynthesis, and negative regulation of programmed cell death (PCD) categories were differentially expressed. (Table 2-7, and Figure 2-8C and D). Validation of Differentially Expressed Genes using qrt-pcr To validate the results of RNA-seq data, seven genes that had a distinct expression pattern in the RNA seq study in both the scion/rootstock combinations were selected (Table 2-8). These genes are encoding BAP2, BRI1, NPR3, MIPS2, JAZ8, PMEI and LTP1. All seven genes validated RNA seq results of with respect to leaves of the asymptomatic treatment. Also, the expression level changes were not significantly different between RNA-seq and qrt-pcr Log2 FC (Figure 2-9A to F). In leaves of the symptomatic treatment, qrt -PCR amplified BAP2, BRI1, NPR3, MIPS2, JAZ8, LTP1, and PMEI transcripts expression levels validated RNA-seq generated expression pattern (Figure 2-9A to G). The genes selected to validate the DGE analysis in roots of the symptomatic treatments were BAP2 and JAZ8. All three genes showed a similar pattern of gene expression using qrt-pcr as that of RNA-seq (Figure 2-9A, and E). 70

71 Discussion HLB is the most serious disease ever affecting Florida s citriculture. In the absence of a definite cure for the disease, HLB-tolerant cultivars can potentially be a solution to increase tree survival and plant productivity under endemic HLB spread. The VAL/CAN plants selected in this study showed an enhanced plant sustainability and better fruit quality under pressure of HLB. In Florida, Valencia and Hamlin sweet oranges are important scion varieties for juice production. Higher Brix and BAR, and lower acidity percentage fruits fetch a higher price in the market. In year 2015, fruit harvested from asymptomatic VAL/SW combination had the highest Brix value followed by symptomatic VAL/CAN, asymptomatic VAL/CAN, and symptomatic VAL/SW Brix values. Although there was no significant difference in the Brix of VAL fruit harvested from the different combinations and treatments, fruit harvested from the symptomatic VAL/SW had significantly higher acidity as compared to fruit harvested from the symptomatic VAL/CAN combination. Higher acidity percentage of symptomatic VAL/SW fruit seems to be an effect of HLB infection. Both the treatments in VAL/CAN combination had higher Brix and BAR values of fruit suggesting that CAN rootstock can possibly maintain the optimum juice quality when the plant is CaLas-infected. Higher Ct values and high Brix of VAL/CAN also indicate that VAL/CAN combination is probably less affected by CaLas-infection. In contrast, lower Ct and low Brix in symptomatic VAL/SW indicate that at the advanced stage of CaLas-infection, VAL/SW combination had low fruit juice quality as compared to VAL/CAN. The use of NGS technologies is not limited to the model organisms; rather its use is expanding in studies of non-model organisms too. The transcriptomic analyses of HLB-citrus interactions are mostly studied using the microarray technique (Albrecht and 71

72 Bowman, 2008; Febres et al., 2012; Aritua et al., 2013). In recent years, a few studies of RNA-seq based transcriptome analysis of HLB-affected leaves, roots and fruit have also been published (Martinelli et al., 2013; Wang et al., 2016; Zhong et al., 2016). The use of RNA-seq technology in this study explored the differential gene regulation between combinations and treatments. Results obtained from all three functional analysis software: MapMan, Pathway Studio, and Blast2GO showed similarity in their output. In the comparison between leaves of the asymptomatic VAL/SW and VAL/CAN, response to wounding and chitin, JA and ET metabolism, negative regulators of PCD, response to water deprivation, and secondary metabolism functional categories were overrepresented by the GO analysis. The overrepresentation of wounding and chitin response and JA-ET signaling pathways suggests the CaLas-infected plants were responding to the herbivore attack. Involvement of JA-ET pathways and chitin responses in the herbivore attacks was also reported in Arabidopsis (Foyer et al., 2015). CaLas, HLB putative causal agent, is vectored by the psyllid insect Diaphorina citri Kuwayama. Psyllids feed on the plant vascular system and transmit CaLas into the plant cells. Therefore, the GO term enrichment showing wounding response, chitin response, and JA-ET signaling pathways suggest that many DGE changes were in response to the phloem-feeding of psyllids. DGE analysis of leaves and roots of symptomatic VAL/SW and VAL/CAN combinations overrepresented the flavonoid biosynthesis pathway, lignin metabolism, cell wall degrading enzymes, and pectin methyl esterases pathways suggesting that genes associated with cell wall modification were significantly altered in their expression 72

73 level at an advanced stage of HLB disease development. Blast2GO biological functional analysis showed an overrepresentation of protein phosphorylation and proteolysis functional categories in leaves of symptomatic VAL/CAN. Protein phosphorylation action triggers the conformational changes in proteins that alter biological properties (Ubersax and Ferrell, 2007). Proteolysis plays a vital role in cleansing the plant system during stress responses (Vierstra, 1996). Taken together, GO enrichment of the phosphorylation and proteolysis term in the symptomatic leaves of VAL/CAN suggests that at symptomatic stage CaLas-infected VAL leaves had differential changes at the post-transcriptional level. MapMan analysis of functional categories showed significant differential regulation of BR metabolism-related genes in roots of the symptomatic treatment comparison between the VAL/CAN and VAL/SW combinations. BR hormone is involved in plant growth and defense response (Nakashita et al., 2003; Haubrick and Assmann, 2006). External application of BR has been shown to induce defense in the CaLasinfected greenhouse and field plants (Canales et al., 2016). Significant reprogramming of BR metabolism associated genes in roots of the symptomatic treatment between the VAL/CAN and VAL/SW combinations suggests the potential involvement of BR in response to HLB. SA-mediated systemic acquired resistance (SAR) signaling pathway, leucine rich receptor kinase, MAPK cascade, and plant oxidative burst associated genes were also significantly reprogrammed in the transcriptomic comparisons between VAL/SW and VAL/CAN combinations in both the treatments. The overrepresentation of SAR mediated signaling and plant hypersensitive reaction functional categories indicates the 73

74 activation of plant defense. Functional analysis of DEGs also overrepresented water deprivation and abscisic acid (ABA) response pathways in leaves and roots of the symptomatic and asymptomatic treatments in both scion/rootstock combinations. ABA is associated with drought response (Fernando and Schroeder, 2016). The GO analysis showed that ABA signaling functional category was overrepresented which Indicates CaLas-infected plants show a response to water deprivation and salt stress. Overall analysis of functional categories showed that plant defense to HLB was activated by activating genes related to SA-mediated signaling, JA and ET-dependent signaling, protein kinases, and secondary metabolite pathways. Whereas, genes involved in nitrate, metal, and sugar transporters suggest development-associated genes were also differentially regulated in response to rootstock differences. The functional analysis of DGE also highlighted the involvement of ABA and BR signaling pathway genes in response to rootstock differences in CaLas-infected plants. Functional analysis of genes differentially expressed between VAL/SW and VAL/CAN plants showed DGE in response to asymptomatic and symptomatic stages of HLB development. The genes that are contributing to the differential reprogramming are not discussed in this Chapter. The detailed analysis of upregulation and downregulation of genes involved in the functional categories is discussed in Chapter 3, Chapter 4, and Chapter 5. 74

75 Table 2-1. Experimental treatments and combinations Rootstock Rootstock Parents Scion Treatments based on visual observations of HLB symptoms Swingle; 2n (SW) Grapefruit X Trifoliate orange Valencia sweet orange (VAL) Symptomatic VAL/SW Asymptomatic VAL/SW Putative HLBtolerant candidate; 2n (CAN) HBP Pummelo X Cleopatra Mandarin Valencia sweet orange (VAL) Slightly Symptomatic VAL/CAN Asymptomatic VAL/CAN Approximately 7- year old trees planted in the Lee Family s Alligator Grove east of St. Cloud, FL. Table 2-2. Comparison pairs used for differential gene expression analysis in leaves and roots of the experimental scion/rootstock combinations Leaves Roots Asymptomatic VAL/SW vs. Asymptomatic Asymptomatic VAL/SW vs. Asymptomatic VAL/CAN VAL/CAN Symptomatic VAL/SW vs. Symptomatic Symptomatic VAL/SW vs. Symptomatic VAL/CAN VAL/CAN Table 2-3. Analysis of HLB and CTV detection in the experimental samples Experiment Sample ID Rootstock Scion Treatment HLB detection (2014 April) HLB detection (2015 December) CTV detection (2015 December) Leaves Roots Leaves Leaves 13 SW VAL Symptomatic Pos Pos Pos Pos 14 SW VAL Symptomatic Pos Pos Pos Pos 15 SW VAL Symptomatic Pos Pos Pos Pos 19 SW VAL Asymptomatic Neg Pos Pos Pos 20 SW VAL Asymptomatic Neg Pos Pos Pos 21 SW VAL Asymptomatic Neg Pos Pos Neg 5 CAN VAL Symptomatic Pos Neg Pos Neg 6 CAN VAL Symptomatic Pos Pos Pos Neg 7 CAN VAL Asymptomatic Pos Pos Pos Neg 22 CAN VAL Asymptomatic Neg Neg Pos Neg 23 CAN VAL Asymptomatic Pos Pos Pos Pos 24 CAN VAL Asymptomatic Neg Pos Pos Pos Pos, PCR positive; Neg, PCR negative 75

76 Table 2-4. Reads obtained from the sequencing run Experimental Sample ID Plant tissue Rootstock Scion Treatment NEB index* No. of reads 13B Leaves SW VAL Symptomatic i504-i702 45,474,082 13D Roots SW VAL Symptomatic i508-i703 38,740,620 14A Leaves SW VAL Symptomatic i505-i702 41,910,010 14D Roots SW VAL Symptomatic i504-i701 30,066,970 15B Leaves SW VAL Symptomatic I506-i702 41,476,846 15D Roots SW VAL Symptomatic i505-i701 42,976,370 19B Leaves SW VAL Asymptomatic i507-i702 43,050,176 19D Roots SW VAL Asymptomatic i506-i701 36,758,134 20B Leaves SW VAL Asymptomatic i508-i702 30,822,930 20D Roots SW VAL Asymptomatic i507-i701 40,494,122 21A Leaves SW VAL Asymptomatic i501-i703 28,336,160 21D Roots SW VAL Asymptomatic i508-i701 74,596,402 5A Leaves CAN VAL Symptomatic i501-i701 38,546,288 5C Roots CAN VAL Symptomatic i502-i703 10,798,204 6A Leaves CAN VAL Symptomatic i502-i701 35,288,378 6D Roots CAN VAL Symptomatic i503-i703 12,792,946 7B Leaves CAN VAL Symptomatic i ,952,680 7C Roots CAN VAL Asymptomatic i504-i703 13,831,332 22B Leaves CAN VAL Asymptomatic i501-i702 49,335,730 22C Roots CAN VAL Asymptomatic i505-i703 49,720,036 23B Leaves CAN VAL Asymptomatic i502-i702 53,907,670 23C Roots CAN VAL Asymptomatic i506-i703 30,115,828 24B Leaves CAN VAL Asymptomatic i503-i702 42,682,422 24D Roots CAN VAL Asymptomatic i507-i ,236 * New England Biolabs Inc developed Illumina index used for multiplexing the samples for the sequencing run 76

77 Table 2-5. Functional analysis of significant DEGs in leaves of the asymptomatic treatment between VAL/CAN and VAL/SW Pathway studio MapMan Biological functional category p-value Bin Name elements* p-value response to chitin 1.07E-76 transport E-04 response to wounding 3.15E-49 RNA.regulation of transcription.wrky domain transcription factor family E-04 respiratory burst involved in defense response 3.72E-48 RNA.regulation of transcription. PHOR intracellular signal transduction 2.41E-34 misc.gdsl-motif lipase response to jasmonic acid stimulus 8.09E-34 signalling protein targeting to membrane 1.58E-33 secondary metabolism.flavonoids response to ethylene stimulus 2.48E-33 signalling.calcium regulation of plant-type hypersensitive response 2.72E-33 misc jasmonic acid mediated signaling pathway 3.42E-30 stress ethylene biosynthetic process 5.21E-30 hormone metabolism. ethylene negative regulation of programmed cell death 8.53E-28 development. storage proteins defense response to fungus 2.05E-27 secondary metabolism.flavonoids.dihydroflavonols response to water deprivation 8.61E-27 AP2/EREBP, APETALA2/Ethyleneresponsive element binding protein family jasmonic acid biosynthetic process 8.40E-23 signalling.receptor kinases.s-locus glycoprotein like negative regulation of defense response 1.15E-22 hormone metabolism.ethylene.signal transduction response to fungus 4.16E-22 protein.postranslational modification.kinase response to high light intensity 6.01E-22 transport.abc transporters and multidrugresistance systems abscisic acid mediated signaling pathway 1.03E-21 DNA.unspecified salicylic acid mediated signaling pathway 3.85E-21 signalling.receptor kinases systemic acquired resistance, salicylic acid mediated signaling pathway 2.02E-20 signalling.in sugar and nutrient physiology Classification of the measured parameter into a set a functional category in the MapMan analysis tool. Top 20 (based on p value) biological functional categories are presented that were identified in Pathway studio and MapMan (Wilcoxon rank sum test), *No. of DGE present 77

78 Table 2-6. Functional analysis of significant DEGs in leaves of the symptomatic treatment between VAL/CAN and VAL/SW Pathway studio MapMan Biological functional category p-value Bin Name elements* p-value response to heat 3.91E-53 stress.abiotic.heat E-09 response to high light intensity 2.14E-52 stress E-07 response to hydrogen peroxide 2.33E-41 stress.abiotic E-07 protein folding 3.02E-31 secondary metabolism.flavonoids E-05 response to wounding 1.60E-29 cell wall.pectin*esterases 7 2.2E-03 response to water deprivation 1.98E-25 cell E-03 response to chitin 2.23E-23 secondary metabolism E-03 lignin biosynthetic process 3.53E-21 lipid metabolism.lipid degradation E-03 lysophospholipases response to cold 1.74E-19 cell wall E-03 response to karrikin 2.77E-19 signalling. receptor kinases E-03 response to salt stress 3.47E-18 secondary 4 9.5E-03 metabolism.flavonoids.chalcones response to red light 8.74E-18 secondary metabolism E-03 flavonoids.dihydroflavonols chloroplast 9.98E-18 misc.protease inhibitor/seed 6 9.7E-03 storage/lipid transfer protein (LTP) family protein response to endoplasmic reticulum 1.19E-17 transport.amino acids E-02 stress jasmonic acid mediated signaling 6.79E-17 cell wall.degradation 8 1.4E-02 pathway response to sucrose stimulus 8.73E-17 signalling E-02 defense response to bacterium 1.89E-16 misc.glutathione S transferases 4 2.1E-02 response to jasmonic acid stimulus 3.76E-16 signalling.receptor kinases.leucine 2 2.2E-02 rich repeat XII plasma membrane 6.54E-16 RNA.regulation of transcriptionmybrelated transcription factor family 5 2.8E-02 Classification of the measured parameter into a set a functional category in the MapMan analysis tool. Top 20 (based on p value) biological functional categories are presented that were identified in Pathway Studio and MapMan (Wilcoxon rank sum test), *No. of DGE present. 78

79 Table 2-7. Functional analysis of significant DEGs in roots of the symptomatic treatment between VAL/CAN and VAL/SW Biological functional category Pathway studio p-value Bin Name MapMan elements* p-value response to chitin 2.25E-52 metal handling.binding, chelation and storage negative regulation of programmed cell death 5.12E-40 RNA.regulation of transcription response to ethylene stimulus 7.53E-38 hormone metabolism.brassinosteroid protein targeting to membrane 2.60E-37 RNA.regulation of transcription.wrky domain transcription factor family regulation of plant-type hypersensitive response 4.27E-37 transport. nitrate response to wounding 9.30E-37 misc jasmonic acid mediated signaling pathway 1.34E-35 DNA.unspecified defense response to fungus 5.25E-34 transport.metal response to jasmonic acid stimulus 1.52E-31 transporter.sugars.sucrose endoplasmic reticulum unfolded protein response respiratory burst involved in defense response 1.93E E-29 RNA.regulation of transcription.ap2/erebp, APETALA2/Ethylene-responsive element binding protein family RNA.regulation of transcription.hb, Homeobox transcription factor family MAPK cascade 8.57E-28 Gluconeogenese/ Glyoxylate cycle systemic acquired resistance, salicylic acid hormone metabolism.brassinosteroid.synthesisdegradation 1.77E-27 mediated signaling pathway negative regulation of defense response 3.87E-27 hormone metabolism.gibberelin abscisic acid mediated signaling pathway 2.31E-26 Hormone metabolism. brassinosteroid. synthesisdegradation.sterols.other salicylic acid biosynthetic process 8.79E-26 RNA.regulation of transcription. Aux/IAA family salicylic acid mediated signaling pathway 5.59E-24 protein. degradation. serine protease response to water deprivation 2.71E-23 misc. myrosinases-lectin-jacalin intracellular signal transduction 1.24E-21 RNA regulation of hydrogen peroxide metabolic process 2.07E-19 protein. degradation.aspartate protease ethylene biosynthetic process 2.68E-19 metal handling. acquisition Classification of the measured parameter into a set a functional category in the MapMan analysis tool. Top 20 (based on p value) biological functional categories are presented that were identified in Pathway Studio and MapMan (Wilcoxon rank sum test), *No. of DGE present. 79

80 Table 2-8. Genes used for validation of RNA-sequencing results Gene Identification Abbr. Primer sequence* Ciclev m.g/ F- 5 -GCTTCTCTTCGCGGTTAGAT -3 PMEI Pectin methyl esterase inhibitor R- 5 -CAGTTGTGGTTTGGTTGATAG-3 Ciclev m.g/BON associated F- 5 -TTCGTCACCATCACCACATAC-3 BON protein 2 R- 5 -TCCGGGACTACAGGTTCTAAT-3 Ciclev m.g/Lipid transfer protein F- 5 -CATGTGAGCAAGTGACAATCTG-3 LTP1 1 R- 5 -CTAGGATAAGAATTAAGGGCGTACT-3 Ciclev m.g/BR leucine rich F- 5 -CTCCGGGTCCTTTGTCTGATATT-3 BRI receptor R- 5 -ACCGAATCTACTGGGAACTTTAC-3 Ciclev m.g/LOC /Nonexpressor of protein 3 R- 5 -CCATCGGATTCCTCATTTC-3 F- 5 -AGGTTCTCAGCCTCCGGATTA-3 NPR3 F- 5 -GCCTTCTTCCTTATTCCGACTC-3 Ciclev m.g/Zim domain JAZ 8 JAZ8 R- 5 -GCTAAGCTCCTGTGCTTTCT-3 Ciclev m/Myo-inositol phosphate F- 5 -TGTTTGGAGGATGGGACATTAG-3 MIPS2 synthase 2 R- 5 -GCATACAAGGTGGAAGGAGAA-3 Glyceradehyde-3-phosphate GAPC F- 5 -GGAAGGTCAAGATCGGAATCAA-3 dehydrogenase C2 R- 5 -CGTCCCTCTGCACfGATGACTCT-3 *F; forward primer sequence, R; reverse primer sequence, Abbreviations used in the texts 80

81 A B C D Figure 2-1. VAL/CAN combination field trees. A) symptomatic (Year 2015), B) symptomatic (Year 2016). C) asymptomatic (Year 2015), D) asymptomatic (Year 2016) 81

82 A B C D Figure 2-2. VAL/SW combination field trees. A) symptomatic (Year 2015), B) symptomatic (Year 2016), C) asymptomatic (Year 2015), D) asymptomatic (Year 2016) 82

83 Brix and BAR Brix and BAR A Juice quality_ BRIX BAR Acid % Sympt_VAL/SW Asympt_VAL/SW Asympt_VAL/CAN Combinations B Juice quality_2016 BRIX BAR Acid % Sympt_VAL/SW Asympt_VAL/SW Asympt_VAL/CAN Combinations Figure 2-3. Juice qualityt analysis. A) Year 2015, B) Year Sympt: symptomatic; Asympt: asymptomatic, BAR: Brix-acid ratio. A 83

84 A B Figure 2-4.RNA quality and quantity analysis. A) Leaf RNA B) Root RNA. Electrophoresis gel based quality/quantity analysis of leaf and root RNA tissue. The quality of RNA is based on 260/280 ratio obtained from NanoDrop technique, and RNA quantity is expressed in ng/µl. Each lane contains 50 ng RNA. Leaf and root samples have different identifications. Leaves are designated either A or B, and roots are designated either C or D followed by sample ID. 84

85 Figure 2-5. Read mapping statistics. Raw reads were trimmed and indexed, and then mapped to the C. clementina genome for downstream analysis. 85

86 A B C Figure 2-6. Display of Volcano plot of using CummeRbund. A) leaves of asymptomatic VAL/SW (X19B) and VAL/CAN (X22B), B) leaves of symptomatic treatment VAL/SW (X5C) and VAL/CAN (X19C), C) roots of symptomatic treatment VAL/SW (X5C) and VAL/CAN (X19) 86

87 A C B D Figure 2-7. Blast2GO functional analysis of DEGs in leaves (A and B) and roots (C and D) of the asymptomatic VAL/CAN and VAL/SW combinations. Biological (bp) functional categories identified (Top 10 and p value < 10-6 ) in upregulated (up) and downregulated (down) genes in the VAL/CAN as compared to the VAL/SW. Graphics are adapted from Blast2GO statistics results. L, leaves; R, roots 87

88 A C B D Figure 2-8. Blast2GO functional analysis of DEGs in leaves (A and B) and roots (C and D) of the symptomatic VAL/CAN and VAL/SW combinations. Biological (bp) functional categories identified (Top 10 and p value < 10-6 ) in upregulated (up) and downregulated (down) genes in the VAL/CAN as compared to the VAL/SW. Graphics are adapted from Blast2GO statistics results. L, leaves; R, root 88

89 A E B F C G D Figure 2-9. qrt-pcr based DEGs validation. A) BAP2, B) BRI1, C) JAZ8, D) LTP1, E) NPR3, F) MIPS2, G) PMEI 89

90 CHAPTER 3 DIFFERENTIAL EXPRESSION ANALYSIS OF HORMONAL METABOLISM- ASSOCIATED GENES IN LEAVES AND ROOTS OF TWO SCION/ROOTSTOCK COMBINATIONS AT ASYMPTOMATIC AND SYMPTOMATIC STAGES OF HLB DISEASE Introduction As sessile organisms, plants constantly need to cope up with their surrounding environment for growth and survival. Evolutionary, plants have developed the sensory system to sense the environmental changes through plant growth regulators (PGRs). PGRs or plant hormones are vital sensing chemicals that are important in plant growth and defense. Plant hormones have a significant role in stress signal perception, transduction, and response. Hence, hormones are crucial for plant growth and sustainability. PGRs are low molecular weight chemicals that are secreted by specialized cells and that affect the metabolism or behavior of other cells possessing functional receptors. The well-known PGRs are auxins (AU), gibberellins (GA), cytokinins (CT), abscisic acid (ABA), ethylene (ET), salicylic acid (SA), jasmonic acid (JA), brassinosteroids (BR), and strigolactone (ST) (McCourt, 1999). Some signaling peptides are also acknowledged as PGRs (Wang and Irving, 2011). AU, CT, GA, and BR are highly regulated in plant growth (Werner et al., 2001; Liscum and Reed, 2002; Birnbaum and Benfey, 2004). Whereas, SA, JA, and ET are plant defense associated hormones (Pieterse et al., 2009). BRs and ABA are implicated in the plant development as well as plant defenses (Haubrick and Assmann, 2006; Cao et al., 2011). The studies on plant functional biology and genetics revealed that the PGRs are multitasking chemicals, either working individually or in combination with other hormones, which supports the concept of hormonal crosstalk (McCourt, 1999; Swarup et al., 2002; 90

91 Pieterse et al., 2012; Vanstraelen and Benková, 2012). The crosstalk between PGRs is limited not only to the plant growth and development. Hormonal signaling is also involved in plant immunity, nutrient uptake and transport, and metal homeostasis (Rubio et al., 2009; Krouk et al., 2011). Interdependence of AU and BR showed the regulatory network and negative feedback mechanism between AU and BR response pathways in seedling growth of Arabidopsis thaliana (Nemhauser et al., 2004). The interplay between GA and BR in development and defense signaling is discussed by Lieselotte et al., JA and SA -induced antagonism and its molecular differences in the necrotrophic and biotrophic pathogen infection is evident in many plant species (Kunkel and Brooks, 2002; Pieterse et al., 2009; Leon-Reyes et al., 2010). The developmental role of AU, CT, GA, ABA, and ET is well known. However, these PGRs are also involved in the plant defense signaling (Cao et al., 2011; Giron et al., 2013). Hormonal homeostasis is a key to performing plant functions at an optimum level. Environmental imbalances, physiological changes, and plant growth initiate the hormonal changes in the plant. Hormonal regulation hierarchy starts with signal perception followed by hormone synthesis, signal transduction and the response activation (Figure 3-1). Hormonal concentration is subsequently regulated by negative feedback mechanisms that include negative regulation of hormone synthesis, catabolism or sequestration (Woodward and Bartel, 2005; Brenner et al., 2012). The hormonal activation input signal is perceived by a binding receptor which initiates the signal transduction. Each PGR has its specific receptor kinase (RK), signal transduction ligands, protein-coupled receptors (G-coupled proteins) to bring a hormone into action (Wang and Irving, 2011). Examples of RKs are CT specific cytokinin regulated kinase 1 91

92 (CRK1) (Brenner et al., 2012), ET specific ethylene regulated 1 (ETR1) (Gallie, 2015), GA specific gibberellin insensitive dwarf 1 (GID1), ABA specific -regulatory components of ABA receptor/pyrabactin resistance protein1/pyr-like proteins (RCARs/PYR1/PYLs) (Raghavendra et al., 2010), and BR specific brassinosteroid insensitive 1 (BRI1) (Haubrick and Assmann, 2006). Once the hormone is perceived by RKs, the next step is signal transduction. Signaling transduction activates the functional transcription factors (TFs) such as AU activated auxin response factors (ARFs), JA-dependent MYC, GA-dependent phytochrome interacting factor (PIF) and ET activated ethylene response factor 1 (ERF1). The hormone specific TFs activate downstream hormonal responses. Regulation of hormonal turnover is controlled by repressor proteins which downregulate the hormonal response. Hence, the repression proteins are either repressed or degraded by S-phase kinase associated protein1 (SKP1), cullin and F-box protein complexes (SCF) upon hormonal reception (Woodward and Bartel, 2005; Raghavendra et al., 2010; Brenner et al., 2012). In different hormonal signaling pathways, SCFmediated degradation of repressor proteins is characterized. In AU signaling, the SCF TIR1 complex represses the auxin/indole-3-acetic acid (AUX/IAA) hormonal receptor repressor. TIR1 is an auxin specific F-box protein which co-represses AUX/IAA repression and thereby activates AU response element (AuxRE) and ARFs (Woodward and Bartel, 2005; Grones and Friml, 2015; Li et al., 2016). In GA signaling, DELLA proteins are the receptor repressors that downregulate the GA response. GID1 is an F- box protein and a GA receptor. In the presence of GA, GID binds to DELLA and degrades it. Once the DELLA is deactivated, GA-responsive PIF genes induce the GAactivated growth response (Frgerioi et al., 2006). Jasmonate zim-domain (JAZ) proteins 92

93 are negative regulators of MYC transcription factors. JAZ repression is removed by coronitine-insensitive 1 (COI1) and SCF complex which then activates JA-responsive genes (Wasternack and Hause, 2013). Similarly, the role of other hormone-specific receptors, SCF complex and responsive genes are well characterized by screening mutants in A. thaliana (Wang and Irving, 2011). The activity of hormonal receptor suppressor is crosslinked or controlled by other hormonal receptors, repressors, TFs, and responsive genes. One of the impacts of hormone crosstalk is a plant growth and defense trade-off (Wei Wang et al., 2012; Denancé et al., 2013; Huot et al., 2014). Hormonal regulations in plants are a highly coordinated and synchronized process to maintain a balance between plant growth and defense. Transcriptomic studies of CaLas-infected susceptible and HLB-tolerant citrus cultivars underscored the role of PGRs in the HLB-citrus interaction (Canales et al., 2016; Fan et al., 2012; Martinelli et al., 2013; Albretch and; Bowman, 2011). The regulatory network analysis of diverse CaLas-infected scion/rootstock combination at various stages of infection showed the altered expression levels of AU, GA, ABA, ET, JA, and SA-regulated genes. In HLB-citrus interaction studies, AU, GA, JA, and SAregulated functional categories were overrepresented by gene ontology (GO) algorithms (Zheng and Zhao, 2013). In CaLas-infected plants, the JA and ET-response activated ENDOCHITINASE gene expression was found to be repressed at the early stage (5 weeks after infection) of infection in Valencia sweet orange (Citrus sinensis [L.] Osbeck) scion grafted onto Cleopatra rootstock (Albrecht and Bowman, 2012b). Whereas, CaLas-B232 (Thailand strain) and Citrus Tristeza Virus (CTV) infected Valencia overexpressed the JA-responsive LOX2 encoding gene (Fu et al., 2016). SA- 93

94 induced CONSTITUTIVE DISEASE RESISTANCE (CDR1) was upregulated in the leaves of non-grafted HLB-tolerant US-897 rootstock (Albrecht and Bowman, 2012b). GA-induced cell organization and biogenesis related gene encoding GA-responsive GAS1 protein homolog GASA was overexpressed in rootstock US-897 leaves (Albrecht and Bowman, 2012b). Transcriptomic analysis of fruit harvested from CaLas-infected Valencia /Swingle combination was reported by Martinelli et al., HLBsymptomatic fruit showed overexpression of AU biosynthesis and signal transduction related transcripts. Also, transcripts encoding ET biosynthesis, receptors, and responsive genes such as ETR1, ERF1, ERF2 and ethylene forming enzyme (EFE) were induced in the HLB-symptomatic Valencia fruit. However, GA and CT metabolism related ENT-KAURENOIC ACID HYDROXYLASE 2 (KAO2) and GASA1 genes were downregulated in the CaLas-infected Valencia fruit. In addition, transcript encoding LOX2 was also suppressed. Overall, transcriptomic studies revealing HLB-citrus interaction showed that hormonal changes in the CaLas-infected plants vary with the scion and rootstock cultivars (Albrecht and Bowman, 2012b; Nwugo et al., 2013a; Zhong et al., 2016). Differential gene expression (DGE) changes in the HLB-affected plants are tissue and stage of infection specific (Fan et al., 2012; Martinelli et al., 2012; Zheng and Zhao, 2013). Field and laboratory -based experiments provide evidence that exogenous hormonal treatments can manipulate transcriptome of CaLas-infected plants to promote growth and induce defense (Shen et al., 2013; Canales et al., 2016). Knowledge of hormonal changes is a key to understanding the HLB-citrus interaction. The goal of comparative transcriptomic analysis between Valencia grafted onto the putative HLB-tolerant candidate rootstock 42x (CAN) or the HLB- 94

95 susceptible commercial rootstock Swingle citrumelo is to emphasize the vital role of citrus rootstocks in modulating the scion hormonal network in response to CaLasinfection. Materials and Methods Plant Material Two combinations of scion and rootstocks were used in this experiment (Table 3-1). Field grown seven-year-old experimental plants were planted in the Lee Family's Alligator Grove, east of St. Cloud, Florida. The one combination of trees was Valencia' (VAL) sweet orange grafted onto a putative HLB-tolerant candidate (CAN) rootstock. The CAN rootstock (46x ) is a hybrid of Hirado Buntan Pink' pummelo (HBP) (Citrus maxima Merr.) and Cleopatra mandarin (Citrus reticulata Blanco.). The 2 nd combination was Valencia' (VAL) scion grafted onto standard commercial Swingle citrumelo (SW) rootstock. Swingle is a hybrid of grapefruit (Citrus paradisi [Macf.]) and trifoliate orange (Poncirus trifoliata [L.] Raf). Each combination of VAL/CAN and VAL/SW, plants were divided into two treatments based on the visible presence of HLBlike symptoms (Table 3-2). Highly infected and symptomatic trees in each combination grouped into the symptomatic treatment, whereas, trees with very few or no visible symptoms were grouped into the asymptomatic treatment. All biological replicates in each treatment and combination were tested using quantitative PCR (qpcr) based CaLas detection, and enzyme-linked immunosorbent assay (ELISA) assisted CTV detection. Sampling, RNA extraction, and RNA sequencing A detailed protocol of sampling, RNA extraction, and RNA sequencing is explained in Chapter 2. In brief, differentially expressed genes (DEGs) in the pairwise 95

96 comparison between asymptomatic VAL/CAN and VAL/SW combinations, and symptomatic VAL/CAN, and VAL/SW combinations were obtained using RNA-seq Tuxedo pipeline (Chapter 2). The significant DEGs in leaves and roots were annotated to the C. clementina genome database in the Phytozome V1.0 (Goodstein et al., 2012). Functional categories of the significantly expressed genes were identified using A. thaliana annotation in the Phytozome server and MapMan (Thimm et al., 2004) software (Chapter 2.). Blast2GO algorithm (Conesa et al., 2005) also used to identify molecular, cellular, and biological functional categories. An overview of DEGs was developed using PageMan analysis tool in the MapMan software (Usadel et al., 2009). Results HLB Detection and RNA Sequencing Output The results of qrt PCR-based HLB detection and RNA-seq output in all combinations and treatments are discussed in Chapter 2. Differential Expression of Hormonal Regulation-Associated Genes in Leaves and Roots of Asymptomatic VAL/CAN and VAL/SW Combinations Leaf samples The results of DGE studies combined with gene functional analysis tools showed a significantly different regulation of genes involved in all the hormonal pathways in the comparison of asymptomatic treatment between VAL/CAN and VAL/SW combinations in leaves. In the asymptomatic treatment, CaLas-infected leaves of VAL/CAN had a greater number of AU and BR metabolism related genes that were significantly upregulated as compared to the asymptomatic leaves of VAL/SW combination (Figure 3-2), whereas, SA, JA, ET, AU and ABA hormonal signaling genes were strongly 96

97 upregulated in the leaves of asymptomatic of VAL/SW as compared to leaves of asymptomatic VAL/CAN combination. Leaves of asymptomatic VAL/CAN combination significantly upregulated genes encoding BR biosynthesis STEROL METHYL ESTERASE (SMT1), BRI1, HERCULES RECEPTOR KINASE (HERK1), and BR KINASE INHIBITOR (BKI1) as compared to leaves of the asymptomatic VAL/SW combination (Table 3-3). Genes involved in different steps of AU signaling and homeostasis were also upregulated in the asymptomatic VAL/CAN combination. These include genes encoding AU regulators such IAA14, AUX/IAA, MYO-INOSITOL-1-PHOSPAHTE SYNTHASE-2 (MIPS2), UDP- GLUCOSYL TRANSFERASE 74B1 (UGT74B1) and AU responsive genes such as SAUR-like, ARF8, and ARF16 (Table 3-3). Leaves collected from asymptomatic VAL/CAN also significantly upregulated genes encoding JA biosynthesis LOX2 and SA biosynthesis phenylalanine ammonia lyase 1 (PAL1). Genes encoding ET-responsive SHN1 TF also significantly overexpressed in leaves of the asymptomatic VAL/CAN combination. In the asymptomatic treatment, leaves collected from VAL/CAN also showed significant upregulation of genes encoding cytokinin-dependent response regulators ARR9, Che-Y like two components responsive regulator, GIBBERELLIN DEGRADING GA 2-OXIDASE (GA2OX), and the ABA biosynthesis gene NINE-CIS- EPOXYCAROTENOID DIOXYGENASE 5 (NCED5) (Table 3-3). In the asymptomatic VAL/SW leaves, AU dependent IAA-LEUCINE RESISTANT (ILR)-like 6 (ILL6), IAA-ALANINE RESISTANT 3 (IAR3), and genes encoding glucosinolate biosynthesis CYP83B1 and R2R3-Myb77 transcription factor were overexpressed. JA and ET response genes were highly upregulated in leaves of the 97

98 asymptomatic VAL/SW combination (Table 3-4). ET biosynthesis EFE, and genes encoding ET receptors such as ERT2, ERF5, ERF12, ERF13, ET signal transduction apetala 2 (AP2) domain containing, REDOX RESPONSIVE TRANSCRIPTION FACTORS (RRTF1), and ET-responsive ELEMENT BINDING PROTEIN (EBP) were significantly upregulated in leaves of the asymptomatic VAL/SW as compared to leaves of the asymptomatic VAL/CAN. JA biosynthesis gene ALLENE OXIDE CYCLASE 3 (AOC3), and genes encoding LOX3 and JA response transcription factors WRKY30 and WRKY 33, ZIM domain containing JA repressors such as JAZ1, JAZ8 and JAZ10 and JA catabolic turnover CYP94C1 were upregulated in the VAL leaves of asymptomatic VAL/SW combination as compared to VAL leaves of asymptomatic VAL/CAN in the range of 2-4 log2 fold change (log2 FC) (Table 3-4). Asymptomatic VAL/SW leaves also showed significant upregulation of ABA signaling genes at higher expression levels compared to the asymptomatic VAL/CAN combination (Table 3-4). These include genes encoding ABA-responsive GRAM domain containing TFs, F-box EMPFINDLICHER IM DUNKELROTEN LICHT 1-LIKE PROTEIN 3 (EDL-3), abscisic acid 8'-hydroxylase (CYP707A1) and type 2C protein phosphatases (PP2Cs). Genes encoding transcription factors involved in SA activated resistance (SAR) pathway, WRKY70 and thioredoxin family proteins GRX480, were overexpressed in the asymptomatic VAL grafted onto the SW rootstock as compared to the asymptomatic VAL grafted onto CAN rootstock. Root samples The comparative gene expression analysis of roots collected from the asymptomatic treatment of VAL/CAN and VAL/SW did not show differential expression of many hormonal metabolism associated genes. 98

99 Differential Expression of Hormonal Regulation-Associated Genes in Leaves and Roots of Symptomatic VAL/CAN and VAL/SW Combinations Leaf samples Leaves sampled from symptomatic CaLas-infected VAL grafted onto the CAN rootstock significantly upregulated BR and CT -metabolism and -response related genes. Whereas CaLas-infected VAL grafted onto SW rootstock showed significant upregulation of ET, JA and AU signaling genes (Figure 3-2). In leaves of the symptomatic VAL/CAN combination, genes encoding sterol biosynthesis- CYCLOARTENOL SYNTHASE 1 (CAS1), STEROL METHYLTRANSFERASE1 (SMT1) and DWF5 were overexpressed as compared to leaves of symptomatic VAL/SW combination (Figure 3-2). In addition, genes encoding BR SIGNALING KINASE-2 (BSK2) and BR ENHANCED EXPRESSION 3 (BEE3) were upregulated in leaves of VAL/CAN as compared to the VAL/SW in the symptomatic treatment (Table 3-5). CT and GA metabolism related genes increased in their expression level in leaves of the symptomatic VAL/CAN as compared to symptomatic VAL/SW. These included CYTOKININ OXIDASE 7 (CKX7) gene, and genes encoding chase domain containing histidine kinase; WOODEN LEG (WOL), and CT-two component system associated ARR4, APRR2, and APRR10 (Table 3-4). Genes involved in SA, JA, and ET -hormones activated plant defense responses did not upregulate at their higher expression level in the symptomatic VAL/CAN combination (Figure 3-2). However, transcripts encoding PAL1 which is a key enzyme in the SAR signaling pathway were upregulated. Genes involved in the ET or JA biosynthesis did not overexpress significantly in the symptomatic VAL/CAN combination; whereas, 99

100 genes encoding in the AU response regulators were significantly upregulated (Table 3-5). In the symptomatic treatment, VAL/SW overexpressed a greater number of JA, ET, AU, and ABA metabolism associated genes (Table 3-6). In the same treatment, VAL/SW leaves did not show a significant increase in expression level of genes involved in SA metabolism related genes except WRKY70 TF. However, SA-responsive SIGMA FACTOR BINDING PROTEIN1 (SIB1) upregulated by 2 log2 FC. The gene encoding negative regulator of NON-EXPRESSOR OF PATHOGENESIS-RELATED 1 (NPR1), NPR3, was also overexpressed in the leaves collected from the symptomatic VAL/SW combination. Genes involved in the JA, ET, AU, and ABA -biosynthesis, regulation, and negative regulation were substantially increased in their expression level in the symptomatic VAL/SW combination (Table 3-6). BASIC CHITINASE (HCHIB) is a pathogenicity related (PR) protein which the expression level upregulates in response to higher expression of JA and ET. Expression of HCHIB significantly upregulated in the leaves of the VAL/SW as compared to leaves of VAL/CAN in the symptomatic treatment. Notably, transcripts encoding JA repressors JAZ1 and JAZ8 were also strongly induced in the leaves of the symptomatic VAL/SW combination (Table 3-6). Root samples In the symptomatic treatment, CaLas-infected roots collected from the CAN and SW rootstock showed significant differences in the BR, CT, GA, SA, JA, and ABA - regulated genes. Fibrous roots collected from the VAL/CAN combination showed significant upregulation of a higher number of genes involved in BR, GA, and ABA metabolism (Table 3-7). Roots collected from VAL/CAN combination, also, showed significant overexpression of defense associated SA, JA and ET hormonal signaling and 100

101 responsive genes as compared to roots collected from VAL/SW, in the symptomatic treatment. In the same treatment, CAN roots showed significant upregulation of genes encoding negative regulators of JA, SA, and GA hormonal response (Table 3-7). These negative regulators are JAZ1, JAZ8, JAZ10, NPR3, GIBBERELLIC ACID INSENSITIVE (GAI), and RGA like-3 (RGL-3) proteins. Roots collected from symptomatic VAL/CAN significantly overexpressed genes encoding transcriptional activators of SA, JA, and ABA as compared to roots collected from symptomatic VAL/SW combination. These transcription activators are THIOREDOXIN PROTEINS (GRX480), MYC and PYL4 (Table 3-7). Roots collected from the symptomatic VAL/CAN combination showed significant overexpression of the genes involved in the BR-mediated hormonal signaling pathway as compared to the roots collected from the symptomatic VAL/SW combination (Figure 3-2). These genes are encoding BR biosynthesis SQUALENE MONOOXYGENASE (XF1), BAS1, CAS1 and BRI like kinase receptors (Table 3-7). In the symptomatic treatment, CaLas-infected SW roots overexpressed JA, ET, AU, and ABA metabolism associated genes as compared to CAN roots. In the symptomatic treatment, transcripts of JA biosynthesis AOC4 and JA responsive PR4 were significantly upregulated in roots collected from VAL/SW as compared to VAL/CAN roots (Table 3-8). Genes encoding AU transporter such as EFFLUX PINFORMED 4 (PIN4) and LIKE-AUXIN RESISTANT-1 (LAX1), AU regulator GH3.1 and IAA29 significantly upregulated in the roots of symptomatic VAL/SW as compared to symptomatic VAL/CAN (Table 3-8). The ABA transcription activator ABSCISIC ACID -DEFICIENT 4 (ABA4) transcripts were also significantly overexpressed in roots of the symptomatic VAL/SW combination. In the symptomatic treatment, VAL/SW roots 101

102 significantly upregulated ET RESPONSE SENSOR-1 (ERS1) and ET degradation ACC OXIDASE (ACO) transcripts. Discussion The comparative gene expression analysis in leaves of the HLB-asymptomatic treatment between VAL grafted onto putative tolerant CAN and the susceptible SW rootstock showed an abundance of significant differentially regulated genes in the category of hormonal metabolism. However, roots collected from CAN and SW rootstocks in the same treatment did not show significant expression of genes involved in the hormonal regulations. To confirm the results, the RNA-seq protocol and data analysis of comparison between asymptomatic -VAL/CAN and -VAL/SW roots were performed twice., However, results again showed very few DEGs. CAN and SW rootstocks have different genotypes; therefore, it is expected that the DGE analysis should have generated more numbers of differentially regulated genes in response to CaLas infection. However, the consistent results of DGE analysis of the asymptomatic VAL/CAN and VAL/SW roots suggests that the lower number of DEGs may not be a result of genetic differences between two rootstocks, rather it is probably a result of the encountered technical difficulty to obtain good quality RNA from the roots. The read mapping data also showed that roots collected from samples 24D and 19D which represent biological replicate in asymptomatic VAL/CAN and VAL/SW combinations, respectively, had very low number of reads paired and mapped. Also, sample 24D also showed the lowest number of reads (0.1 million). Transcriptome analysis of VAL leaves in the asymptomatic VAL/CAN combination showed significant upregulation of genes encoding AU conjugators such as MIPS, AU transporter, AU responsive ARFs, BR biosynthesis, BR kinase receptors as 102

103 compared to asymptomatic VAL/SW leaves. Upregulation of BR and AU metabolism associated genes in the asymptomatic VAL/CAN suggests that CAN rootstock reprograms the AU and BR metabolism in VAL scion at the asymptomatic stage of CaLas-infection. Pathogen-infected plants are marked with enhanced AU metabolism (Ludwig-Müller, 2015). Also, pathogens manipulate AU and GA pathways to facilitate cell wall expansion to spread the pathogen infection (Ma and Ma, 2016). Induction of genes encoding AU response repressors, AUX/IAA regulators and negative regulators of GA biosynthesis in asymptomatic VAL/CAN combination suggests the possible mechanism of CAN rootstock in inhibiting AU and GA -induced cell expansion, and CaLas infection spread in the scion. Whereas, SW rootstock significantly upregulated AU biosynthesis genes such as ILL6, IAR3, and transcripts encoding CYP83B1 which increase the AU pool in the plant. Overexpression of these genes in asymptomatic VAL/SW combination supports the previous studies that showed the potential involvement of pathogen-induced AU production (Mutka et al., 2013; Castillo-Lizardo et al., 2015). Cytokinin is an important hormone that regulates shoot and root morphogenesis (Werner et al., 2001). VAL leaves collected from asymptomatic VAL/CAN combination showed significant upregulation of genes encoding positive and negative CT response regulators suggesting CT-mediated growth or defense responses were induced in the asymptomatic VAL/CAN combination. Whereas, genes encoding ABA TFs such as C-REPEAT BINDING FACTOR 4 (CBF4) and ABA-responsive drought tolerance PP2C were significantly upregulated in leaves of the asymptomatic treatment of VAL/SW. Upregulation of CBF4 and PP2C genes was observed in plant drought adaptation (Singh et al., 2015; Haake et al., 2016). CaLas-infected plants have 103

104 shown reduced root growth (Graham et al., 2013) which may reduce the uptake of nutrients and water from the rhizosphere. The highly upregulated CBF4 and PP2C transcripts in the asymptomatic VAL/SW suggest that reduction in the root growth might activate drought tolerance signaling in plants. Plant defense hormone genes encoding SA-, and ET- response regulator, and JA biosynthesis and response regulators were upregulated in leaves of the asymptomatic VAL/SW combination as compared to leaves of the asymptomatic VAL/CAN, in which ET and SA responses were strongly indicated through upregulation of genes encoding transcription regulators such as ERF1, ERF13, AP2, RRTF1, EBP, GRX480 and WRKY 70. JA biosynthesis LOX3 gene was significantly upregulated in leaves of the VAL/SW symptomatic treatment when compared to VAL/CAN leaves. However, the JAZ8 gene (encoding a Jasmonate Zim domain JAZ protein) was significantly upregulated in the asymptomatic VAL/SW leaves when compared to asymptomatic VAL/CAN leaves. Jasmonate Zim domain JAZ proteins play an important role in repressing JA activated signaling cascade through interaction between JAZ and COI1 (Wasternack and Hause, 2013). The JA and ET signaling pathways induce upon necrotrophic and herbivore infection (Simms and Rausher, 1987). The upregulated JAZs encoding genes in the VAL/SW combination suggest the suppression of the JA pathway and activation of the ET signaling cascade. However, significant upregulation of gene encoding LOX3 is still need to invistage to understand its potential role in asymptomatic VAL/SW leaves. A significant upregulation of many ET-regulated genes supports the HLB-associated leaf senescence events in the plants. Ethylene production is found to be increased in the biotrophic and necrotrophic pathogen virulence 104

105 (Weingart et al., 2001). The strongly upregulated transcripts of ET regulators suggest that CaLas-infection overproduces or promotes the plant to overexpress the ET signaling pathway in the VAL/SW combination. NPR3 gene, a negative regulator of NPR1, was also significantly overexpressed in leaves of the asymptomatic VAL/SW combination. The SAR exhibited by upregulating SA-mediated defense pathway was potentially suppressed by its negative regulator known as NPR3. Altogether, comparison of DGE analysis between leaves of asymptomatic VAL/CAN and VAL/SW suggests that in leaves of asymptomatic VAL/SW, there is a critical reprogramming between SA-JA-ET hormone metabolism to suppress the CaLas infection but, CaLas might be targeting the ET signaling pathway to manipulate plant induced defense and facilitates the pathogen infection. Also, CaLas infection induced AU and ABA metabolism genes in the HLB presymptomatic VAL/SW combination. Defense associated SA, JA and ET hormonal response related genes were not significantly upregulated in asymptomatic VAL/CAN leaves as compared to leaves collected from asymptomatic VAL/SW. However, CAN rootstock promoted the BR-dependent signaling pathway and downregulated AU production in the VAL scion grafted onto it at the asymptomatic stage of the CaLas-infection, suggesting that BR-AU interactions might play an important role that is potentially altering the VAL gene expression in VAL/CAN combination at the asymptomatic stage of HLB. Transcriptomic analysis of CaLas-infected leaves collected from the symptomatic treatment of VAL/CAN and VAL/SW combinations showed genes involved in the hormonal metabolism were significantly upregulated in the VAL leaves as well as roots collected from CaLas-infected CAN and SW rootstocks. The abundance of DEGs in the 105

106 leaves and roots shows that trees at the symptomatic stage of CaLas-infection respond differentially compared to those at the asymptomatic stage. DGE analysis of hormone metabolism and regulation related genes at the advanced stage of CaLas-infection showed that CAN and SW rootstock could differentially reprogram the hormone regulation in the leaves and roots of the grafted plants. Putative HLB-tolerant CAN rootstock differentially upregulated BR, CT, and GA metabolism related genes in roots, and reprogrammed the BR, CT, and AU metabolic signaling related genes in the VAL scion. The symptomatic VAL/CAN combination showed upregulation of AU response regulators ARFs as well as AU repressor IAAs. Whereas, the CaLas-infected symptomatic VAL/SW combination showed overexpression AU biosynthesis genes that can increase AU pool in the plants, and therefore, can facilitate spread of bacteria via cell expansion. Altogether, this suggests that the CAN rootstock probably attains AU homeostasis in HLB-symptomatic leaves and roots by upregulating AU response regulators and repressors as compared to upregulated AU biosynthesis genes in the VAL/SW combination. Genes encoding BR and CT receptor kinases were overexpressed in leaves and roots of the symptomatic VAL/CAN as compared to symptomatic VAL/SW, suggesting that CAN rootstock activates the BR and CT -dependent immunity and growth responses in leaves and roots at the symptomatic stage of HLB-affected plants. Jasmonic acid hormone metabolism related genes were not significantly upregulated in the leaves of the symptomatic VAL/CAN as compared to symptomatic VAL/SW. However, the JAinduced MYC2, a gene involved in the JA defense signaling branch, overexpressed in the symptomatic VAL/CAN roots. In addition, roots collected from symptomatic 106

107 VAL/CAN combination showed the upregulation of genes encoding the negative regulators of JA, SA, and GA. The activation of the MYC associated response in the roots is attributed to the interaction between JA repressor JAZ and GA repressor DELLA proteins as reviewed in the hormones crosstalk literature (De Bruyne et al., 2014). In the absence of GA, DELLA interacts with JAZ which released the JAZ induced JA repression. The suppression of JAZ leads to the activation of MYC signaling pathway (Navarro et al., 2008). MYC singling branch activates the JA signaling and suppression of SA-induced responses (Pieterse et al., 2009). Also, JA and ABA-induced MYC and PYL4 TFs have a synergistic effect on the herbivore resistance (Lackman et al., 2011). Therefore, upregulation of transcripts encoding MYC and PYL4 TFs, and overexpression of DELLA indicate that VAL/CAN plants had defense activated for herbivore (psyllid) attack. The symptomatic treatment of VAL/SW combination modulated JA, ET, AU, and ABA hormonal metabolism associated genes in the VAL leaves and SW roots. SAmediated signaling pathway genes were not upregulated at higher expression levels in either of the tissues suggesting that JA-ET dependent SA antagonism suppressed the SA pathway (Pieterse et al., 2012). Moreover, overexpression of AU and ABA metabolism pathway genes supported the downregulation of SA-dependent defenses as studied by De Torres Zabala et al. (2009). Upregulation of genes involved in the ABA metabolism and signaling pathway in the symptomatic treatment of the VAL/SW roots as compared to symptomatic VAL/CAN roots supports the HLB induced root growth reduction, and possible water deficiency associated abiotic stress in the plant. The water deficiency associated abiotic stress might activate ABA signaling in the 107

108 symptomatic VAL/SW roots. Antagonism between ABA and SA has also been reported when overexpression of NCED5, an ABA biosynthesis gene, accumulated JA and lowered SA content in Arabidopsis (Fan et al., 2009). In symptomatic VAL/SW roots, the significant upregulation of NCED5 suggests that ABA might play an important role in downregulating SAR response. Auxin-responsive GH3 is an important modulator in AU homeostasis that inactivates AU by conjugating excess AU with amino acids (Staswick, 2005). Higher expression of transcripts encoding GH3.1 in the roots of the symptomatic treatment of the VAL/SW as compared to symptomatic VAL/CAN roots suggests that VAL/SW is possibly controlling excess AU accumulation. Overall, the DGE analysis of the genes involved in hormonal metabolism regulation suggests that susceptibility of the SW rootstock is significantly attributed to the AU accumulation, water deficiency induced ABA responses and ET metabolism overexpression. Although ET is associated with the plant defenses to the pathogen, the overwhelming ET induces response imbalances between JA-SA interaction. Also, ABA and AU -induced responses strongly downregulate SA-regulated defense in the advanced stage of symptom development. The JA activated pathway seems to be a part of SA-JA hormonal crosstalk and response to psyllid attack. The improved tolerance induced by the CAN rootstock can be attributed to the substantial upregulation of BR metabolism and AU homeostasis in the presymptomatic and symptomatic stages of CaLas infection. Crosstalk between DELLA and JAZ proteins significantly bypass the JA suppression and activates MYC branch. The upregulation of JA-induced MYC also explains the suppression ET and SA signaling pathway in the symptomatic VAL/CAN roots. Overall, the data showed that CAN rootstock reprograms the VAL scion by 108

109 attaining AU homeostasis, and BR-CT-JA regulated growth and defense responses under CaLas infection. Whereas SW is unable to defy AU and ET -induced reactions in the CaLas-infected plant, at least in the infection phases studied. In conclusion, DGE analysis of two different scion-rootstock combinations study showed that rootstock can differentially regulate the scion transcriptome in response to the HLB infection, affecting overall tree tolerance potentially through hormonal regulations. 109

110 Table 3-1. Experimental treatments and scion/rootstock combinations Treatments based on visual Rootstock Rootstock Parents Scion observations of HLB symptoms Swingle; 2n (SW) putative HLBtolerant candidate; 2n (CAN) Grapefruit X Trifoliate orange HBP Pummelo X Cleopatra Mandarin Valencia sweet orange (VAL) Valencia sweet orange (VAL) Symptomatic VAL/SW Slightly Symptomatic VAL/CAN Asymptomatic VAL/SW Asymptomatic VAL/CAN Approximately 7- year old trees planted in the Lee Family s Alligator Grove east of St. Cloud, FL. Table 3-2. Comparison pairs used for DGE analysis in leaves and roots of the experimental scion/rootstock combinations Leaves Roots Asymptomatic VAL/CAN vs. Asymptomatic Asymptomatic VAL/CAN vs. VAL/SW Asymptomatic VAL/SW Symptomatic VAL/CAN vs. Symptomatic Symptomatic VAL/CAN vs. Symptomatic VAL/SW VAL/SW. 110

111 Table 3-3. Differentially expressed hormonal metabolism-associated genes significantly upregulated in leaves of asymptomatic VAL/CAN combination as compared to asymptomatic VAL/SW leaves Arabidopsis Associated C.clementina_ID log2 FC Arabidopsis-define Gene hormone Functional description AT3G45140 Ciclev m.g lipoxygenase 2 JA JA Biosynthesis LHY Ciclev m.g Homeodomain-like superfamily protein AU Circadian clock regulation TT4 Ciclev m.g Chalcone and stilbene synthase family protein AU negative regulator of AU transport AT3G09610 Ciclev m.g Homeodomain-like superfamily protein AU Circadian clock MIPS2 Ciclev m.g myo-inositol-1-phosphate synthase 2 AU Auxin storage and transport MYB3 Ciclev m.g myb domain protein 3 AU Myb domain transcription factor SHN1 Ciclev m.g Integrase-type DNA-binding superfamily protein ET Cutin Biosynthesis TINY2 Ciclev m.g Integrase-type DNA-binding superfamily protein ET putative transcription factor F20P5.26 Ciclev m.g myb-like transcription factor family protein AU Myb transcription factor TT7 Ciclev m.g Cytochrome P450 Flavonoid synthesis, Convert AU superfamily protein naringenin to eriodictyol Type-A response regulators seem ARR9 Ciclev m.g response regulator 9 CT to act as negative regulators of the cytokinin signaling AT1G08810 Ciclev m.g myb domain protein 60 JA Stomatal regulation T12P18.14 Ciclev m.g RING/U-box superfamily protein AU Proteasome/ protein degradation ARF16 Ciclev m.g auxin response factor 16 AU Auxin responsive gen IAA14 Ciclev m.g indole-3-acetic acid inducible 14 AU IAA/AUX gene family NRT1.1 Ciclev m.g nitrate transporter 1.1 AU Nitrate transporter ATAUX2-11 Ciclev m.g AUX/IAA transcriptional regulator family protein AU Auxin response repressor F10A5.21 Ciclev m.g SAUR-like auxin-responsive protein family AU Auxin responsive gene 111

112 Table 3-3. Continued Arabidopsis Gene C.clementina_ID log2 FC Arabidopsis-define GLIP1 Ciclev m.g GDSL lipase 1 ET GPRI1 Ciclev m.g GBF\'s pro-rich regioninteracting factor 1 ZFP8 Ciclev m.g zinc finger protein 8 SA BRI1 Ciclev m.g Leucine-rich receptor-like protein kinase family protein Associated hormone SA BR Functional description Ethylene signaling, antimicrobial resistance Chloroplast development/regulation of chlorophyll biosynthesis transcription factor required for the initiation of inflorescence trichomes to GA and CT BR Signaling AT1G68530 Ciclev m.g ketoacyl-CoA synthase 6 JA long chain fatty acid biosynthesis, Wax biosynthesis BKI1 Ciclev m.g BRI1 kinase inhibitor 1 BR Negative regulator of brassinosteroid signaling F27J15.20 Ciclev m.g Duplicated homeodomain-like superfamily protein AU Transcription factors IAA14 Ciclev m.g indole-3-acetic acid inducible IAA/AUX gene family, repressor of AU AU 14 responsive gene MBG8.12 Ciclev m.g Major facilitator superfamily protein SA folate transport GA2OX8 Ciclev m.g gibberellin 2-oxidase 8 GA inactivates GA SMT1 Ciclev m.g sterol methyltransferase 1 BR Sterol Biosynthesis (cycloartenol to 24- methylene cycloarteno) MGH6.18 Ciclev m.g Major facilitator superfamily affinity carnitine transporter involved in the ET protein active cellular uptake of carnitine HB-2 Ciclev m.g homeobox protein 2 AU Transcription factor EXPA1 Ciclev m.g expansin A1 GA loosening and extension of cell wall TCP14 Ciclev m.g TEOSINTE BRANCHED, cycloidea and PCF (TCP) 14 CT affect internode length COP1 Ciclev m.g Transducin/WD40 repeat-like repressor of photomorphogenisis and GA superfamily protein activates etiolation in dark ARF8 Ciclev m.g auxin response factor 8 AU Promote JA production and flower maturation HERK1 Ciclev m.g hercules receptor kinase 1 BR required for cell elongation during vegetative growth BR independent manner 112

113 Table 3-3. Continued Arabidopsis Gene C.clementina_ID log2 FC Arabidopsis-define SHN2 Ciclev m.g AT1G06620 Ciclev m.g APRR2 Ciclev m.g Integrase-type DNA-binding superfamily protein 2-oxoglutarate (2OG) and Fe(II)-dependent oxygenase superfamily protein CheY-like two-component responsive regulator family protein BKI1 Ciclev m.g BRI1 kinase inhibitor 1 BR Associated hormones ET JA CT Functional description Cutin Biosyntehsis Oxido-reductase Ripening and chlorophyll development Negative regulator of brassinosteroid signaling F27J15.20 Ciclev m.g Duplicated homeodomain-like superfamily protein AU Transcription factors IAA14 Ciclev m.g indole-3-acetic acid inducible IAA/AUX gene family, repressor of AU 14 AU responsive gene MBG8.12 Ciclev m.g Major facilitator superfamily protein SA folate transport GA2OX8 Ciclev m.g gibberellin 2-oxidase 8 GA inactivates GA SMT1 Ciclev m.g sterol methyltransferase 1 BR Sterol Biosynthesis (cycloartenol to 24-methylene cycloarteno) MGH6.18 Ciclev m.g affinity carnitine transporter involved Major facilitator superfamily ET in the active cellular uptake of protein carnitine HB-2 Ciclev m.g homeobox protein 2 AU Transcription factor EXPA1 Ciclev m.g expansin A1 GA loosening and extension of cell wall TCP14 Ciclev m.g TEOSINTE BRANCHED, cycloidea and PCF (TCP) 14 CT affect internode length COP1 Ciclev m.g Transducin/WD40 repeat-like repressor of photomorphogenisis and GA superfamily protein activates etiolation in dark ARF8 Ciclev m.g auxin response factor 8 AU Promote JA production and flower maturation HERK1 Ciclev m.g hercules receptor kinase 1 BR required for cell elongation during vegetative growth BR independent manner 113

114 Table 3-4. Differentially expressed hormonal metabolism-associated genes significantly upregulated in leaves of asymptomatic VAL/SW combination as compared to asymptomatic VAL/CAN leaves Arabidopsis Associated C.clementina_ID log2 FC* Arabidopsis-define Gene hormones Functional description CBF4 Ciclev m.g C-repeat-binding factor 4 ABA ABA induce abiotic stress related TF F12K2.11 Ciclev m.g F-box family protein ABA WRKY33 Ciclev m.g WRKY DNA-binding protein 33 SA JA regulated transcription factor AT2G31945 Ciclev m.g hypothetical protein ET hypothetical protein CBF4 Ciclev m.g C-repeat-binding factor 4 ABA ABA induce abiotic stress related TF CYP83B1 Ciclev m.g cytochrome P450, family 83, glucosinolate biosynthesis, elavated ET subfamily B, polypeptide 1 auxins, JA suppressor EDL3 Ciclev m.g EID1-like 3 ABA F3H9.15 Ciclev m.g hypothetical protein ABA hypothetical protein SIB1 Ciclev m.g sigma factor binding protein 1 SA Hyperactive defense _SA accumulation F22D22.22 Ciclev m.g Acyl-CoA N-acyltransferases GCN5-related N-acetyltransferase-like ET (NAT) superfamily protein protein T5J17.9 Ciclev m.g Cupredoxin superfamily protein SA L-ascorbate oxidase/oxidation-reduction DREB2C Ciclev m.g Integrase-type DNA-binding superfamily protein ABA abiotic tolerance JAZ8 Ciclev m.g jasmonate-zim-domain protein 8 ET JA biosynthesis suppressor MYB15 Ciclev m.g myb domain protein 15 AU ABA induced drought/ stress tolerance ERF1 Ciclev m.g ethylene response factor 1 ET Ethylene transcription factor F22D22.22 Ciclev m.g Acyl-CoA N-acyltransferases GCN5-related N-acetyltransferase-like ET (NAT) superfamily protein protein CYP81D8 Ciclev m.g cytochrome P450, family 81, subfamily D, polypeptide 8 ET response to karrikin ERF-1 Ciclev m.g ethylene responsive element Involved in the regulation of gene ET/ABA binding factor 1 expression T19D16.6 Ciclev m.g Protein kinase superfamily protein ET/ABA putative receptor-like protein kinase WRKY40 Ciclev m.g WRKY DNA-binding protein 40 SA/JA Suppress PAMP induce ROS and seedling growth inhibition/suppress PTI JAZ1 Ciclev m.g jasmonate-zim-domain protein 1 SA/JA/ABA JA repressor gene AT3G57450 Ciclev m.g JA/ABA uncharacterized protein RRTF1 Ciclev m.g redox-responsive transcription involved in the regulation of gene ET factor 1 expression by stress factors 114

115 Table 3-4. Continued Arabidopsis Associated C.clementina_ID log2 FC Arabidopsis-define Gene hormones Functional description K18I23.10 Ciclev m.g ET uncharacterized protein ATAF1 Ciclev m.g NAC (No Apical Meristem) domain transcriptional regulator ABA superfamily protein CCR3 Ciclev m.g CRINKLY4 related 3 ET Serine/threonine-protein kinase CYP94C1 Ciclev m.g cytochrome P450, family 94, subfamily C, polypeptide 1 AU JA Lle catabolic turn over ERF1 Ciclev m.g ethylene response factor 1 ET key integrator of ethylene and jasmonate signals in the regulation defenses. JAZ1 Ciclev m.g jasmonate-zim-domain protein 1 SA JA respressor gene T1F9.17 Ciclev m.g F-box family protein ET F-box protein AT5G57150 Ciclev m.g basic helix-loop-helix (bhlh) DNAbinding superfamily protein JA transcription factor bhlh35 WRKY40 Ciclev m.g WRKY DNA-binding protein 40 SA/JA/ABA Suppress PAMP induce ROS and seedling growth inhibition/suppress PTI F18A17.4 Ciclev m.g Protein kinase family protein with Protein kinase family protein with leucinerich repeat domain ET/ABA leucine-rich repeat domain ERF13 Ciclev m.g ethylene-responsive element components of stress signal transduction ET/AU binding factor 13 pathways WRKY33 Ciclev m.g WRKY DNA-binding protein 33 SA JA regualted transcription factor MPA22.3 Ciclev m.g ARM repeat superfamily protein ET U-box domain-containing protein 21; Functions as an E3 ubiquitin ligase TT7 Ciclev m.g Cytochrome P450 superfamily Flavonoid synthesis, Convert naringenin to AU protein eriodictyol HERK1 Ciclev m.g hercules receptor kinase 1 BR Receptor-like protein kinase required for cell elongation during vegetative growth PLA-2 Ciclev m.g alpha/beta-hydrolases superfamily protein ET JAZ1 Ciclev m.g jasmonate-zim-domain protein 1 SA JA siganling respressor T32G9.25 Ciclev m.g ET uncharacterized protein F13A10.15 Ciclev m.g P-loop containing nucleoside The enzyme involved in the ethylene triphosphate hydrolases ET biosynthesis. superfamily protein CYP707A1 Ciclev m.g cytochrome P450, family 707, subfamily A, polypeptide 1 ABA ABA Biosynthesis 115

116 Table 3-4. Continued Arabidopsis Associated C.clementina_ID log2 FC Arabidopsis-define Gene hormones Functional description T32G9.25 Ciclev m.g ET uncharacterized protein F13A10.15 Ciclev m.g P-loop containing nucleoside Enzyme involved in the ethylene triphosphate hydrolases ET biosynthesis. superfamily protein CYP707A1 Ciclev m.g cytochrome P450, family 707, subfamily A, polypeptide 1 ABA ABA Biosynthesis CYP94C1 Ciclev m.g cytochrome P450, family 94, subfamily C, polypeptide 1 JA/ABA/AU JA Lle catabolic turn over EFE Ciclev m.g ethylene-forming enzyme ET/SA activating ethylene biosynthesis HAI2 Ciclev m.g highly ABA-induced PP2C gene 2 ABA negative regulator of PCD, SA, JA, and ET LOX3 Ciclev m.g lipoxygenase 3 SA Linolenic acid MAPKKK19 Ciclev m.g mitogen-activated protein kinase mitogen-activated protein kinase kinase ET kinase kinase 19 kinase 19 F3G5.22 Ciclev m.g C2H2 and C2HC zinc fingers ET zinc finger of Arabidopsis thaliana 11 AP2C1 Ciclev m.g superfamily protein Protein phosphatase 2C family protein DIC2 Ciclev m.g dicarboxylate carrier 2 ET ABA May be involved in protecting plant cells against oxidative stress damage AT5G26170 Ciclev m.g WRKY DNA-binding protein 50 JA Mediate SA and repress JA EBP Ciclev m.g ethylene-responsive element involved in the regulation of gene CT binding protein expression by stress factors ERF13 Ciclev m.g ethylene-responsive element binding factor 13 ABA ERF13 Ciclev m.g ethylene-responsive element involved in the regulation of gene AU binding factor 13 expression by stress factors BT1 Ciclev m.g BTB and TAZ domain protein 1 AU substrate-specific adapter of an E3 ubiquitin-protein ligase complex MYBR1 Ciclev m.g myb domain protein r1 AU/ABA Confers resistance to abiotic stresses dependent of ABA STZ Ciclev m.g salt tolerance zinc finger AU involved in jasmonate (JA) early signaling response. ERF12 Ciclev m.g ERF domain protein 12 ET Ehtylene resposnive gene GRX480 Ciclev m.g Thioredoxin superfamily protein SA SA-responsive gene 116

117 Table 3-4. Continued Arabidopsis Gene C.clementina_ID log2 FC Arabidopsis-define MQL5.25 Ciclev m.g AT1G32640 Ciclev m.g ERF5 Ciclev m.g ILL6 Ciclev m.g myb-like transcription factor family protein Basic helix-loop-helix (bhlh) DNAbinding family protein ethylene responsive element binding factor 5 IAA-leucine resistant (ILR)-like gene 6 KCS11 Ciclev m.g ketoacyl-CoA synthase 11 BR Associated hormones GA JA ET AU Functional description Transcription factor Transcription factor components of stress signal transduction pathways IAA storage Cutin/wax synthesis to prevent water loss and pathogen atatck F14D16.17 Ciclev m.g SA uncharacterized protein T6G21.2 Ciclev m.g Polynucleotidyl transferase, ribonuclease H-like superfamily ET/ABA protein expression AT4G16760 Ciclev m.g acyl-coa oxidase 1 JA It is a component of the CCR4 complex involved in the control of gene May be involved in the biosynthesis of jasmonic acid AATP1 Ciclev m.g AAA-ATPase 1 SA Proteosome T23A1.8 Ciclev m.g Protein kinase superfamily protein SA kinase 3 AATP1 Ciclev m.g AAA-ATPase 1 SA Preoteosome MWD22.13 Ciclev m.g Integrase-type DNA-binding ethylene-responsive transcription factor ET superfamily protein ERF105 AOC3 Ciclev m.g allene oxide cyclase 3 ET/ABA production of 12-oxo-phytodienoic acid (OPDA), a precursor of jasmonic acid F14G6.20 Ciclev m.g ET uncharacterized protein F12L6.31 Ciclev m.g Protein of unknown function (DUF506) ET uncharacterized protein NPR3 Ciclev m.g NPR1-like protein 3 SA SA modulator AT5G35735 Ciclev m.g Auxin-responsive family protein SA Auxine responsive gene PKT3 Ciclev m.g peroxisomal 3-ketoacyl-CoA thiolase 3 ABA WRKY70 Ciclev m.g WRKY DNA-binding protein 70 SA SA responsive gene ATERDJ3A Ciclev m.g DNAJ heat shock N-terminal domain-containing protein ABA 117

118 Table 3-4. Continued Arabidopsis Gene C.clementina_ID log2 FC Arabidopsis-define Associated hormones Functional description F2O15.22 Ciclev m.g zinc finger (C3HC4-type RING finger) family protein ET C3H4 type zinc finger protein AOS Ciclev m.g allene oxide synthase SA JA biosyntehsis * The negative sign in the column of log2 FC indicates comparative downregulation of the gene expression level in VAL/CAN and upregulation in VAL/SW combination. 118

119 Table 3-5. Differentially expressed hormonal metabolism-associated genes significantly upregulated in leaves of the symptomatic VAL/CAN combination as compared to symptomatic VAL/SW leaves Arabidopsis Associated C.clementina_ID log2 FC Arabidopsis-define Functional description Gene hormones CKX7 Ciclev m.g cytokinin oxidase 7 CT degradation of cytokinin WRKY75 Ciclev m.g WRKY DNA-binding protein 75 ET Interacts specifically with the W box a frequently occurring elicitor- responsive cis-acting element IAA19 Ciclev m.g indole-3-acetic acid inducible 19 AU Auxin response repressor IAA14 Ciclev m.g indole-3-acetic acid inducible 14 AU Auxin response repressor HCT Ciclev m.g hydroxycinnamoyl-coa shikimate/quinate hydroxycinnamoyl transferase AUX/IAA transcriptional regulator family protein AU lignin bisynthesis, flavonoid accumulation/auxin tranport ARR4 Ciclev m.g response regulator 4 SA Modulates red light signaling through its interaction with the phytochrome B photoreceptor AIR3 Ciclev m.g Subtilisin-like serine Au resonsive root gene, activated by NAC AU endopeptidase family protein transcription factor T12H17.10 Ciclev m.g SAUR-like auxin-responsive protein family AU Auxin response gene KAO2 Ciclev m.g ent-kaurenoic acid hydroxylase 2 GA a key step in gibberellins (GAs) biosynthesis IAA8 Ciclev m.g indoleacetic acid-induced protein 8 AU Auxin response repressor ABF2 Ciclev m.g abscisic acid responsive elements-binding factor 2 ABA nvolved in ABA and stress responses and acts as a positive component of glucose signal transduction. ATAUX2-11 Ciclev m.g AU Auxin response repressor KCS6 Ciclev m.g ketoacyl-CoA synthase 6 JA an essential step of the cuticular wax production F24J1.30 Ciclev m.g Homeodomain-like superfamily protein CT BSK2 Ciclev m.g BR-signaling kinase 2 BR BR-signaling kinase 2 F12K2.11 Ciclev m.g F-box family protein ABA F-box family protein ATAUX2- AUX/IAA transcriptional regulator Ciclev m.g family protein AU Auxin response repressor NDL1 Ciclev m.g N-MYC downregulated-like 1 AU Auxine transport and hence root architcture RPN10 Ciclev m.g regulatory particle non-atpase growth develop via proteosome of ABA AU 10 signaling protein ABI5/DPBF1 119

120 Table 3-5. Continued Arabidopsis Gene C.clementina_ID log2 FC Arabidopsis-definition APRR2 Ciclev m.g CheY-like two-component responsive regulator family protein ARF8 Ciclev m.g auxin response factor 8 AU ARR11 Ciclev m.g response regulator 11 CT CYP707A2 Ciclev m.g cytochrome P450, family 707, subfamily A, polypeptide 2 ABA Associated hormones CT Functional description Promote JA production and flower maturation ABA hydroxylation, Control ABA during mid-maturation 120

121 Table 3-6. Differentially expressed hormonal metabolism-associated genes significantly upregulated in leaves of the symptomatic VAL/SW combination as compared to symptomatic VAL/CAN leaves Arabidopsis Hormones C.clementina_ID log2 FC * Arabidopsis-definition Gene associated Functional description LOX2 Ciclev m.g lipoxygenase 2 JA JA biosynthesis JAZ1 Ciclev m.g jasmonate-zim-domain protein 1 SA JA repressor LOX2 Ciclev m.g lipoxygenase 2 JA JA biosynthesis EXPA1 Ciclev m.g expansin A1 GA loosening and extension of cell wall JAZ8 Ciclev m.g jasmonate-zim-domain protein 8 JA Repressor of jasmonate responses ERF1 Ciclev m.g ethylene response factor 1 JA Binds to the GCC- box pathogenesisrelated promoter element AT4G08850 Ciclev m.g Leucine-rich repeat receptor-like protein kinase family protein WRKY40 Ciclev m.g WRKY DNA-binding protein 40 SA ET Supress PAMP induce ROS and seedling growth inhibition/supress PTI WRKY33 Ciclev m.g WRKY DNA-binding protein 33 SA JA response postive regulator. SIB1 Ciclev m.g sigma factor binding protein 1 SA Hyperactive defense _SA accumulation DREB2C Ciclev m.g Binding to the C-repeat/DRE element Integrase-type DNA-binding ABA mediates high salinity- and abscisic superfamily protein acid-inducible transcription JAZ1 Ciclev m.g jasmonate-zim-domain protein 1 SA JA repressor ERF1 Ciclev m.g ethylene response factor 1 JA Binds to the GCC- box pathogenesisrelated promoter element TCTP Ciclev m.g translationally controlled tumor Involved in calcium binding and AU protein microtubule stabilization ACS6 Ciclev m.g aminocyclopropane-1- Ethylene synthesis, local gene mediated AU carboxylic acid (acc) synthase 6 and basal resistance ATERDJ3A Ciclev m.g DNAJ heat shock N-terminal domain-containing protein ABA DNA J domain-containing protein AP2C1 Ciclev m.g Protein phosphatase 2C family protein ABA Protein phosphatase that negatively regulates defense respones. I OPCL1 Ciclev m.g OPC-8:0 CoA ligase1 JA Contributes to jasmonic acid biosynthesis by initiating the betaoxidative chain shortening of its precursors. AT3G60690 Ciclev m.g SAUR-like auxin-responsive SA Auxin response gene protein family Rap2.6L Ciclev m.g related to AP2 6l ET 121

122 Table 3-6. Continued Arabidopsis Hormones C.clementina_ID log2 FC Arabidopsis-definition Gene associated Functional description EDL3 Ciclev m.g EID1-like 3 ABA F box protein, ABA regulation CYP81D8 Ciclev m.g cytochrome P450, family 81, ET subfamily D, polypeptide 8 PYL4 Ciclev m.g PYR1-like 4 ABA required for ABA- mediated responses such as stomatal closure and germination inhibition F5H14.15 Ciclev m.g Integrase-type DNA-binding ET superfamily protein CYP94C1 Ciclev m.g cytochrome P450, family 94, JA JA Lle catabolic turn over subfamily C, polypeptide 1 MFB16.16 Ciclev m.g SAUR-like auxin-responsive AU Auxin response gene protein family ATAF1 Ciclev m.g NAC (No Apical Meristem) JA domain transcriptional regulator superfamily protein T29M8.8 Ciclev m.g Integrase-type DNA-binding ET superfamily protein T1F9.17 Ciclev m.g F-box family protein ET F5E6.17 Ciclev m.g Plant neutral invertase family ET protein CBF4 Ciclev m.g C-repeat-binding factor 4 ABA freezing tolerance and cold acclimation, drought adaptations PYR1 Ciclev m.g Polyketide cyclase/dehydrase and lipid transport superfamily protein ABA Inhibits the activity of group-a protein phosphatases type 2C (PP2Cs) when activated by ABA T1J8.15 Ciclev m.g UDP-Glycosyltransferase ET superfamily protein TT4 Ciclev m.g Chalcone and stilbene synthase family protein AU negative regulator of AU transport/ flavonoid accumulation/chalcone synthase_naringin ; Acts as a transcriptional activator ERF-1 Ciclev m.g ethylene responsive element binding factor 1 ABA F18A17.4 Ciclev m.g Protein kinase family protein with ABA leucine-rich repeat domain JAZ1 Ciclev m.g jasmonate-zim-domain protein 1 SA JA repressor WRKY33 Ciclev m.g WRKY DNA-binding protein 33 SA JA regulate transcription factor 122

123 Table 3-6. Continued Arabidopsis Gene C.clementina_ID log2 FC Arabidopsis-definition Hormones associated Functional description CYP83B1 Ciclev m.g cytochrome P450, family 83, subfamily B, polypeptide 1 JA PR4 Ciclev m.g pathogenesis-related 4 JA JA responsive gene WRKY30 Ciclev m.g WRKY DNA-binding protein 30 ET MEE59 Ciclev m.g maternal effect embryo arrest 59 ET EBF1 Ciclev m.g EIN3-binding F box protein 1 ET AP22.63 Ciclev m.g ARM repeat superfamily protein SA EBP Ciclev m.g ethylene-responsive element binding protein CT ABP1 Ciclev m.g endoplasmic reticulum auxin binding protein 1 AU glucasinolate biosynthesis, elavated auxins, JA suppressor Auxin binding protein involved in cell elongation and cell division/receptor of AU LOX3 Ciclev m.g lipoxygenase 3 SA JA responsive genes LECRKA4.2 Ciclev m.g lectin receptor kinase a4.1 ABA involved in negative regulation of abscisic acid response in seed germination ERF-1 Ciclev m.g ethylene responsive element binding factor 1 ABA T11A7.19 Ciclev m.g Integrase-type DNA-binding superfamily protein ET F3F20.16 Ciclev m.g basic helix-loop-helix (bhlh) DNA-binding superfamily protein ET MWD22.13 Ciclev m.g Integrase-type DNA-binding superfamily protein ET ABI2 Ciclev m.g MKD10.10 Ciclev m.g Protein phosphatase 2C family protein Methylenetetrahydrofolate reductase family protein PYL11 Ciclev m.g PYR1-like 11 ABA ABA JA Repressor of the abscisic acid (ABA) signaling pathway that regulates numerous ABA responses proline dehydrogenase 2; Converts proline to delta-1-pyrroline-5-carboxylate Receptor for ABA, required for ABAmediated responses WRKY70 Ciclev m.g WRKY DNA-binding protein 70 SA SA acid transcription factor Signal transduction histidine Acts as a redundant negative regulator of EIN4 Ciclev m.g kinase, hybrid-type, ethylene ET ethylene signaling sensor 123

124 Table 3-6. Continued Arabidopsis Gene C.clementina_ID log2 FC Arabidopsis-definition Hormones associated Functional description DHS2 Ciclev m.g deoxy-d-arabino-heptulosonate 7-phosphate synthase CT LOX5 Ciclev m.g PLAT/LH2 domain-containing lipoxygenase family protein JA WRKY70 Ciclev m.g WRKY DNA-binding protein 70 SA SA acid transcription factor regulate root architecture, leniolate biosynthesis Ciclev m.g NPR1-like protein 3 SA SA modulator NPR3 * The negative sign in the column of log2 FC indicates comparative downregulation of the gene expression level in VAL/CAN and upregulation in VAL/SW combination. 124

125 Table 3-7. Differentially expressed hormonal metabolism-associated genes significantly upregulated in roots of the symptomatic VAL/CAN combination as compared to symptomatic VAL/SW roots Arabidopsis Hormones C.clementina_ID log2 FC Arabidopsis-definition Gene associated JAZ8 Ciclev m.g jasmonate-zim-domain protein 8 JA CYP94C1 Ciclev m.g cytochrome P450, family 94, subfamily C, polypeptide 1 JA NPR3 Ciclev m.g NPR1-like protein 3 SA SA modulator JAZ1 Ciclev m.g jasmonate-zim-domain protein 1 JA/SA JA repressor Functional description JAZ1 Ciclev m.g jasmonate-zim-domain protein 1 JA/SA JA repressor CYP83B1 Ciclev m.g cytochrome P450, family 83, subfamily B, polypeptide 1 AU MYC2 Ciclev m.g Basic helix-loop-helix (bhlh) DNA-binding JA, ABA transcriptional JA family protein activator ERF13 Ciclev m.g ethylene-responsive element binding factor 13 SA BAS1 Ciclev m.g Cytochrome P450 superfamily protein BR GAI Ciclev m.g GRAS family transcription factor family protein GA/SA Della protein AOS Ciclev m.g allene oxide synthase JA JA biosynthesis gene F1M20.4 Ciclev m.g Leucine-rich repeat protein kinase family protein JA RGL1 Ciclev m.g RGA-like 1 GA/SA Della protein DHS1 Ciclev m.g deoxy-D-arabino-heptulosonate 7- phosphate synthase 1 JA JAZ10 Ciclev m.g jasmonate-zim-domain protein 10 JA HERK2 Ciclev m.g hercules receptor kinase 2 BR NCED5 Ciclev m.g nine-cis-epoxycarotenoid dioxygenase 5 ABA GRX480 Ciclev m.g Thioredoxin superfamily protein SA BKI1 Ciclev m.g BRI1 kinase inhibitor 1 BR AT5G59845 Ciclev m.g Gibberellin-regulated family protein GA F14G6.12 Ciclev m.g Auxin efflux carrier family protein AU PYL4 Ciclev m.g PYR1-like 4 ABA RR5 Ciclev m.g response regulator 5 GA GA20OX1 Ciclev m.g oxoglutarate (2OG) and Fe(II)-dependent oxygenase superfamily protein GA 125

126 Table 3-7. Continued Arabidopsis Hormones C.clementina_ID log2 FC Arabidopsis-definition Gene associated F12K2.11 Ciclev m.g F-box family protein ABA T11I11.4 Ciclev m.g F-box family protein BR AIR12 Ciclev m.g auxin-responsive family protein AU DREB2C Ciclev m.g Integrase-type DNA-binding superfamily protein ABA CYP711A1 Ciclev m.g cytochrome P450, family 711, subfamily A, polypeptide 1 AU RGA1 Ciclev m.g GRAS family transcription factor family protein GA GRX480 Ciclev m.g Thioredoxin superfamily protein SA MYC2 Ciclev m.g Basic helix-loop-helix (bhlh) DNA-binding family protein GA2OX6 Ciclev m.g gibberellin 2-oxidase 6 GA PSBO2 Ciclev m.g photosystem II subunit O-2 JA RGA1 Ciclev m.g GRAS family transcription factor family protein GA ERF1 Ciclev m.g ethylene response factor 1 JA ATAF1 Ciclev m.g NAC (No Apical Meristem) domain transcriptional regulator superfamily protein JA LTA2 Ciclev m.g oxoacid dehydrogenases acyltransferase family protein JA BAS1 Ciclev m.g Cytochrome P450 superfamily protein BR ARF16 Ciclev m.g auxin response factor 16 AU GH3.1 Ciclev m.g Auxin-responsive GH3 family protein BR ARF2 Ciclev m.g auxin response factor 2 AU GAI Ciclev m.g GRAS family transcription factor family protein GA/SA ERF13 Ciclev m.g ethylene-responsive element binding factor 13 JA JAR1 Ciclev m.g Auxin-responsive GH3 family protein JA SA Functional description Della protein JA, ABA transcriptional activator Della protein Della protein 126

127 Table 3-8. Differentially expressed hormonal metabolism-associated genes significantly upregulated in roots of the symptomatic VAL/SW combination as compared to symptomatic VAL/CAN roots Arabidopsis Gene NCED4 C.clementina_ID log2 FC* Arabidopsis-definition Ciclev m.g nine-cis-epoxycarotenoid dioxygenase 4 PIN4 Ciclev m.g Auxin efflux carrier family protein SA Hormones associated ABA4 Ciclev m.g abscisic acid (aba)-deficient 4 ABA F24J13.5 Ciclev m.g Domain of unknown function (DUF220) SA AOC4 Ciclev m.g allene oxide cyclase 4 AU ARF19 Ciclev m.g auxin response factor 19 AU ASA1 Ciclev m.g anthranilate synthase alpha subunit 1 AU SERK1 Ciclev m.g somatic embryogenesis receptor-like kinase 1 AU PAP1 Ciclev m.g phytochrome-associated protein 1 ET HK5 Ciclev m.g histidine kinase 5 CT DFL1 Ciclev m.g Auxin-responsive GH3 family protein AU F24J1.30 Ciclev m.g Homeodomain-like superfamily protein AU OEP16-1 Ciclev m.g outer plastid envelope protein 16-1 AU MGH6.18 Ciclev m.g Major facilitator superfamily protein JA K19P17.6 Ciclev m.g Serine/threonine-protein kinase WNK (With No Lysine)- related JA F17H15.18 Ciclev m.g Leucine-rich receptor-like protein kinase family protein SA CYP83B1 Ciclev m.g cytochrome P450, family 83, subfamily B, polypeptide 1 AU F12K2.11 Ciclev m.g F-box family protein GA LAX1 Ciclev m.g like AUXIN RESISTANT 1 SA KAO2 Ciclev m.g ent-kaurenoic acid hydroxylase 2 GA IAA8 Ciclev m.g indoleacetic acid-induced protein 8 AU F5A9.17 Ciclev m.g anthranilate synthase beta subunit 1 AU ARF4 Ciclev m.g auxin response factor 4 AU CYP714A1 Ciclev m.g cytochrome P450, family 714, subfamily A, polypeptide 1 CT * The negative sign in the column of log2 FC indicates comparative downregulation of the gene expression level in VAL/CAN and upregulation in VAL/SW combination. ABA 127

128 Figure 3-1. Graphical presentation of Hormonal regulation in plants. (adapted from Plant physiology and development, sixth edition,2015) 128

129 Figure 3-2. Graphical presentation of DEGs involved in hormonal metabolism. Hormonal metabolism overview in the PageMan depicting DGE in leaves and roots of HLB-asymptomatic and -symptomatic treatment in VAL/CAN and VAL/SW combinations. Log2 FC are indicated as a gradient between blue (up-regulated in VAL/CAN combination) and red (up-regulated and VAL/SW combination. Asymp; Asymptomatic, Sympt; symptomatic 129

130 CHAPTER 4 DIFFERENTIAL EXPRESSION OF PLANT IMMUNITY AND DEFENSE-ASSOCIATED GENES IN LEAVES AND ROOTS OF TWO SCION/ROOTSTOCK COMBINATIONS AT ASYMPTOMATIC AND SYMPTOMATIC STAGES OF HLB DISEASE Introduction Plants live in a complex environment where they interact with a variety of organisms. Some plant associations with organisms are beneficial, whereas some are detrimental. The compatible or incompatible interaction between plants and pathogens leads to the activation of plant defense responses. Immunity responses to plant pathogens categorize into non-host specific and host-specific interactions. Non-host specific defense involves modulating plant structures and/or secreting chemical signals to stop the entry of the pathogen or to alert the neighboring plant tissue to prepare itself for the impending pathogen infection. The structural modifications include thickening of the leaf surface, the closing of stomata to stop a pathogen entry, and secretion of volatiles or phenolics to deter the plant pathogens. The interaction of a plant innate immunity system with a host-specific pathogen is a two-branched system. The first component of the plant immunity is called PAMPtriggered immunity (PTI). PTI is the interaction between the pattern recognition receptors (PRR) that are located the plant membrane and pathogen-associated molecular patterns (PAMP). A classic example to understand PRR induced PAMP recognition is elucidated in the model plants. The interaction between Pseudomonas syriangae induced flagellin 2 (FLG2) and the flagellin sensitive 2 (FLS2) transmembrane receptor kinase was reported in Arabidopsis thaliana (Chinchilla, 2006). Failure or weakened PTI induces the second component of the plant innate immunity which is known as effector-triggered immunity (ETI) (Jones and Dangl, 2006). Effectors and 130

131 elicitors are host-specific pathogen virulence factors that target the plant's immunity. Interactions between resistance (R) genes of the plants and avirulence (AVR or elicitor) genes of the pathogens determine the compatibility of the pathogen with its host. The successful interaction of R-AVR gene products results in resistance, whereas failure of this interaction leads to susceptibility to the disease. The ETI responses activate nuclear R genes that result in a hypersensitive reaction (HR). Within few hours of the infection, the HR results in programmed cell death (PCD) that stops the spread of pathogen within the plant. Genomics studies of R genes have identified the R gene-encoded signature protein domains (Jones and Dangl, 2006; Wu and Zhou, 2013). These include a nuclear binding site (NBS), leucine-rich repeats (LRR) and a Toll-interleukin 1 receptor (TIR). The interaction between ETI and the effector-triggered susceptibility (ETS) determines plant survival. Plant defense strategies also involve hormonal crosstalk (Pieterse et al., 2012). Modulation of salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) hormones through a network of transcription factors (TFs), secondary messengers, and regulatory factors contribute to plant immunity (Pieterse et al., 2009). The antagonism between SA and JA responses has been reviewed extensively (Reymond and Farmer, 1998; Pieterse et al., 2009; Robert-Seilaniantz et al., 2011). The SA-induced defense signaling pathway results in systemic acquired resistance (SAR) which is effective mostly against biotrophs and hemibiotrophs. Whereas, JA and ET activate induced systemic resistance (ISR) is effective against necrotrophs and herbivores (Simms and Rausher, 1987; Dam and Oomen, 2008). The role of SA, JA, and ET is not restricted to biotrophs or necrotrophs, but SAR and ISR signaling components can interchange 131

132 between the SA, JA, and ET-induced immunity responses through hormonal crosstalk (Pieterse et al., 2009; Pieterse et al., 2012). The functional genomics of the plant growth hormones: auxins (AU), cytokinins (CT), and gibberellins (GA) showed that these hormones are key in the plant defense responses too (Navarro et al., 2008; Bari and Jones, 2009; Giron et al., 2013). The critical role of hormone-associated receptor kinases such as the BR RECEPTOR BR INSENSITIVE 1 (BRI1), ET RECEPTOR ET REGULATED 1 (ETR1), and CT RECEPTOR CT RESPONSE 1 (CRE1) in the plant defense and growth was studied using the A. thaliana model plant (Huot et al., 2014). Abscisic acid (ABA) was also found to be involved in defense response (Cao et al., 2011). Nakashita et al. (2003) reported brassinosteroid (BR)-induced disease resistance to tobacco mosaic virus (TMV) in tobacco (Nicotiana tabcum cv. Xanthi) and to P.syringae in rice (Oryza Sativa L). JA and ET-regulated plant defenses were also found in response to herbivore attack (Vos et al., 2005; Pieterse et al., 2012; Foyer et al., 2015). Studies of phloem-feeding insects (PFI)-plant interactions showed that PFI infestation can modulate SA, JA, and ET hormone regulated genes expression, and thereby influence the different defense signaling pathways (Thompson and Goggin, 2006; Foyer et al., 2015). Transcriptomic reprogramming PFI-infected host plants are reviewed in A. thaliana, Sorghum bicolor, Nicotiana attenuate, Oryza sativa, Malus domestica, Lycopersium esculantum (Vos et al., 2005; Foyer et al., 2015). Hormonal or innate plant immune responses are perceived, amplified, and transduced to activate defense via various signaling molecules. The category of signal receptor molecules is comprised of receptor-like kinases (RLKs), receptor-like proteins (RLPs), PRRs, mitogen-activated protein kinase (MAPK) cascades, secondary 132

133 messenger molecules such as calcium (Ca 2+ ), cyclic nucleotide gated channels (CNGC), reactive oxygen species (ROS), and ROS scavengers and, lipids. RLKs do not function only as receptors but also act as a surveillance system to detect potential threats to plants (Zhou et al., 2017). RLKs are phosphorylated by an enzyme kinase upon recognition of PAMP, MAMP (Microbe-associated molecular pattern) or herbivoreassociated molecular patterns (HAMP). Phosphorylation initiates signal transduction. RLKs are also critical in defense-induced hormonal signaling (Wu and Zhou, 2013). WALL-ASSOCIATED KINASE 1 (WAK1) proteins are the receptors activated by herbivores or by PFI induced wounding (Ferrari et al., 2013). Wounding in plants leads to damage associated molecular pattern (DAMP) alarming signals. During pathogen induced wounding or PFI interaction with plants, cell wall degrading enzymes are secreted that act as threat signal for the host plant. In this scenario, plants activate DAMP signals. DAMP signals are exhibited as secretion of oligogalacturonide (OGs) such as chitinase, glucanase, callose deposition enzymes, and production of ROS. WAK1 recognized the OG induced DAMP signals to activate downstream defense signaling (Ferrari et al., 2013). The signals received through receptors are then amplified through a MAPK cascade, Ca 2+ signaling pathways, changes in the cell wall ph, ROS production and lipid signaling. Calcium is the most ubiquitous second messenger in the plants. Calcium-dependent protein kinase (CDPK), Calmodulin (CaM), CaM-like protein (CML), glutamate-like receptors (GLRs), CNGCs are tightly interdependent in the Ca 2+ mediated signal propagation in the pathogen infected plants (Ma et al., 2009; Schulz et al., 2013). The plasma membranes are primarily made up of lipids. Activation of hydrolytic enzymes that break the bonds between lipids are 133

134 implicated as messenger molecules in a variety of cellular processes. Among these enzymes, acyl hydrolyzes and phospholipases (PLAs) A, C, and D lead to the conformational changes in plasma membrane upon pathogen infection, and confer nonhost specific defense responses (Canonne et al., 2011). The different strategies of plant defense discussed above have been reported in various Citrus diseases as well. However, the defense mechanisms involved in citrus - huanglongbing (HLB) interaction are still unclear because of limitations in culturing HLB causing putative bacteria Candidatus Liberibacter asiaticus (CaLas). The epidemic of HLB in the Florida-citrus groves is destructive. The current HLB control practices are only partially successful, and a temporary solution. Some citrus and related genera such as certain pummelos (Citrus maxima Merr.), lemons (Citrus limon L. [Burm.] f.), trifoliate orange (Poncirus trifoliata [L.] Raf), and finger lime (Microcitrus australasica F. Muell.) show tolerance to HLB. However, these species might have limited commercial viability (Folimonova et al., 2009). In contrast, the commercially viable scions Valencia,' and Hamlin' sweet oranges (Citrus sinensis [L.] Osbeck), and rootstocks such as Cleopatra mandarin (Citrus reticulata Blanco), Swingle citrumelo (Citrus paradisi x Poncirus trifoliata) are not HLB-tolerant or resistant. Therefore, for a long term HLB-solution, new combinations of two or more of the tolerant citrus species and/or cultivars will be required to produce HLB-tolerant/resistant and commercially profitable citrus varieties. Some of such improved hybrids have been created in the University of Florida (UF) and U.S Department of Agriculture (USDA) sponsored citrus breeding programs and are being analyzed for their performance under CaLas-infection. The comparative transcriptomic and proteomic analyses between CaLas-infected and healthy plant 134

135 tissues among different citrus varieties, including newly improved citrus hybrids, showed the reprogramming of defense-associated genes (Albretch and Bowman, 2008; Fan et al., 2011; Fan et al., 2012; Wang et al., 2016). Comparative analysis between CaLasinfected susceptible and tolerant cultivars showed modulation in the expression levels of genes encoding scavengers of superoxide, PHLOEM PROTEIN 2B (PP2B), DEFECTIVE INDUCED RESISTANCE 1(DIR1), RECEPTOR-LIKE PROTEIN KINASE 33 (RLP33), PLANT DEFENSIN, OSMOTIN-like protein (OSM), CHITINASE, HEAT SHOCK PROTEINS (HSPs), THAUMATIN-like proteins, MIRACULIN-like proteins, glutathione transferases, secondary metabolites involved in the glycosylation and cell wall modification, and SA-JA-ET dependent signaling regulators (Albretch and Bowman, 2008; Fan et al., 2011; Martinelli et al., 2013; Zheng and Zhao, 2013; Wang et al., 2016; Zhong et al., 2016). Altogether, this suggests that CaLas infection does not induce pathogen-specific plant immunity in these citrus cultivars but induces nonspecific plant defenses. Biological and economic tolerance to HLB is an urgent need to reduce HLB severity as necessary for sustainable and profitable citrus fruit production. The improved candidate (CAN) hybrid rootstock (46x ; Hirado Buntan Pink (HBP) pummelo x Cleopatra mandarin) used in this study was selected based on the better juice quality and reduced HLB symptoms of the Valencia' scion grafted onto it. This suggests that improved CAN rootstock has potential to address the need of biological and economic viability of HLB-tolerance. The goal of the comparative transcriptomic study is to analyze the differential reprogramming of defense related genes that were induced in the CaLas-infected Valencia' scion due to the rootstock differences, comparing the CAN to the susceptible commercial Swingle citrumelo (SW). 135

136 Materials and Methods Plant Material Two combinations of scion and rootstocks were used in this experiment (Table 4-1). Field grown seven-year-old experimental plants were planted in the Lee Family's Alligator Grove, east of St. Cloud, Florida. The one combination of trees was Valencia' (VAL) sweet orange grafted onto putative HLB-tolerant candidate (CAN) rootstock. The CAN rootstock is a hybrid between HBP pummelo and Cleopatra' mandarin. The 2 nd combination was Valencia' (VAL) scion grafted onto Swingle citrumelo (SW) rootstock. Swingle is a hybrid of grapefruit (Citrus paradisi [Macf.]) and trifoliate orange. Each combination of VAL/CAN and VAL/SW plants was divided into two treatments based on the visible presence of HLB-like symptoms (Table 4-2). Highly infected and symptomatic trees in each combination grouped into the symptomatic treatment, whereas trees with fewer symptoms or no visible symptoms were grouped into the asymptomatic treatment. All biological replicates in each treatment and combination were tested using quantitative PCR (qpcr) based CaLas detection and enzyme-linked immunosorbent assay (ELISA) -assisted citrus tristeza virus (CTV) detection. Sampling, RNA extraction, and RNA sequencing A detailed protocol of sampling, RNA extraction, and RNA sequencing is explained in Chapter 2. In brief, differentially expressed genes (DEGs) in the pairwise comparison between asymptomatic VAL/CAN and VAL/SW combinations, and symptomatic VAL/CAN, and VAL/SW combinations were obtained using RNA-Seq Tuxedo pipeline (Table 4-2). The significant differentially regulated genes in leaves and roots tissue were annotated to the C. clementina genome database in Phytozome V1.0 (Goodstein et al., 2012). Functional categories of the DEGs were identified using A. 136

137 thaliana annotation in the Phytozome server and MapMan sottware (Thimm et al., 2004) (Chapter 2). The Blast2GO algorithm (Conesa et al., 2005) was also used to identify molecular, cellular, and biological functional categories. An overview of the DEGs was developed using PageMan analysis tool in the MapMan software (Usadel et al., 2009). Results HLB Detection and RNA Sequencing Output The results of qrt PCR-based HLB detection and RNA-Seq output in all combinations and treatments are presented in Chapter 2. Differential Expressed of Defense-Associated Genes in Leaves and Roots of Asymptomatic VAL/CAN and VAL/SW combinations Leaf samples The results of transcriptome studies combined with Mapman-Pageman analysis tools showed that asymptomatic treatment VAL/CAN leaves did not show a significant increase in the expression levels of genes associated with defense signaling and innate immunity as compared to VAL/SW leaves. Leaves collected from CaLas-infected asymptomatic VAL/CAN showed upregulation of a few genes related to non-host resistance and PFI induced defense. Among these, genes encoding ET-induced-GDSL LIPASE-LIKE 1 (GLIP1), BRI1 receptor kinase, LIPID TRANSFER PROTEIN (LTP), MILDEW RESISTANCE LOCUS O-like (MLO-like), CIPK 20, and PP2-B15 were significantly upregulated more than 1 log2 fold change (log2 FC) (Table 4-3). In the asymptomatic treatment, genes involved in the secondary metabolite flavonoid and terpenoid biosynthesis pathway were significantly overexpressed in leaves of the VAL/CAN combination as compared to leaves of VAL/SW (Figure 4-3). These genes are encoding TRANSPARENT TESTA 4 (TT4), TRANSPARENT TESTA 7 (TT7), 137

138 TERPENE SYNTHASE 14 (TPS14), FLAVANONE-3 HYDROXYLASE (F3H) and UGT72B1. In the asymptomatic VAL/CAN combination, genes encoding PECTIN METHYLESTERASE 44 (PME 44), glucosinolate UGT74B1 and cytochrome P450 superfamily CYP98A3 were also significantly upregulated (Table 4-3). Leaves collected from the asymptomatic VAL/SW combination showed a greater number of significantly upregulated defense signaling and innate immunity-related genes as compared to leaves collected from asymptomatic VAL/SW (Figure 4-1 and 4-3). These genes are encoding proteins involved in secondary metabolite biosynthesis pathways, PAMP-induced receptor kinases, WRKY TFs, PATHOGENESIS RELATED (PR) proteins, Ca 2+ mediated signaling components, MAPK cascade, glutamate receptor-like GRLs, HSPs, and PHOSPHOLIPASE A2. Transcripts encoding the ETI triggered NUCLEOTIDE BINDING-adaptor shared by APAF-1, R proteins and CED-4 (NB-ARC) domain containing disease resistance, and leucine-rich repeat (LRR) family proteins were, also, significantly overexpressed in asymptomatic VAL/SW leaves. Gene encoding TÓXICOS EN LEVADURA (ATL2), a RING-H2 family protein that responds to elicitors, was also significantly upregulated in the asymptomatic VAL/SW leaves as compared to leaves collected from asymptomatic VAL/CAN (Table 4-4). Asymptomatic VAL/SW leaves showed a significant overexpression of genes encoding SA-induced RECEPTOR LECTIN KINASE (RLK), RLP33, RLK1 and GLR2.7 in the range of 2 to 4 log2 FC (Table 4-4). Genes encoding of defense signal amplifier MAPKs such as MPK3, MKK4, MAPKK5, MAPKKK13, MAPKKK18, and MAPKKK19 were also significantly overexpressed in the asymptomatic treatment of VAL/SW leaves. In the Ca 2+ mediated signaling branch of defense, genes encoding CPK32, CML37, and calmodulin binding 138

139 family protein EDA39 were significantly upregulated in the range of 1 to 3 log2 FC in the leaves of asymptomatic VAL/SW combination as compared to leaves collected from asymptomatic VAL/CAN (Table 4-4). Genes involved in the SA, JA, and ET -mediated defense were significantly overexpressed in the asymptomatic VAL/SW leaves as compared to asymptomatic VAL/CAN leaves (Figure 4-3). Asymptomatic VAL/SW leaves showed significant upregulation of genes encoding SA-induced WRKY70, WRKY50, thioredoxin superfamily GRX480, SIGMA FACTOR BINDING PROTEIN 1 (SIB1), a positive regulator of JA-dependent defense WRKY33, and JA-responsive PR4 and BASIC CHITINASE as compared to the asymptomatic VAL/CAN leaves. The nonhost resistance induced Ca 2+ dependent PENETRATION 3 (PEN3) upregulated in the asymptomatic VAL/SW leaves. In the category of enzymes, transcripts encoding PLA 2A, cytochrome 450 family monooxygenase CYP83B1, beta-glucosidase, UDP glucosyltransferase UGT73B4, glutathione transferases and beta 1,3 glucan hydrolyses were significantly overexpressed in the asymptomatic VAL/SW leaves (Figure 4-3). Asymptomatic VAL/SW leaves, also, significantly upregulated transcripts encoding negative regulator of SA-induced plant defense NPR3 gene. Negative regulators of HRinduced programmed cell death genes were also strongly overexpressed in the asymptomatic VAL/SW combination. These included genes encoding for MAC/Perforins domain-containing protein CAD and NSL1 proteins, BON ASSOCIATED PROTEIN 2 (BAP2), and ABA-induced PP2C (Table 4-4). In addition, genes encoding mitogenactivated MKK4, WRKY 40, WRKY 48 and WRKY 60 were co-expressed strongly in asymptomatic VAL/SW leaves as compared to asymptomatic VAL/CAN leaves (Table 4-4). 139

140 Root samples Roots collected from asymptomatic VAL/CAN and VAL/SW combination did not show many DEGs in the defense category (Figure 4-1). However, roots collected from the CAN rootstock overexpressed transcripts encoding cell wall degrading enzymes BETA-GALACTOSIDASE 12 and BETA-XYLOSIDASE 1. Differentially Expression of Defense-Associated Genes in Leaves and Roots of Symptomatic VAL/CAN and VAL/SW combinations Leaf Samples Rootstock genetic differences showed differential regulation of defenseassociated genes in symptomatic VAL scion. Leaves collected from the symptomatic VAL/CAN combination showed a low number of defense-associated genes those were significantly upregulated as compared to symptomatic VAL/SW leaves. These genes are encoding LTP, PHENYLALANINE AMMONIA LYASE (PAL), ZINC FINGER PROTEIN 7, a protein from pectin methylesterase inhibitor (PMEI) family, and COPPER/ZINC SUPEROXIDE DISMUTASE 2 (CSD2). Symptomatic VAL/CAN leaves also showed significant upregulation of multiple transcripts encoding aspartyl proteases, the SA-positive regulator MYB-like HTH transcriptional regulator family protein ASYMMETRIC LEAVES 1(AS1), NB-ARC DOMAIN CONTAINING DISEASE RESISTANCE, and CHITINASE A. (Table 4-5). Genes involved in the lignin biosynthesis and flavonoid networking were also significantly upregulated in symptomatic VAL/CAN leaves as compared to symptomatic VAL/SW leaves (Table 4-5). Leaves collected from the symptomatic VAL/SW combination showed strong overexpression of plant immunity and defense-induced genes as compared to 140

141 symptomatic VAL/CAN leaves (Figure 4-1 and 4-4). Symptomatic VAL/SW leaves showed upregulation of genes encoding pathogen signaling and ETI-induced numerous NB-ARC domain, TIR-containing disease resistance proteins, and LRR receptors (Table 4-6). Symptomatic VAL/SW leaves significantly overexpressed transcripts encoding signaling kinases such as RECEPTOR KINASE 3 (RPK3), RECEPTOR-LIKE PROTEIN 56 (RLP 56) RECEPTOR LECTIN KINASE (RLK), MALECTIN-like LRR, RLP1, RLP 13, CYSTEINE-RICH RLK 3, GRL 2.7, GRL 3.6, WAK, and RLP 33 (Table 4-6). Leaves collected from symptomatic VAL/SW tees also showed overexpression of the transcripts encoding biotic stress induced mitogen-activated and secondary defense messengers (Table 4-6). In the category of secondary defense messengers, transcripts of Ca 2+ dependent lipid binding CaLB domain containing protein, and LIPID-DEPENDENT PHOSPHOLIPASE 2A, PHOSPHOLIPASE C2, and non-specific PHOSPHOLIPASE C3 were upregulated in symptomatic VAL/SW leaves (Table 4-6). Leaves collected from symptomatic VAL/SW combination also upregulated genes encoding enzyme in the defense responses. These include oxidoreductase family proteins, peroxidase superfamily proteins ascorbate peroxidase and 2-oxoglutarate (2OG), Fe(ii)-dependent oxygenase superfamily proteins, serine protease inhibitors, aspartyl proteases, betaglucosidases, glutathione transferases, callose forming cytochrome P450 monooxygenase CYP83B1, LTP1, and ET-induced GDSL-like lipase superfamily protein (Table 4-6 and Figure 4-4). Hormone activated defense and signaling genes were also significantly upregulated in HLB-symptomatic VAL/SW combination (Figure 4-4). Transcripts encoding hormonal activated WRKY 70, SIB1, WRKY 33 and BASIC CHITINASE were significantly upregulated in symptomatic VAL/SW leaves as 141

142 compared to symptomatic VAL/CAN leaves (Table 4-6). In the symptomatic treatment, VAL/SW leaves showed significant upregulation of multiple genes encoding enzymes or proteins in the phenylpropanoid activated lignin biosynthesis pathway as compared to VAL/CAN leaves (Table 4-6). Genes encoding HSPs were overexpressed in the symptomatic VAL/SW leaves. Highly upregulated HSPs were found in the symptomatic VAL/SW leaves treatment were HSP20, HSP70T.2, HSP90.1, HSP17.6A, and HSP22. Leaves collected from symptomatic VAL/SW also showed significant overexpression of genes encoding negative regulators of hormones response, HR induced cell death, JA signaling, and MAPK regulators. These negative regulators are NPR3, BAP2, JAZ proteins, WRKY40, and histidine kinase PP2C. Root Samples Comparative transcriptomic analysis in the symptomatic treatment between VAL/CAN and VAL/SW roots showed significant upregulation of transcripts encoding ETI-induced TIR-NBS-LRR (Ciclev m.g), NB-ARC (Ciclev m.g) and LRR (Ciclev m.g) domain containing disease resistance genes in VAL/CAN roots. Symptomatic VAL/CAN roots also significantly overexpressed transcripts encoding protein kinases, MAPKKK19, MAPKKK18, MAPKKK15, members of lectin protein kinase family and cysteine-rich RLK10 (Table 4-7). However, symptomatic VAL/CAN roots did not show upregulation of many hormone activated defenseassociated genes except the thioredoxin GRX480 gene. Symptomatic VAL/CAN roots also showed significant overexpression of transcripts encoding enzymes that are involved in cell wall modification, flavonoid biosynthesis, and ROS scavenging, as well as glutathione transferases, cytochrome P450 monooxygenases, phospholipases and protease inhibitors as compared to symptomatic VAL/SW roots (Figure 4-5). Among 142

143 these, transcripts encoding BETA-XYLOSIDASE 1, an enzyme involved in defense response by callose deposition CYP83B1, PMEI protein, GLUTATHIONE S- TRANSFERASE TAU 8, and PHOSPHOLIPASE A 2A were strongly upregulated (Table 4-7). Roots collected from the symptomatic of VAL/CAN combination showed significant overexpression of genes encoding negative regulators of defense as compared to symptomatic VAL/SW roots. These genes are encoding NPR3, JAZ proteins (JAZ1 and JAZ10), RPM INTERACTING PROTEIN 4 (RIN4), and BAP2 (Table 4-7) In the symptomatic treatment, VAL/SW roots showed significant upregulation of genes encoding various NB-ARC domain containing (Ciclev m.g, Ciclev m.g, Ciclev m.g, Ciclev m.g), and TIR-NBS-LRR class (Ciclev m.g) disease resistance proteins as compared to the VAL/CAN roots (Table 4-8). Transcripts encoding kinase superfamily proteins such as cysteinerich RLK (CSRLK29 and CSRLK10), lectin proteins, and LRR signaling receptor kinase family proteins (II, III, IV, V, VI, VIII-1, X, XII, XII, and XIV) were also significantly overexpressed in the symptomatic VAL/SW roots as compared to the symptomatic VAL/CAN roots (Figure 4-1). Genes involved in the plant basal defense were also highly upregulated in the symptomatic VAL/SW roots as compared to the symptomatic VAL/CAN roots. Among these, genes encoding STRICTOSIDINE SYNTHASE-LIKE-4 (SSL-4), PATHOGENESIS-RELATED 4 (PR4), OSMOTIN 34 (OSM34), and a homolog of EP3-CHITINASE strongly overexpressed in the symptomatic VAL/SW roots (Table 4-8). In the category of enzyme-induced defense responses, many genes encoding glutathione S-transferase Tau and Phi family, and NAD(P)-linked oxidoreductase family were strongly overexpressed in roots of the symptomatic VAL/SW combination. Many 143

144 genes encoding pleiotropic drug resistance and multidrug resistance-associated proteins (PDR4, PDR9, MDR3, and MDR11) also upregulated in the symptomatic VAL/SW roots. In response to the presence of CaLas, defense mechanisms were strongly expressed in the symptomatic SW roots (Figure 4-1 and 4-2). However, genes encoding negative response of the defense carbonic anhydrase (CA2) and RIN4 were also significantly overexpressed in symptomatic VAL/SW roots (Table 4-8). Discussion Differential gene expression (DGE) analysis of VAL leaves, and roots sampled from both VAL/SW and VAL/CAN combinations showed differential transcriptomic reprogramming in response to the asymptomatic and symptomatic stages of HLB disease development. In the asymptomatic treatment, VAL/SW showed a greater number plant defense and immunity response genes upregulated in leaves as compared to the VAL/CAN leaves (Figure 5-1 and 5-2). The DGE analysis of the asymptomatic root tissue did not show significant expression differences of defenseassociated genes. As discussed in Chapter 3, in the symptomatic treatment, VAL/CAN leaves did not show upregulation of hormone-induced genes that are involved in the biotic responses. However, ET-induced GLIP1, and BR-induced receptor kinase transcripts were overexpressed in the asymptomatic VAL/CAN leaves (Table 4-3). The Role of GLIP is discussed in activating systemic signaling in interaction with fungal and bacterial pathogens (Kwon et al., 2009). The role of BR in plant growth has been reviewed in many crops, and BRI-induced defense responses are discussed in many studies (Nakashita et al., 2003; Albrecht et al., 2012; Choudhary et al., 2012). Therefore, upregulation of GLIP and BRI1 in the asymptomatic VAL/CAN tissues suggests plant defense activation. The interaction between BR and SA showed that BR 144

145 antagonizes the SA-induced NPR1 gene expression (De Vleesschauwer et al., 2012). Significant downregulation of SA-induced defense genes expression levels, and increased expression level of BRI1 in the asymptomatic VAL/CAN leaves indicates that BR may play an important role to improve HLB tolerance in VAL/CAN plants. CaLas bacterial infection in the plants is vectored by psyllids. Psyllids feed on the citrus trees by puncturing the phloem tissue which helps the bacterial spread. The wound created by psyllid-feeding in the vascular tissue is sensed as a potential threat through HAMP or DAMP signaling. HAMP- or DAMP-induced defense strategies involve cell wall modifications such as secretion of PMEs and secondary metabolite-induced defenses that fight against the pathogen attack (Redovnikovic et al., 2008; Foyer et al., 2015). Overexpression of PME 44 and glucosinolate UGT74B1 in the asymptomatic VAL/CAN leaves as compared to asymptomatic VAL/SW leaves suggests that the CAN rootstock induces wounding defense in response to psyllid feeding in the VAL. Plant flavonoids are important for physiological processes. In addition, flavonoids also contribute to the plant defense responses to herbivore infestation or pathogen infection (Gould, 2004). Herbivore attack also triggers Ca 2+ dependent defenses (Schulz et al., 2013; Foyer et al., 2015). Upregulation of transcripts encoding the proteins in flavonoid biosynthesis pathway, cell wall modification enzymes, and Ca 2+ dependent protein kinase suggests that the CAN rootstock is strongly reacting to the psyllid attack at the asymptomatic stage of HLB. Leaves collected from the asymptomatic treatment of VAL/SW showed upregulation of PTI-induced genes. Receptor kinases which are part of PTI system can recognize potential threats to the plants through their signaling components (Zhou et al., 145

146 2017). Upregulation of genes encoding RLK in the asymptomatic VAL/SW leaves as compared to asymptomatic VAL/CAN leaves suggests that SW has a robust threat recognition system which strongly activated defense mechanisms in the asymptomatic VAL/SW combination. In addition, overexpression of genes involved in the defense signal cascade such as Ca 2+ (Schulz et al., 2013), MAP kinase (Pedley and Martin, 2005), phospholipase (Canonne et al., 2011) and HSPs (Martinelli et al., 2012) supports the strong defense of SW rootstock towards HLB. Glutamate receptors are key players in the disease priming (Manzoor et al., 2013; Mousavi et al., 2013). Overexpression of transcripts encoding glutamate receptors in the asymptomatic VAL/SW combination suggests that SW has an active signaling system which is priming the non-infected portion of the tree. In the asymptomatic treatment, SA-JA-ET regulated defense response genes were upregulated in VAL/SW leaves as compared VAL/CAN leaves. Among these, SAR signaling thioredoxin, WRKY TFs and JA positive regulator WRKY33 genes were overexpressed. SIB1 is a SA-regulated protein that confers resistance to P. syriangae (Chalovich and Eisenberg, 2005). Herbivore-induced CHITINASE and PR4 transcript upregulation is reported in HLB transcriptomics analyses (Fan et al., 2011; Sharma et al., 2011). Genes encoding SIB1, CHITINASE, and PR4 were, also, found to be upregulated in the asymptomatic VAL/SW leaves as compared to asymptomatic VAL/CAN leaves suggesting SA and JA induced defenses in the VAL/SW leaves. Genes encoding cell modifying beta-glucosidase (Morant et al., 2008), and 1,3 glucan hydrolyse (Levy et al., 2007) have a crucial role in activating chemical defense against herbivores. Significant upregulation of transcripts encoding beta-glucosidase and 1,3 glucan hydrolyse in asymptomatic VAL/SW leaves indicated 146

147 that in the asymptomatic VAL/SW trees defense was also exhibited by modifying cell walls structures. In the asymptomatic treatment, the ETI signature NB-ARC and LRR domain-encoding genes were also overexpressed in VAL/SW leaves as compared to VAL/CAN leaves. ETI- induced defense is mostly R-gene mediated defense. There is no R-AVR interaction yet identified in the HLB-citrus interaction, although asymptomatic VAL/SW showed overexpression of ETI and PTI-associated genes as well as upregulation of negative regulators of HR induced cell death. The activation of genes involving in PTI-, ETI-induced defense suggests that at an early stage of CaLas infection, VAL/SW trees initiate a strong defense response. In asymptomatic VAL/SW leaves, the gene encoding NPR3, a negative regulator NPR1, was also significantly upregulated, indicating the possible downregulation of NPR1 dependent defense. However, more analyses are required to conclude the role of NPR3 upregulation in defense downregulation in CaLas infected VAL/SW leaves. Transcriptomic analysis of HLB symptomatic VAL/CAN and VAL/SW leaves showed similar results as the asymptomatic treatment, but with the stronger overexpression of the genes. Symptomatic VAL grafted onto the putative HLB-tolerant CAN rootstock showed increased expression of genes encoding redox enzymes. Reactive oxygen species (ROS) are a byproduct of oxidation burst reaction in the basal defense mechanism (Glazebrook, 2005). However, ROS are harmful to the plant. Therefore, to neutralize ROS effect, plant activates oxidoreductase enzymes. In the symptomatic treatment, VAL/CAN leaves showed upregulation of genes encoding thioredoxins, dismutases, glutathione transferases and glutaredoxins as compared to VAL/SW leaves. PAL is a key enzyme in phenylpropanoid pathway which is important in 147

148 secondary metabolite biosynthesis pathways such as lignin, flavonoids, and phytoalexins (Hahlbrock and Scheel, 1989). These secondary metabolites have a significant role in plant defenses (Besseau et al., 2007). Significant upregulation of PAL and PMEI in the symptomatic VAL/CAN leaves suggests that CAN rootstock triggered the secondary metabolite-induced plant defense via cell wall modification in the HLBsymptomatic VAL. In the symptomatic VAL/CAN combination, roots showed upregulation of transcripts encoding NB-ARC and LRR protein domains, and MAPK cascade suggesting that at the symptomatic stage VAL/CAN may potentially activate ETI-induced defense. Comparative transcriptomic analysis of leaves and roots sampled from the symptomatic VAL/SW combination showed significant upregulation of PTI-induced receptors in response to HLB. In the symptomatic VAL/SW combination, nonhostspecific defense responses exhibited by upregulating transcripts encoding detoxification enzymes, activation of secondary messengers such as MAPK, Ca 2+ -Calmodium dependent stress response, HSPs, secondary metabolite activated cell wall modification enzymes, multidrug associated antibiotic resistance proteins, and many ETI-induced genes. The overall transcriptomic data of symptomatic leaves and roots collected from the VAL/SW combination showed that SW was overreacting to the CaLas-infection in the scion by activated PTI- and ETI-activated defense responses. CaLas-infected plants trigger callose deposition. Callose deposition is a strategy to stop pathogen spread in the plants (Koh et al., 2012). However, in HLB-citrus interaction, overwhelming callose depositions not only blocks the spread of CaLas but also causes significant blockage of the phloem, leading to an inadequate food supply to 148

149 the sink tissue (Achor et al., 2010; Fan et al., 2012). The monooxygenase p 450 CYP83B1 gene is involved in the callose synthesis. VAL/SW combination strongly upregulated CYP83B1 in asymptomatic as well as symptomatic VAL, and callose degrading 1,3- glucan hydrolase transcripts were upregulated only in the asymptomatic VAL grafted onto SW. Whereas, the CAN rootstock triggered CYP83B1 expression only in the symptomatic roots. The overexpression of transcripts encoding CYP83B1 and 1,3-glucan hydrolase in the asymptomatic VAL/SW treatment suggests that SW can regulate the callose production and turnover at the asymptomatic stage only. At an advanced stage of CaLas-infection, SW is unable to repair plants from uncontrolled callose depositions and leads to the physical damage to the phloem. Whereas, the CAN rootstock promotes callose synthesis at an advanced stage of CaLas infection only. Also, expression of the PP2 gene at the asymptomatic stage supports that the CAN rootstock is not hypersensitive to the CaLas infection, rather it is gradually deploying its defense strategies based on the stage of HLB disease development in the plant. Altogether, combined data suggests that SW is highly sensitive to the CaLas infection, and VAL scion grafted on the SW overact to the disease causing metabolic imbalances detrimental to the tree. Upregulation transcripts encoding negative regulators of HR reaction such as CAD, BON, WRKY60, WRKY40, and NPR3 in the asymptomatic VAL/SW combination suggests that SW was controlling the overwhelming plant defense response in VAL by activating the negative controller of the defense. In this study, VAL grafted onto the CAN and SW rootstocks showed many commonalities in HLB-defense responses. These commonalities were activation of genes encoding PTI receptors-induced signaling, detoxification enzymes, LTP 149

150 superfamily genes, callose depositions, chitinase, and secondary metabolite-triggered pathways. However, the levels of gene expression were substantially higher in the VAL/SW combination in the asymptomatic stage of CaLas-infection and enhanced further in the symptomatic stage of infection. Moreover, VAL grafted onto SW strongly upregulated R genes encoding NB-ARC and LRR domain. Whereas, the putative HLBtolerant CAN rootstock upregulated a low level of ETI involved defense genes and defense triggered by secondary metabolite pathways such as cell wall modifying pectin methylesterases, lignin biosynthesis and detoxification processes at an optimum level. In summary, transcriptomic analysis of VAL grafted onto CAN and SW rootstock at two stages of HLB-infection showed that in the absence of disease-specific resistance, SW activates many branches of non-host specific resistance at a higher intensity. Whereas, the CAN rootstock exhibits a more balanced level of induction of defense-associated genes, which may help the plant to invest adequate energy resources needed for routine growth and sustainability. 150

151 Table 4-1. Experimental treatments and combinations Rootstock Rootstock Parents Scion Swingle; 2n (SW) putative HLBtolerant candidate; 2n (CAN) Grapefruit X Trifoliate orange HBP Pummelo X Cleopatra Mandarin Valencia sweet orange (VAL) Valencia sweet orange (VAL) Treatments based on visual observations of HLB symptoms Symptomatic VAL/SW Slightly Symptomatic VAL/CAN Approximately 7-year old citrus trees in the Lee Family s Alligator Grove east of St. Cloud, FL. Asymptomatic VAL/SW Asymptomatic VAL/CAN Table 4-2. Comparison pairs used for differential gene expression analysis in leaves and roots of the experimental scion/rootstock combinations Leaves Roots Asymptomatic VAL/CAN vs. Asymptomatic VAL/CAN vs. Asymptomatic VAL/SW Asymptomatic VAL/SW Symptomatic VAL/CAN vs. Symptomatic Symptomatic VAL/CAN vs. Symptomatic VAL/SW VAL/SW 151

152 Table 4-3. Differentially expressed immunity and defense-associated genes significantly upregulated in leaves of asymptomatic VAL/CAN combination as compared to asymptomatic VAL/SW leaves Arabidopsis Gene C. Clementina_ID Log2 FC Arabidopsis-define Functional description CYP98A3 Ciclev m.g cytochrome P450, family 98, subfamily A, polypeptide 3 Essential for lignin biosysntheis CIPK20 Ciclev m.g CBL-interacting protein kinase 20 Calcium signaling TT4 Ciclev m.g Chalcone and stilbene synthase family protein flavonoid accumulation/chalcone synthase PP2-B15 Ciclev m.g Phloem protein 2-B15 MJE7.13 Ciclev m.g Lipid transfer protein SHN1 Ciclev m.g Integrase-type DNA-binding superfamily protein cutin Biosyntehsis TINY2 Ciclev m.g Integrase-type DNA-binding superfamily protein putative transcription factor F20P5.26 Ciclev m.g myb-like transcription factor family protein TPS14 Ciclev m.g terpene synthase 14 TT7 Ciclev m.g Cytochrome P450 superfamily protein Flavonoid syntehsis, Convert naringenin to eriodictyol AT1G08810 Ciclev m.g myb domain protein 60 T12P18.14 Ciclev m.g RING/U-box superfamily protein GLIP1 Ciclev m.g GDSL lipase 1 MLO-12 Ciclev m.g Seven membrane MLO family protein SHN2 Ciclev m.g Integrase-type DNA-binding superfamily protein PAL1 Ciclev m.g PHE ammonia lyase 1 AT1G06620 Ciclev m.g oxoglutarate (2OG) and Fe(II)-dependent oxygenase superfamily protein UGT74B1 Ciclev m.g UDP-glucosyl transferase 74B1 ZFP8 Ciclev m.g zinc finger protein 8 BRI1 Ciclev m.g Leucine-rich receptor-like protein kinase family protein AT1G68530 Ciclev m.g ketoacyl-CoA synthase 6 Wax metabolism PME44 Ciclev m.g pectin methylesterase 44 BKI1 Ciclev m.g BRI1 kinase inhibitor 1 152

153 Table 4-4. Differentially expressed immunity and defense-associated genes significantly upregulated in leaves of asymptomatic VAL/SW combination as compared to asymptomatic VAL/CAN leaves Arabidopsis Gene C. Clementina_ID Log2 FC * Arabidopsis-define PLA2A Ciclev m.g phospholipase A 2A PUB23 Ciclev m.g plant U-box 23 BAP2 Ciclev m.g BON association protein 2 WRKY33 Ciclev m.g WRKY DNA-binding protein 33 CYP83B1 Ciclev m.g cytochrome P450, family 83, subfamily B, polypeptide 1 GLR2.7 Ciclev m.g glutamate receptor 2.7 SIB1 Ciclev m.g sigma factor binding protein 1 T5J17.9 Ciclev m.g Cupredoxin superfamily protein MYB15 Ciclev m.g myb domain protein 15 CML37 Ciclev m.g calmodulin like 37 BAP2 Ciclev m.g BON association protein 2 JAZ10 Ciclev m.g jasmonate-zim-domain protein 10 WRKY40 Ciclev m.g WRKY DNA-binding protein 40 AT3G14470 Ciclev m.g NB-ARC domain-containing disease resistance protein JAZ1 Ciclev m.g jasmonate-zim-domain protein 1 EDA39 Ciclev m.g calmodulin-binding family protein CML37 Ciclev m.g calmodulin like 37 JAZ1 Ciclev m.g jasmonate-zim-domain protein 1 WRKY40 Ciclev m.g WRKY DNA-binding protein 40 F18A17.4 Ciclev m.g Protein kinase family protein with leucine-rich repeat domain MPK3 Ciclev m.g mitogen-activated protein kinase 3 WRKY33 Ciclev m.g WRKY DNA-binding protein 33 K14A3.2 Ciclev m.g Protein kinase superfamily protein AT4G08850 Ciclev m.g Leucine-rich repeat receptor-like protein kinase family protein JAZ1 Ciclev m.g jasmonate-zim-domain protein 1 MLO6 Ciclev m.g Seven transmembrane MLO family protein HSP90.1 Ciclev m.g heat shock protein 90.1 SYP121 Ciclev m.g syntaxin of plants

154 Table 4-4. Continued Arabidopsis C. Clementina_ID Log2 FC Arabidopsis-define Gene AT4G08850 Ciclev m.g Leucine-rich repeat receptor-like protein kinase family protein F13E7.22 Ciclev m.g ARM repeat superfamily protein PTR3 Ciclev m.g peptide transporter 3 F19K19.4 Ciclev m.g Protein kinase superfamily protein AT4G08850 Ciclev m.g Leucine-rich repeat receptor-like protein kinase family protein MAPKKK19 Ciclev m.g mitogen-activated protein kinase kinase kinase 19 AT4G08850 Ciclev m.g Leucine-rich repeat receptor-like protein kinase family protein T19F6.150 Ciclev m.g alpha/beta-hydrolases superfamily protein AT3G14470 Ciclev m.g NB-ARC domain-containing disease resistance protein MYBR1 Ciclev m.g myb domain protein r1 CPK32 Ciclev m.g calcium-dependent protein kinase 32 AT1G73805 Ciclev m.g Calmodulin binding protein-like WRKY11 Ciclev m.g WRKY DNA-binding protein 11 GRX480 Ciclev m.g Thioredoxin superfamily protein YLS9 Ciclev m.g Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family WRKY48 Ciclev m.g WRKY DNA-binding protein 48 MAPKKK18 Ciclev m.g mitogen-activated protein kinase kinase kinase 18 PR4 Ciclev m.g pathogenesis-related 4 ATL6 Ciclev m.g RING/U-box superfamily protein HSPRO2 Ciclev m.g ortholog of sugar beet HS1 PRO-1 2 T6G21.2 Ciclev m.g Polynucleotidyl transferase, ribonuclease H-like superfamily protein CLH1 Ciclev m.g chlorophyllase 1 RLK Ciclev m.g receptor lectin kinase NSL1 Ciclev m.g MAC/Perforin domain-containing protein SNAP33 Ciclev m.g soluble N-ethylmaleimide-sensitive factor adaptor protein 33 T23A1.8 Ciclev m.g Protein kinase superfamily protein RHD2 Ciclev m.g NADPH/respiratory burst oxidase protein D PTR3 Ciclev m.g peptide transporter 3 AOC3 Ciclev m.g allene oxide cyclase 3 154

155 Table 4-4. Continued Arabidopsis Gene C. Clementina_ID Log2 FC Arabidopsis-define PTR3 Ciclev m.g peptide transporter 3 MES1 Ciclev m.g methyl esterase 1 BAP2 Ciclev m.g BON association protein 2 F12L6.31 Ciclev m.g Protein of unknown function (DUF506) NPR3 Ciclev m.g NPR1-like protein 3 WRKY70 Ciclev m.g WRKY DNA-binding protein 70 AT3G09830 Ciclev m.g Protein kinase superfamily protein THI1 Ciclev m.g thiazole biosynthetic enzyme, chloroplast (ARA6) (THI1) (THI4) F14N23.22 Ciclev m.g Ankyrin repeat family protein AT4G08850 Ciclev m.g Leucine-rich repeat receptor-like protein kinase family protein PEN3 Ciclev m.g ABC-2 and Plant PDR ABC-type transporter family protein AT3G07720 Ciclev m.g Galactose oxidase/kelch repeat superfamily protein CAD1 Ciclev m.g MAC/Perforin domain-containing protein PLDBETA1 Ciclev m.g phospholipase D beta 1 MEE62 Ciclev m.g Leucine-rich repeat protein kinase family protein ATL2 Ciclev m.g TOXICOS EN LEVADURA 2 OBP2 Ciclev m.g Dof-type zinc finger DNA-binding family protein AOS Ciclev m.g allene oxide synthase AT3G14470 Ciclev m.g NB-ARC domain-containing disease resistance protein AT3G14470 Ciclev m.g NB-ARC domain-containing disease resistance protein MKK4 Ciclev m.g mitogen-activated protein kinase kinase 4 NDR1 Ciclev m.g Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family FC1 Ciclev m.g ferrochelatase 1 * The negative sign in the column of Log2 FC indicates comparative downregulation of the gene expression level in VAL/CAN and upregulation in VAL/SW combination. 155

156 Table 4-5. Differentially expressed immunity and defense-associated genes significantly upregulated in leaves of the symptomatic VAL/CAN combination as compared to symptomatic VAL/SW leaves C. Clementina_ID Arabi ID Gene name Bin Name Ciclev m.g AT3G53980 LTP Ciclev m.g AT5G07990 TT7 Secondary metabolism. flavonoids. dihydroflavonols. flavonoid 3''-monooxy genase Log2 FC Arabidopsis_define Bifunctional inhibitor/ lipid-transfer protein Cytochrome P450 superfamily protein Ciclev m.g AT1G11530 CXXS1 redox.thioredoxin C-terminal cysteine residue is changed to a serine 1 Ciclev m.g AT5G05270 K18I23.7 secondary metabolism. Chalcone-flavanone flavonoids.chalcones isomerase family protein Ciclev m.g AT2G37040 PAL1 secondary metabolism. phenylpropanoids.lignin biosynthesis.pal PHE ammonia lyase 1 Ciclev m.g AT2G28190 CSD2 redox. dismutases and catalases copper/zinc superoxide dismutase 2 Ciclev m.g AT1G23200 PMEI Plant invertase/pectin methylesterase inhibitor Ciclev m.g AT1G61820 Bglu 46 misc.gluco-, galacto- and mannosidases beta glucosidase 46 Ciclev m.g AT2G37040 PAL1 phenylpropanoids.lignin biosynthesis.pal PHE ammonia lyase 1 Ciclev m.g AT4G39230 T22F8.130 secondary metabolism. NmrA-like negative transcriptional flavonoids.isoflavonols regulator family protein Ciclev m.g AT4G34050 CCoAOMT1 S-adenosyl-L-methionine-dependent phenylpropanoids methyltransferases lignin biosynthesis.ccoaomt superfamily protein Ciclev m.g AT2G44310 F4I1.12 signalling.calcium Calcium-binding EF-hand family protein Ciclev m.g AT1G CL3 phenylpropanoids.lignin biosynthesis.4cl coumarate:CoA ligase 3 Ciclev m.g AT3G55120 TT5 secondary metabolism.flavonoids. chalcones.chalcone isomerase Ciclev m.g AT4G27280 M4I22.90 signalling.calcium Chalcone-flavanone isomerase family protein Calcium-binding EF-hand family protein Ciclev m.g AT3G51240 F3H flavonoids. dihydroflavonols flavanone 3-hydroxylase Ciclev m.g AT2G37040 PAL1 phenylpropanoids.lignin biosynthesis.pal PHE ammonia lyase 1 Ciclev m.g AT1G03020 F10O3.16 redox.glutaredoxins Thioredoxin superfamily protein Ciclev m.g AT2G37040 PAL1 secondary metabolism. phenylpropanoids.lignin biosynthesis.pal PHE ammonia lyase 1 156

157 Table 4-5. Continued C. Clementina_ID Arabi ID Gene Log2 Bin Name name FC Arabidopsis_define Ciclev m.g AT1G64500 F1N19.7 redox.glutaredoxins glutaredoxin family protein Ciclev m.g AT3G CL2 lignin biosynthesis.4cl coumarate:CoA ligase 2 Ciclev m.g AT5G28840 GME redox.ascorbate and glutathione.ascorbate.gme gdp-d-mannose 3\',5\'-epimerase Ciclev m.g AT2G28470 BGAL8 misc.gluco-, galacto- and mannosidases.beta-galactosidase beta-galactosidase 8 Ciclev m.g AT5G10250 DOT3 signalling.light Phototropic-responsive NPH3 family protein Ciclev m.g AT5G54490 PBP1 signalling.calcium pinoid-binding protein 1 Ciclev m.g AT5G23400 K19M13.1 stress.biotic.pr-proteins Leucine-rich repeat (LRR) family protein Ciclev m.g AT4G39330 CAD9 phenylpropanoids.lignin biosynthesis.cad cinnamyl alcohol dehydrogenase 9 Ciclev m.g AT2G14820 NPY2 signalling.light Phototropic-responsive NPH3 family protein Ciclev m.g AT5G48930 HCT phenylpropanoids.lignin biosynthesis.hct hydroxycinnamoyl-coa shikimate/ Ciclev m.g AT4G27570 T29A15.60 secondary UDP-glycosyltransferase metabolism.flavonoids.anthocyanins superfamily protein Ciclev m.g AT1G59960 F23H11.27 secondary NAD(P)-linked oxidoreductase Ciclev m.g AT5G54010 K19P17.18 metabolism.flavonoids.chalcones secondary metabolism.flavonoids.dihydroflavonols Ciclev m.g AT5G02010 ROPgEF7 signalling.g-proteins superfamily protein UDP-glycosyltransferase superfamily protein RHO guanyl-nucleotide exchange factor 7 Ciclev m.g AT3G18080 BGLU44 misc.gluco-, galacto- and mannosidases B-S glucosidase 44 Ciclev m.g AT2G36570 F1O11.20 signalling.receptor kinases.leucine rich Leucine-rich repeat protein kinase repeat III family protein Ciclev m.g AT1G18250 ATLP-1 stress.biotic Pathogenesis-related thaumatin superfamily protein Ciclev m.g AT2G15220 F15A23.4 stress.biotic Plant basic secretory protein (BSP) Ciclev m.g AT4G03100 F4C21.2 signalling.g-proteins Rho gtpase activating protein with PAK-box/P21-Rho-binding domain Ciclev m.g AT3G45290 MLO3 stress.biotic.signalling.mlo-like Seven transmembrane MLO family protein 157

158 Ciclev m.g AT5G24090 CHIA stress.biotic.pr-proteins chitinase A Table 4-5. Continued C. Clementina_ID Arabi ID Gene name Bin Name Log2 FC Arabidopsis_define Ciclev m.g AT3G49260 iqd21 signalling.calcium IQ-domain 21 Ciclev m.g AT3G16920 CTL2 stress.biotic chitinase-like protein 2 Ciclev m.g AT5G13930 TT4 secondary Chalcone and stilbene metabolism.flavonoids.chalcones synthase family protein naringenin-chalcone synthase myb-like HTH Ciclev m.g AT2G AS transcriptional regulator family protein Ciclev m.g AT1G50180 F14I3.19 stress.biotic.pr-proteins NB-ARC domaincontaining disease resistance protein Ciclev m.g AT4G01070 gt72b1 Ciclev m.g AT5G61480 PXY secondary metabolism.flavonoids.dihydroflavonols signalling.receptor kinases.leucine rich repeat XI Classification of the measured parameter into a set a functional category in the MapMan analysis tool. UDP-glycosyltransferase superfamily protein Leucine-rich repeat protein kinase family protein 158

159 Table 4-6. Differentially expressed immunity and defense-associated genes significantly upregulated in leaves of the symptomatic VAL/SW combination as compared to symptomatic VAL/CAN leaves C. Clementina_ID Arabi ID Gene name Bin Name Log2 FC* Arabidopsis_define Ciclev m.g AT2G38870 T7F6.4 stress.biotic Serine protease inhibitor, potato inhibitor I-type family protein Ciclev m.g AT1G02850 BGLU11 misc.gluco-, galacto- and mannosidases beta glucosidase 11 Ciclev m.g AT1G17020 SRG1 secondary metabolism.flavonoids senescence-related gene 1 Ciclev m.g AT2G38870 T7F6.4 stress.biotic Serine protease inhibitor, potato inhibitor I-type family protein Ciclev m.g AT3G09640 APX2 redox.ascorbate and glutathione.ascorbate ascorbate peroxidase 2 Ciclev m.g AT3G12500 HCHIB stress.biotic basic chitinase Ciclev m.g AT1G17860 F2H15.9 stress.biotic.pr-proteins.proteinase Kunitz family trypsin and inhibitors.trypsin inhibitor protease inhibitor protein Ciclev m.g AT3G12500 HCHIB stress.biotic basic chitinase Ciclev m.g AT2G34930 F19I3.16 stress.biotic.pr-proteins disease resistance family protein / LRR family protein Ciclev m.g AT1G17020 SRG1 secondary metabolism. flavonoids.flavonols senescence-related gene 1 Ciclev m.g AT4G35160 T12J5.30 secondary metabolism.phenylpropanoids O-methyltransferase family protein Ciclev AT5G GDSL-like lipas superfamily Ciclev m.g AT2G34930 F19I3.16 stress.biotic.pr-proteins disease resistance family protein / LRR family protein Ciclev m.g AT5G42380 CML37 signalling.calcium calmodulin like 37 Ciclev m.g AT3G05660 RLP33 stress.biotic.kinases receptor like protein 33 Ciclev m.g AT5G17680 MVA3.30 stress.biotic.pr-proteins disease resistance protein (TIR- NBS-LRR class), putative Ciclev m.g AT5G54160 OMT1 secondary metabolism.phenylpropanoids. lignin biosynthesis.comt O-methyltransferase 1 Ciclev m.g AT1G69730 T6C23.7 signalling.receptor kinases. Wall-associated kinase family wall associated kinase protein Ciclev m.g AT3G47570 AT3G47570 signalling.receptor kinases. Leucine-rich repeat protein leucine rich repeat XII kinase family protein Ciclev m.g AT4G08850 AT4G08850 signalling.receptor kinases. Leucine-rich repeat receptor-like leucine rich repeat XII protein kinase family protein Ciclev m.g AT5G17680 MVA3.30 stress.biotic.pr-proteins disease resistance protein (TIR- NBS-LRR class), putative Ciclev m.g AT3G54800 AT3g54800 signalling.lipids lipid-binding START dpmain 159

160 Table 4-6. Continued C. Clementina_ID Arabi ID Gene name Bin Name Log2 FC Arabidopsis_define Ciclev m.g AT5G06690 WCRKC1 redox.thioredoxin WCRKC thioredoxin 1 Ciclev m.g AT5G54160 OMT1 secondary metabolism.phenylpropanoids. lignin biosynthesis.comt O-methyltransferase 1 Ciclev m.g AT1G70530 CRK3 signalling.receptor kinases.duf cysteine-rich RLK (RECEPTOR-like protein kinase) 3 Ciclev m.g AT3G Ca dependent lipid binding protein Ciclev m.g AT4G21380 RK3 signalling.receptor kinases.slocus glycoprotein like receptor kinase 3 Ciclev m.g AT1G73500 MKK9 signalling.map kinases MAP kinase kinase 9 Ciclev m.g AT5G50120 MPF21.14 signalling.g-proteins Transducin/WD40 repeat-like superfamily protein Ciclev m.g AT3G12500 HCHIB stress.biotic basic chitinase Ciclev m.g AT2G02100 LCR69 stress.biotic low-molecular-weight cysteine-rich 69 Ciclev m.g AT1G07390 RLP1 signalling.receptor kinases.misc receptor like protein 1 Ciclev m.g AT2G20142 AT2g20142 stress.biotic.receptors Toll-Interleukin-Resistance (TIR) domain family protein Ciclev m.g AT2G18750 MSF3.13 signalling.calcium Calmodulin-binding protein Ciclev m.g AT5G39000 MXF12.10 signalling.receptor Malectin/receptor-like protein kinases.catharanthus roseuslike RLK kinase family protein Ciclev m.g AT2G37710 RLK signalling.receptor kinases.misc receptor lectin kinase Ciclev m.g AT5G20050 AT5g20050 signalling.receptor kinases.misc Protein kinase superfamily protein Ciclev m.g AT4G27290 M4I signalling.receptor kinases.slocus glycoprotein like protein S-locus lectin protein kinase family Ciclev m.g AT2G29120 glr2.7 signalling.in sugar and nutrient physiology glutamate receptor 2.7 Ciclev m.g AT1G20510 OPCL1 secondary OPC-8:0 CoA ligase1 metabolism.phenylpropanoids Ciclev m.g AT2G31880 SOBIR1 signalling. receptor kinases.leucine rich repeat XI Ciclev m.g AT5G49290 RLP56 stress. biotic receptor like protein 56 Leucine-rich repeat protein kinase family protein 160

161 Table 4-6. Continued C. Clementina_ID Arabi ID Gene name Bin Name Log2 FC Arabidopsis_define Ciclev m.g AT3G14470 AT3g14470 stress.biotic.pr-proteins NB-ARC domain-containing disease resistance protein Ciclev m.g AT1G07390 RLP1 signalling.receptor kinases.misc receptor like protein 1 Ciclev m.g AT1G56130 T6H22.8 Leucine-rich repeat signalling. receptor kinases transmembrane protein leucine rich repeat VIII-2 kinase Ciclev m.g AT3G08510 PLC2 Ciclev m.g AT4G34050 CCoAOMT1 signalling.phosphinositides. phosphoinositide phospholipase C secondary metabolism. phenylpropanoids. lignin biosynthesis.ccoaomt phospholipase C Ciclev m.g AT3G54420 EP3 stress.biotic S-adenosyl-L-methioninedependent methyltransferases superfamily protein homolog of carrot EP3-3 chitinase Ciclev m.g AT5G42380 CML37 signalling.calcium calmodulin like 37 Ciclev m.g AT3G26740 CCL signalling.light CCR-like Ciclev m.g AT3G14460 AT3g14460 stress.biotic.pr-proteins LRR and NB-ARC domains-containing disease resistance protein Ciclev m.g AT4G21380 RK3 signalling.receptor kinases. S-locus glycoprotein like receptor kinase 3 Ciclev m.g AT1G17020 SRg1 secondary metabolism. flavonoids.flavonols senescence-related gene 1 Ciclev m.g AT4G32690 glb3 redox.heme hemoglobin 3 Ciclev m.g AT3G05660 RLP33 stress.biotic.kinases receptor like protein 33 Ciclev m.g AT5G07990 TT7 secondary metabolism.flavonoids. Cytochrome P dihydroflavonols.flavonoid 3''-monooxygenase superfamily protein Ciclev m.g AT5G25930 F18A17.4 Protein kinase family signalling.receptor kinases.leucine rich protein with leucine-rich repeat XI repeat domain Ciclev m.g AT1G53440 T3F20.24 Leucine-rich repeat signalling.receptor kinases transmembrane protein leucine rich repeat VIII-2 kinase Ciclev m.g AT3G04720 PR4 stress.biotic pathogenesis-related 4 Ciclev m.g AT2G33580 F4P9.35 signalling.receptor kinases.lysine motif Protein kinase superfamily protein 161

162 Table 4-6. Continued C. Clementina_ID Arabi ID Gene name Bin Name Log2 FC Arabidopsis_define Ciclev m.g AT2G23770 F27L4.5 protein kinase family protein / signalling.receptor peptidoglycan-binding LysM domaincontaining protein kinases.lysine motif Ciclev m.g AT4G38660 T9A14.6 stress.biotic Pathogenesis-related thaumatin superfamily protein Ciclev m.g AT3G14470 AT3g14470 stress.biotic.pr-proteins NB-ARC domain-containing disease resistance protein Ciclev m.g AT5G48150 PAT1 signalling.light gras family transcription factor Ciclev m.g AT4G27190 AT4g27190 stress.biotic.pr-proteins Ciclev m.g AT4G37990 ELI3-2 secondary metabolism.phenylpropanoids. lignin biosynthesis.cad Ciclev m.g AT5G17680 MVA3.30 stress.biotic.pr-proteins NB-ARC domain-containing disease resistance protein elicitor-activated gene 3-2 disease resistance protein (TIR-NBS- LRR class), putative signalling.receptor Ciclev m.g AT5G01550 LECRKA lectin receptor kinase a4.1 kinases.misc secondary metabolism. NAD(P)-binding Rossmann-fold Ciclev m.g AT5G58490 MQJ2.6 phenylpropanoids.lignin superfamily protein biosynthesis.ccr1 Ciclev m.g AT4G33050 EDA39 signalling.calcium calmodulin-binding family protein * The negative sign in the column of Log2 FC indicates comparative downregulation of the gene expression level in VAL/CAN and upregulation in VAL/SW combination. Classification of the measured parameter into a set a functional category in the MapMan analysis tool. 162

163 Table 4-7. Differentially expressed immunity and defense-associated genes significantly upregulated in roots of the symptomatic VAL/CAN combination as compared to symptoaatic VAL/SW roots C. Clementina_ID Arabi ID Gene name Bin Name log2 FC Arabidopsis_define Ciclev m.g AT3G09270 ATGSTU8, misc.glutathione S transferases glutathione S-transferase TAU 8 Ciclev m.g AT5G beta-xylosidase 1 Ciclev m.g AT1G45616 RLP6 stress.biotic receptor like protein 6 Ciclev m.g AT3G03305 misc.calcineurin-like phosphoesterase family protein Ciclev m.g AT4G19810 stress.biotic Calcineurin-like metallophosphoesterase superfamily protein Glycosyl hydrolase family protein with chitinase insertion domain NAD(P)-linked oxidoreductase superfamily protein Ciclev m.g AT1G59960 secondary metabolism.flavonoids.chalcones secondary Ciclev m.g AT5G54160 OMT1 metabolism.phenylpropanoids O-methyltransferase 1 lignin biosynthesis.comt Ciclev m.g AT4G12010 stress.biotic.pr-proteins Disease resistance protein (TIR- NBS-LRR class) family Ciclev m.g AT1G78780 stress.biotic pathogenesis-related family protein Ciclev m.g AT1G12740 CYP87A2 misc.cytochrome P cytochrome P450, family 87, subfamily A, polypeptide 2 Ciclev m.g AT2G27690 CYP94C1 misc.cytochrome P cytochrome P450, family 94, subfamily C, polypeptide 1 Ciclev m.g AT4G19010 secondary metabolism. AMP-dependent synthetase and phenylpropanoids ligase family protein Ciclev m.g AT3G52780 PAP20 misc. acid and other Purple acid phosphatases phosphatases superfamily protein secondary metabolism. 2-oxoglutarate (2OG) and Fe(II)- Ciclev m.g AT5G05600 flavonoids dependent oxygenase superfamily anthocyanins protein Ciclev m.g AT3G54420 CHIV,EP3 stress.biotic homolog of carrot EP3-3 chitinase Ciclev m.g AT4G31500 CYP83B1, SUR2 secondary metabolism.cyp83b cytochrome P450, family 83, phenylacetaldoxime subfamily B, polypeptide 1 monooxygenase Ciclev m.g AT5G67080 MAPKKK mitogen-activated protein kinase kinase kinase 19 Ciclev m.g AT2G38870 stress.biotic Serine protease inhibitor, potato inhibitor I-type family protein 163

164 Table 4-7. Continued C. Clementina_ID Arabi ID Gene name Bin Name log2 FC Arabidopsis_define Ciclev m.g AT3G29590 AT5MAT secondary metabolism. flavonoids. anthocyanins.anthocyanin 5-aromatic acyltransferase HXXXD-type acyl-transferase family protein Ciclev m.g AT1G49570 misc.peroxidases Peroxidase superfamily protein Ciclev m.g AT3G03080 misc.oxidases - copper, flavone etc Zinc-binding dehydrogenase family protein Ciclev m.g AT1G22380 AtUGT85A3, misc.udp glucosyl and glucoronyl transferases UDP-glucosyl transferase Ciclev m.g AT1G24620 signalling.calcium Ciclev m.g AT5G36110 CYP716A1 misc.cytochrome P EF hand calcium-binding protein family cytochrome P450, family 716, subfamily A, polypeptide 1 Ciclev m.g AT3G51480 GLR3.6 signalling.in sugar and nutrient physiology glutamate receptor 3.6 Ciclev m.g AT2G46660 CYP78A6 misc.cytochrome P cytochrome P450, family 78, subfamily A, polypeptide 6 Ciclev m.g AT5G21105 redox.ascorbate and glutathione.ascorbate Plant L-ascorbate oxidase Ciclev m.g AT1G53540 stress.abiotic.heat HSP20-like chaperones superfamily protein Ciclev m.g AT5G45920 misc.gdsl-motif lipase SGNH hydrolase-type esterase superfamily protein Ciclev m.g AT1G11530 ATCXXS1, C-terminal cysteine residue is redox.thioredoxin CXXS1 changed to a serine 1 Ciclev m.g AT4G25150 misc.acid and other phosphatases HAD superfamily, subfamily IIIB acid phosphatase Ciclev m.g AT3G02100 misc.udp glucosyl and glucoronyl transferases UDP-Glycosyltransferase superfamily protein Ciclev m.g AT3G48280 CYP71A25 misc.cytochrome P cytochrome P450, family 71, subfamily A, polypeptide 25 Ciclev m.g AT4G35150 misc.o-methyl transferases O-methyltransferase protein Ciclev m.g AT4G35160 secondary metabolism.phenylpropanoids O-methyltransferase protein misc.nitrilases, *nitrile lyases, berberine bridge FAD-binding Berberine family Ciclev m.g AT4G20820 enzymes, reticuline oxidases, troponine protein reductases Ciclev m.g AT5G44390 misc.nitrilases, *nitrile lyases, berberine bridge enzymes, reticuline oxidases, troponine reductases Ciclev m.g AT1G19250 FMO1 misc.oxidases - copper, flavone etc FAD-binding Berberine family protein flavin-dependent monooxygenase 1 164

165 Table 4.7 Continued C. Clementina_ID Arabi ID Gene name Bin Name log2 FC Arabidopsis_define Ciclev m.g AT2G37130 misc.peroxidases Peroxidase superfamily protein Ciclev m.g AT1G24020 MLP423 stress.abiotic.unspecified MLP-like protein 423 Ciclev m.g AT1G23010 LPR1 misc.oxidases - copper, flavone etc Cupredoxin superfamily protein Ciclev m.g AT5G10050 misc.short chain dehydrogenase/reductase NAD(P)-binding Rossmann (SDR) superfamily protein Ciclev m.g AT5G09590 HSC70-5 stress.abiotic.heat mitochondrial HSO70 2 Ciclev m.g AT2G29290 secondary metabolism.n misc.alkaloid-like NAD(P)-binding Rossmannsuperfamily protein Ciclev m.g AT1G05100 MAPKKK18 signalling.map kinases mitogen-activated protein Ciclev m.g AT5G46940 Ciclev m.g AT5G54160 ATOMT1,OMT1 misc.invertase/pectin methylesterase inhibitor family protein secondary metabolism.phenylpropanoids.lignin biosynthesis.comt kinase kinase kinase 18 Plant invertase/pectin methylesterase inhibitor superfamily protein O-methyltransferase 1 Ciclev m.g AT2G32030 misc.gcn5-related N-acetyltransferase Acyl-CoA N-acyltransferases (NAT) superfamily protein Ciclev m.g AT5G05320 misc.oxidases - copper, flavone etc FAD/NAD(P)-binding oxidoreductase family protein Ciclev m.g AT2G43820 SGT1,UGT74F2 misc.udp glucosyl and glucoronyl transferases UDP-glucosyltransferase 74F2 Ciclev m.g AT1G73880 UGT89B1 misc.udp glucosyl and glucoronyl transferases UDP-glucosyl transferase 89B1 Ciclev m.g AT1G30700 misc.nitrilases, *nitrile lyases, berberine bridge FAD-binding Berberine family enzymes, reticuline oxidases, troponine protein reductases Ciclev m.g AT3G11340 misc.udp glucosyl and glucoronyl transferases UDP-Glycosyltransferase superfamily protein Ciclev m.g AT4G15440 CYP74B2,HPL1 misc.cytochrome P hydroperoxide lyase 1 Ciclev m.g AT5G12080 ATMSL10, MSL10 signalling.unspecified mechanosensitive channel of small conductance-like 10 Ciclev m.g AT5G39160 stress.abiotic.unspecified RmlC-like cupins superfamily protein Ciclev m.g AT2G26660 ATSPX2,SPX2 stress.abiotic SPX domain gene 2 Ciclev m.g AT4G01700 stress.biotic Chitinase family protein Ciclev m.g AT4G35290 GLR3.2,GLUR2 signalling.in sugar and nutrient physiology glutamate receptor 2 Ciclev m.g AT2G15760 signalling.calcium Protein of unknown function (DUF1645) 165

166 Table 4-7. Continued C. Clementina_ID Arabi ID Gene name Bin Name log2 FC Arabidopsis_define Ciclev m.g AT2G34930 stress.biotic.pr-proteins disease resistance family protein / LRR family protein Ciclev m.g AT1G30570 HERK2 signalling.receptor kinases. Catharanthus roseus-like RLK hercules receptor kinase 2 Ciclev m.g AT5G54160 ATOMT1 phenylpropanoids.lignin biosynthesis.comt O-methyltransferase 1 Ciclev m.g AT3G14460 stress.biotic.pr-proteins LRR and NB-ARC domainscontaining disease resistance protein Ciclev m.g AT5G MAPKKK mitogen-activated protein kinase kinase kinase 15 Ciclev m.g AT1G20480 secondary metabolism. AMP-dependent synthetase phenylpropanoids and ligase family protein Ciclev m.g AT4G14640 CAM8 signalling.calcium calmodulin 8 Ciclev m.g AT3G54420 ATCHITIV, homolog of carrot EP3-3 stress.biotic ATEP3 chitinase Ciclev m.g AT4G15800 RALFL33 signalling.misc ralf-like 33 Ciclev m.g AT1G28480 GRX480 redox.glutaredoxins Thioredoxin superfamily protein Ciclev m.g AT5G54160 ATOMT1 phenylpropanoids.lignin biosynthesis.comt O-methyltransferase 1 Ciclev m.g AT4G37560 misc.misc Acetamidase/Formamidase family protein Ciclev m.g AT4G21870 stress.abiotic.heat HSP20-like chaperones superfamily protein Ciclev m.g AT2G45510 CYP704A2 misc.cytochrome P cytochrome P450, family 704, subfamily A, polypeptide 2 Ciclev m.g AT3G15354 SPA3 signalling.light SPA1-related 3 Ciclev m.g AT3G47570 signalling. receptor kinases. Leucine-rich repeat protein leucine rich repeat XII kinase family protein 2-oxoglutarate (2OG) and Ciclev m.g AT3G21420 secondary metabolism.flavonoids.flavonols Fe(II)-dependent oxygenase superfamily protein Ciclev m.g AT1G09970 LRR XI-23, signalling. receptor kinases. Leucine-rich receptor-like RLK7 leucine rich repeat XI protein kinase family protein Ciclev m.g AT2G29290 secondary metabolism. NAD(P)-binding Rossmann-fold N misc. alkaloid-like superfamily protein Ciclev m.g AT5G48230 ACAT2, secondary metabolism.isoprenoids.mevalonate EMB1276 Pathway.acetyl-CoA C-acyltransferase acetoacetyl-coa thiolase 2 Ciclev m.g AT4G17190 FPS2 secondary metabolism.isoprenoids.mevalonate farnesyl diphosphate synthase Pathway.farnesyl pyrophosphate synthetase 2 166

167 Table 4-7. Continued C. Clementina_ID Arabi ID Gene name Bin Name log2 FC Arabidopsis_define Ciclev m.g AT4G21390 B120 signalling.receptor kinases. S-locus lectin protein kinase S-locus glycoprotein like family protein Ciclev m.g AT3G25070 RIN4 stress. biotic RPM1 interacting protein 4 Ciclev m.g AT2G45130 ATSPX3,SPX3 stress. abiotic SPX domain gene 3 Ciclev m.g AT3G07320 misc.beta 1,3 glucan hydrolases.glucan O-Glycosyl hydrolases endo-1,3-beta-glucosidase family 17 protein Ciclev m.g AT3G11210 misc.gdsl-motif lipase SGNH hydrolase-type esterase superfamily protein Ciclev m.g AT1G59960 NAD(P)-linked secondary oxidoreductase superfamily metabolism.flavonoids.chalcones protein Ciclev m.g AT2G18950 ATHPT,HPT1 secondary metabolism.isoprenoids.tocopherol biosynthesis.homogentisate phytyltransferase homogentisate phytyltransferase 1 Ciclev m.g AT5G15720 GLIP7 misc.gdsl-motif lipase GDSL-motif lipase 7 Ciclev m.g AT5G64260 EXL2 signalling.in sugar and nutrient physiology EXORDIUM like 2 Ciclev m.g AT5G44400 misc.nitrilases, *nitrile lyases, berberine FAD-binding Berberine bridge enzymes, reticuline oxidases, family protein troponine reductases Ciclev m.g AT2G43290 MSS3 signalling.calcium Ciclev m.g Ciclev m.g Ciclev m.g AT1G22380 AT5G13980 AT3G57630 AtUGT85A3, UGT85A3 Ciclev m.g AT1G17020 ATSRG1,SRG1 Ciclev m.g AT5G56790 misc.udp glucosyl and glucoronyl transferases misc.gluco-, galacto- and mannosidases.alpha-mannosidase misc.udp glucosyl and glucoronyl transferases secondary metabolism.flavonoids.flavonols signalling.receptor kinases.proline extensin like Calcium-binding EF-hand family protein UDP-glucosyl transferase 85A3 Glycosyl hydrolase family 38 protein exostosin family protein senescence-related gene Ciclev m.g AT1G74110 CYP78A10 misc.cytochrome P Ciclev m.g AT1G61560 ATMLO6,MLO6 stress.biotic.signalling.mlo-like Protein kinase superfamily protein cytochrome P450, family 78, subfamily A, polypeptide 10 Seven transmembrane MLO family protein 167

168 Table 4-7. Continued C. Clementina_ID Arabi ID Gene name Bin Name log2 FC Arabidopsis_define Ciclev m.g AT1G55200 signalling.receptor kinases.proline extensin like Protein kinase protein with adenine nucleotide alpha hydrolases-like domain Ciclev m.g AT1G51880 RHS6 signalling.receptor kinases.misc root hair specific 6 Ciclev m.g AT3G11210 misc.gdsl-motif lipase SGNH hydrolase-type esterase superfamily protein Ciclev m.g AT1G59960 secondary NAD(P)-linked oxidoreductase metabolism.flavonoids.chalcones superfamily protein isoprenoids.tocopherol Ciclev m.g AT2G18950 ATHPT,VTE2 biosynthesis. homogentisate phytyltransferase homogentisate phytyltransferase 1 Ciclev m.g AT5G15720 GLIP7 misc.gdsl-motif lipase GDSL-motif lipase 7 Ciclev m.g AT5G64260 EXL2 signalling.in sugar and nutrient physiology EXORDIUM like 2 Ciclev m.g AT5G44400 misc.nitrilases, *nitrile lyases, berberine bridge enzymes, reticuline oxidases, troponine reductases FAD-binding Berberine family protein Ciclev m.g AT2G43290 MSS3 signalling.calcium Calcium-binding EF-hand family protein Ciclev m.g AT1G22380 AtUGT85A3 misc.udp glucosyl and glucoronyl transferases UDP-glucosyl transferase 85A3 Ciclev m.g AT5G13980 misc.gluco-, galacto- and mannosidases.alpha-mannosidase Glycosyl hydrolase family 38 protein Ciclev m.g AT3G57630 misc.udp glucosyl and glucoronyl transferases exostosin family protein Ciclev m.g AT1G17020 ATSRG1, secondary SRG1 metabolism.flavonoids.flavonols senescence-related gene 1 Ciclev m.g AT5G56790 signalling.receptor kinases.proline extensin like Protein kinase superfamily protein Ciclev m.g AT1G74110 CYP78A10 misc.cytochrome P cytochrome P450, family 78, subfamily A, polypeptide 10 Ciclev m.g AT1G61560 ATMLO6, Seven transmembrane MLO family stress.biotic.signalling.mlo-like MLO6 protein Ciclev m.g AT1G55200 signalling.receptor kinases.proline Protein kinase protein with adenine extensin like nucleotide alpha hydrolases-like domain Ciclev m.g AT1G51880 RHS6 signalling.receptor kinases.misc root hair specific 6 Ciclev m.g AT5G11720 misc.gluco-, galacto- and mannosidases.alpha-galactosidase Glycosyl hydrolases family 31 protein 168

169 Table 4-7. Continued C. Clementina_ID Arabi ID Gene name Bin Name log2 FC Arabidopsis_define Ciclev m.g AT4G33420 misc.peroxidases Peroxidase superfamily protein Ciclev m.g AT1G24020 MLP423 stress.abiotic.unspecified MLP-like protein 423 Ciclev m.g AT1G75130 CYP721A1 misc.cytochrome P cytochrome P450, family 721, subfamily A, polypeptide 1 Ciclev m.g AT2G25140 CLPB-M,HSP98.7 stress.abiotic.heat casein lytic proteinase B4 Ciclev m.g AT4G36040 stress.abiotic.heat Ciclev m.g AT4G20840 misc.nitrilases, *nitrile lyases, berberine bridge enzymes, reticuline oxidases, troponine reductases Ciclev m.g AT1G78380 ATGSTU19,GSTU19 misc.glutathione S transferases Ciclev m.g AT5G17540 stress.biotic Ciclev m.g AT5G67150 Ciclev m.g AT1G22380 AtUGT85A3,UGT85A3 Ciclev m.g AT3G21760 HYR1 Ciclev m.g AT2G18950 ATHPT,HPT1VTE2 secondary metabolism.phenylpropanoids misc.udp glucosyl and glucoronyl transferases misc.udp glucosyl and glucoronyl transferases secondary metabolism.isoprenoids.tocopherol biosynthesis.homogentisate phytyltransferase Ciclev m.g AT1G78380 ATGSTU19,GSTU19 misc.glutathione S transferases Ciclev m.g AT5G16970 AER, AT-AER Ciclev m.g AT5G10530 Ciclev m.g AT5G64260 EXL2 Ciclev m.g AT4G15560 CLA1,DEF,DXPS2,DXS Ciclev m.g AT1G28390 misc.oxidases - copper, flavone etc. signalling.receptor kinases.legume-lectin signalling.in sugar and nutrient physiology secondary metabolism.isoprenoids.nonmevalonate Pathway.DXS signalling.receptor kinases.crinkly like Chaperone DnaJ-domain superfamily protein FAD-binding Berberine family protein glutathione S-transferase TAU 19 HXXXD-type acyl-transferase family protein HXXXD-type acyl-transferase family protein UDP-glucosyl transferase 85A3 UDP-Glycosyltransferase superfamily protein homogentisate phytyltransferase 1 glutathione S-transferase TAU alkenal reductase Concanavalin A-like lectin protein kinase family protein EXORDIUM like Deoxyxylulose-5-phosphate synthase Protein kinase superfamily protein 169

170 Table 4-7. Continued C. Clementina_ID Arabi ID Gene name Bin Name log2 FC Arabidopsis_define Ciclev m.g AT2G36800 DOGT1,UGT73C5 stress.biotic don-glucosyltransferase 1 Ciclev m.g AT5G36110 CYP716A1 misc.cytochrome P cytochrome P450, family 716, subfamily A, polypeptide 1 Ciclev m.g AT3G11340 UDPmisc.UDP glucosyl and 1.15 Glycosyltransferase glucoronyl transferases superfamily protein Ciclev m.g AT5G59580 UGT76E1 Ciclev m.g AT2G27500 misc.udp glucosyl and glucoronyl transferases misc.beta 1,3 glucan hydrolases.glucan endo-1,3- beta-glucosidase UDP-glucosyl transferase 76E1 Glycosyl hydrolase superfamily protein Ciclev m.g AT1G59860 stress.abiotic.heat HSP20-like chaperones superfamily protein Ciclev m.g AT3G26300 CYP71B34 misc.cytochrome P cytochrome P450, family 71, subfamily B, polypeptide 34 Ciclev m.g AT1G24620 signalling.calcium EF hand calciumbinding protein family Ciclev m.g AT5G24090 ATCHIA,CHIA stress.biotic.pr-proteins chitinase A Ciclev m.g AT1G06620 redox.ascorbate and glutathione oxoglutarate (2OG) and Fe(II)-dependent oxygenase superfamily protein Ciclev m.g AT5G46080 signalling.receptor kinases.misc Ciclev m.g AT1G59960 secondary metabolism.flavonoids.chalcones Classification of the measured parameter into a set a functional category in the MapMan analysis tool Protein kinase superfamily protein NAD(P)-linked oxidoreductase superfamily protein 170

171 Table 4-8. Differentially expressed hormonal metabolism-associated genes significantly upregulated in roots of the symptomatic VAL/SW combination as compared to symptomatic VAL/CAN roots C. Clementina_ID Arabi ID Gene Name Bin Name Log2 FC* Arabidospsis_define Ciclev m.g AT3G04720 HEL, PR-4,PR4 stress.biotic pathogenesis-related 4 misc.nitrilases, *nitrile lyases, berberine FAD-binding Berberine family Ciclev m.g AT5G44400 bridge enzymes, reticuline oxidases, protein troponine reductases Ciclev m.g Ciclev m.g AT3G51420 AT3G54420 ATSSL4, SSL4 ATCHITIV, ATEP3 secondary metabolism. N misc.alkaloid-like stress.biotic Ciclev m.g AT2G20142 stress.biotic.receptors Ciclev m.g AT1G59960 secondary metabolism. flavonoids.chalcones strictosidine synthase-like Ciclev m.g AT5G51440 stress.abiotic.heat Ciclev m.g AT1G12280 stress.biotic.pr-proteins Ciclev m.g AT5G54160 ATOMT1,OMT1 secondary metabolism. phenylpropanoids.lignin biosynthesis.comt homolog of carrot EP3-3 chitinase Toll-Interleukin-Resistance (TIR) domain family protein NAD(P)-linked oxidoreductase superfamily protein FAD-binding Berberine family protein LRR and NB-ARC domainscontaining disease resistance protein O-methyltransferase 1 Ciclev m.g AT3G09270 ATGSTU8, glutathione S-transferase misc.glutathione S transferases GSTU8 TAU 8 Ciclev m.g AT1G17020 ATSRG1,SRG1 secondary metabolism.flavonoids.flavonols senescence-related gene 1 Ciclev m.g AT2G29420 ATGSTU7, glutathione S-transferase tau misc.glutathione S transferases GSTU7 7 Ciclev m.g AT4G17080 Histone H3 K4-specific signalling.phosphinositides methyltransferase SET7/9 phosphatidylinositol-4-phosphate 5-kinase family protein Ciclev m.g AT4G16660 stress.abiotic.heat Ciclev m.g Ciclev m.g AT1G35710 AT4G27300 signalling.receptor kinases.l eucine rich repeat XII signalling.receptor kinases.s-locus glycoprotein like heat shock protein 70 (Hsp 70) family protein Protein kinase family protein with leucine-rich repeat domain S-locus lectin protein kinase family protein 171

172 Table 4-8. Continued C. Clementina_ID Arabi ID Gene Name Bin Name Log2 FC Arabidospsis_define Ciclev m.g AT5G39120 stress.abiotic.unspecified RmlC-like cupins superfamily protein Ciclev m.g AT4G23180 CRK10 signalling.receptor kinases.duf cysteine-rich RLK (RECEPTORlike protein kinase) 10 Ciclev m.g AT3G26300 CYP71B34 misc.cytochrome P cytochrome P450, family 71, subfamily B, polypeptide 34 Ciclev m.g AT1G78860 misc.myrosinases-lectin-jacalin D-mannose binding lectin protein with Apple-like carbohydratebinding domain Ciclev m.g AT3G22600 Bifunctional inhibitor/lipid-transfer misc.protease inhibitor/seed storage/ protein/seed storage 2S albumin lipid transfer protein (LTP) family protein superfamily protein Ciclev m.g AT5G44390 misc.nitrilases, *nitrile lyases, berberine bridge enzymes, reticuline oxidases, troponine reductases Ciclev m.g AT5G37940 misc.oxidases - copper, flavone etc Ciclev m.g AT2G45550 CYP76C4 misc.cytochrome P Ciclev m.g AT2G20142 stress.biotic.receptors Ciclev m.g AT3G46530 RPP13 stress.biotic Ciclev m.g AT5G54160 OMT1 Ciclev m.g AT3G11340 secondary metabolism.phenylpropanoids. lignin biosynthesis.comt misc.udp glucosyl and glucoronyl transferases FAD-binding Berberine family protein Zinc-binding dehydrogenase family protein cytochrome P450, family 76, subfamily C, polypeptide 4 Toll-Interleukin-Resistance (TIR) domain family protein NB-ARC domain-containing disease resistance protein O-methyltransferase Ciclev m.g AT3G18430 signalling.calcium Ciclev m.g AT2G45400 BEN1 secondary metabolism.flavonoids. flavonols.dihydrokaempferol 4-reductase Ciclev m.g AT1G17020 SRG1 secondary metabolism.flavonoids.flavonols Ciclev m.g AT2G47140 misc.short chain dehydrogenase/reductase (SDR) Ciclev m.g AT4G27290 signalling.receptor kinases. S-locus glycoprotein like UDP-Glycosyltransferase superfamily protein Calcium-binding EF-hand family protein NAD(P)-binding Rossmann-fold superfamily protein senescence-related gene NAD(P)-binding Rossmann-fold superfamily protein S-locus lectin protein kinase family protein 172

173 Table 4-8. Continued C. Clementina_ID Arabi ID Gene Name Bin Name Log2 FC Arabidospsis_define Ciclev m.g AT2G47140 misc.short chain NAD(P)-binding Rossmann-fold dehydrogenase/reductase (SDR) superfamily protein Ciclev m.g AT4G37990 ATCAD8 phenylpropanoids.lignin biosynthesis elicitor-activated gene 3-2 Ciclev m.g AT2G18950,VTE2 isoprenoids.tocopherol biosynthesis. homogentisate phytyltransferase homogentisate phytyltransferase 1 Ciclev m.g AT1G71695 misc.peroxidases Peroxidase superfamily protein Ciclev m.g AT3G09270 GSTU8 misc.glutathione S transferases glutathione S-transferase TAU 8 Ciclev m.g AT4G27190 stress.biotic.pr-proteins NB-ARC domain-containing disease resistance protein Ciclev m.g AT1G66910 signalling. wheat LRK10 like Protein kinase superfamily protein Ciclev m.g AT2G36690 misc.oxidases - copper, flavone etc oxoglutarate (2OG) and Fe(II)- dependent oxygenase superfamily protein Ciclev m.g AT2G29420 GSTU7 misc.glutathione S transferases glutathione S-transferase tau 7 Ciclev m.g AT4G02340 misc.misc alpha/beta-hydrolases superfamily protein Ciclev m.g AT2G47730 GSTF8 misc.glutathione S transferases glutathione S-transferase phi 8 Ciclev m.g AT1G18980 stress.abiotic.unspecified RmlC-like cupins superfamily protein Ciclev m.g AT2G38870 stress.biotic Serine protease inhibitor, potato inhibitor I-type family protein Ciclev m.g AT2G30860 GSTF9 misc.glutathione S transferases glutathione S-transferase PHI 9 Ciclev m.g AT2G18980 misc.peroxidases Peroxidase superfamily protein Ciclev m.g AT3G55700 misc.udp glucosyl and glucoronyl UDP-Glycosyltransferase superfamily transferases protein Ciclev m.g AT1G20030 stress.biotic Pathogenesis-related thaumatin superfamily protein Ciclev m.g AT1G65800 ARK2,RK2 signalling.receptor kinases.s-locus glycoprotein like receptor kinase 2 Ciclev m.g AT5G17680 stress.biotic.pr-proteins disease resistance protein (TIR-NBS- LRR class), putative Ciclev m.g AT4G08850 signalling.receptor kinases. Leucine-rich repeat receptor-like leucine rich repeat XII protein kinase family protein Ciclev m.g AT3G50740 UGT72E1 phenylpropanoids.lignin biosynthesis UDP-glucosyl transferase 72E1 Ciclev m.g AT2G17880 stress.abiotic.heat Chaperone DnaJ-domain superfamily protein 173

174 Table 4-8. Continued C. Clementina_ID Arabi ID Gene Name Bin Name Log2 FC Arabidospsis_define Ciclev m.g AT3G16490 IQD26 signalling.calcium IQ-domain 26 Ciclev m.g AT2G23540 misc.gdsl-motif lipase GDSL-like Lipase/Acylhydrolase superfamily protein Ciclev m.g AT4G21410 CRK29 signalling.receptor kinases.duf cysteine-rich RLK (RECEPTOR-like protein kinase) 29 Ciclev m.g AT3G57330 ACA11 signalling.calcium autoinhibited Ca2+-ATPase 11 Ciclev m.g AT4G37640 ACA2 signalling.calcium calcium ATPase 2 Ciclev m.g AT3G02100 misc.udp glucosyl and glucoronyl UDP-Glycosyltransferase transferases superfamily protein Ciclev m.g AT4G36810 GGPS1 secondary metabolism.isoprenoids.nonmevalonate Pathway.geranylgeranyl geranylgeranyl pyrophosphate synthase 1 pyrophosphate synthase Ciclev m.g AT1G31690 misc.oxidases - copper, flavone etc Copper amine oxidase family protein Ciclev m.g AT3G14630 CYP72A9 misc.cytochrome P Ciclev m.g AT1G30760 Ciclev m.g AT5G22300 AtNIT4,NIT4 Ciclev m.g AT4G14210 PDS3 misc.nitrilases, *nitrile lyases, berberine bridge enzymes, reticuline oxidases, troponine reductases secondary metabolism. glucosinolates. degradation. nitrilase secondary metabolism. isoprenoids.carotenoids. phytoene dehydrogenase cytochrome P450, family 72, subfamily A, polypeptide FAD-binding Berberine family protein nitrilase phytoene desaturase 3 Ciclev m.g AT1G18650 PDCB3 misc.beta 1,3 glucan hydrolases callose-binding protein 3 Ciclev m.g AT4G37000 ACD2, ATRCCR stress.biotic accelerated cell death 2 (ACD2) Ciclev m.g AT3G03080 misc.oxidases - copper, flavone etc Zinc-binding dehydrogenase protein Ciclev m.g AT5G52390 signalling.in sugar and nutrient physiology PAR1 protein Ciclev m.g AT2G28790 stress.abiotic Pathogenesis-related thaumatin superfamily protein Ciclev m.g AT5G26990 stress.abiotic.drought/salt Drought-responsive family protein Ciclev m.g AT5G28840 GME redox.ascorbate and glutathione.ascorbate.gme GDP-D-mannose 3\',5\'-epimerase Ciclev m.g AT5G44400 misc.nitrilases, *nitrile lyases, berberine reticuline oxidases, troponine reductases FAD-binding Berberine family protein 174

175 Table 4-8. Continued C. Clementina_ID Arabi ID Gene Name Bin Name Log2 FC Arabidospsis_define Ciclev m.g AT1G53540 stress.abiotic.heat HSP20-like chaperones superfamily protein Ciclev m.g AT1G68400 signalling.receptor kinases. leucine rich repeat III Ciclev m.g AT3G07500 signalling.light Ciclev m.g AT4G08850 signalling.receptor kinases. leucine rich repeat XII Ciclev m.g AT1G52240 PIRF1,ROPGEF11 signalling.g-proteins Ciclev m.g AT4G27700 misc.rhodanese Ciclev m.g AT5G13930 ATCHS,CHS,TT4 secondary metabolism.flavonoids.chalcones. naringenin-chalcone synthase Ciclev m.g AT4G11650 ATOSM34,OSM34 stress.abiotic osmotin 34 Ciclev m.g AT1G61050 misc.udp glucosyl and glucoronyl transferases stress.biotic.pr-protei Ciclev m.g AT4G27220 Ciclev m.g ns Ciclev m.g AT1G73040 misc.myrosinases-lectin-jacalin leucine-rich repeat transmembrane protein kinase family protein Far-red impaired responsive (FAR1) family protein Leucine-rich repeat receptor-like protein kinase family protein RHO guanyl-nucleotide exchange factor 11 Rhodanese/Cell cycle control phosphatase superfamily protein Chalcone and stilbene synthase family protein alpha 1,4-glycosyltransferase family protein NB-ARC domain-containing disease resistance protein Mannose-binding lectin superfamily protein Leucine-rich repeat protein kinase family protein S-locus lectin protein kinase family protein Protein kinase superfamily protein Ciclev m.g AT3G47570 signalling.receptor kinases. leucine rich repeat XII Ciclev m.g AT4G27290 signalling.receptor kinases.s-locus glycoprotein like Ciclev m.g AT2G23200 signalling. receptor kinases. Catharanthus roseus-like RLK Ciclev m.g AT1G01200 ATRAB-A3, signalling.g-proteins RAB GTPase homolog A3 Ciclev m.g AT4G11650 ATOSM34,OSM34 stress.abiotic osmotin 34 Ciclev m.g AT3G04720 HEL, PR-4,PR4 stress.biotic pathogenesis-related 4 Ciclev m.g AT3G48310 CYP71A22 misc.cytochrome P cytochrome P450, family 71, subfamily A, polypeptide 22 Ciclev m.g AT4G27290 signalling.receptor kinases.s-locus S-locus lectin protein kinase glycoprotein like family protein 175

176 Table 4-8. continued C. Clementina_ID Arabi ID Gene Name Bin Name Log2 FC Arabidospsis_define Ciclev m.g AT2G32520 misc.misc Ciclev m.g AT2G34790 EDA28,MEE23 misc.nitrilases, *nitrile lyases, berberine bridge enzymes, reticuline oxidases, troponine reductases alpha/beta-hydrolases superfamily protein FAD-binding Berberine family protein Ciclev m.g AT3G25070 RIN4 stress.biotic RPM1 interacting protein 4 Ciclev m.g AT1G16030 Hsp70b stress.abiotic.heat heat shock protein 70B Ciclev m.g AT4G38660 stress.biotic Pathogenesis-related thaumatin superfamily protein Ciclev m.g AT2G23910 secondary metabolism. NAD(P)-binding Rossmann-fold phenylpropanoids superfamily protein ATPase E1-E2 type family protein / Ciclev m.g AT3G63380 signalling.calcium haloacid dehalogenase-like hydrolase family protein Ciclev m.g AT5G14130 misc.peroxidases Peroxidase superfamily protein Ciclev m.g AT2G44290 Bifunctional inhibitor/lipid-transfer misc.protease inhibitor/seed storage/lipid protein/seed storage 2S albumin transfer protein (LTP) family protein superfamily protein Ciclev m.g AT5G48930 HCT secondary metabolism.phenylpropanoids.lignin biosynthesis.hct hydroxycinnamoyl-coa shikimate/quinate hydroxycinnamoyl transferase Ciclev m.g AT4G31940 CYP82C4 misc.cytochrome P cytochrome P450, family 82, subfamily C, polypeptide 4 Ciclev m.g AT2G47140 misc.short chain NAD(P)-binding Rossmann-fold dehydrogenase/reductase (SDR) superfamily protein Ciclev m.g AT1G71380 CEL3 misc.gluco-, galacto- and mannosidases.endoglucanase cellulase 3 Ciclev m.g AT1G24020 MLP423 stress.abiotic.unspecified MLP-like protein 423 Ciclev m.g AT4G13700 PAP23 misc.acid and other phosphatases purple acid phosphatase 23 Ciclev m.g AT5G39090 secondary HXXXD-type acyl-transferase family metabolism.flavonoids.anthocyanins protein anthocyanin 5-aromatic acyltransferase Ciclev m.g AT3G09270 GSTU8 misc.glutathione S transferases glutathione S-transferase TAU 8 secondary Chalcone-flavanone isomerase Ciclev m.g AT5G metabolism.flavonoids.chalcones family protein 176

177 Table 4-8. Continued C. Clementina_ID Arabi ID Gene Name Bin Name Log2 FC Arabidospsis_define Ciclev m.g AT1G21550 signalling. calcium Calcium-binding EF-hand family protein Ciclev m.g AT1G71830 SERK1 Signaling.leucine rich repeat II somatic embryogenesis receptor-like kinase 1 Ciclev m.g AT4G22130 SRF8 signalling.leucine rich repeat V STRUBBELIG-receptor family 8 Ciclev m.g AT1G21550 signalling. calcium Calcium-binding EF-hand family protein Ciclev m.g AT5G17770 ATCBR,CBR, redox.misc NADH:cytochrome B5 reductase 1 Ciclev m.g AT5G10610 CYP81K1 misc. cytochrome P cytochrome P450, family 81, subfamily K, polypeptide 1 Ciclev m.g AT1G11000 MLO4 stress.biotic.signalling.mlo-like Seven transmembrane MLO family protein Ciclev m.g AT5G48800 signalling. light Phototropic-responsive NPH3 family protein Ciclev m.g AT3G07410 AtRABA5b signalling.g-proteins RAB GTPase homolog A5B Ciclev m.g AT2G40890 CYP98A3 misc. cytochrome P cytochrome P450, family 98, subfamily A, polypeptide 3 Ciclev m.g AT5G63710 signalling.receptor kinases. Leucine-rich repeat protein leucine rich repeat II kinase family protein Ciclev m.g AT5G53890 PSKR2 signalling..leucine rich repeat X phytosylfokine-alpha receptor 2 Ciclev m.g AT1G15950 CCR1, secondary metabolism. IRX4 phenylpropanoids.lignin biosynthesis.ccr cinnamoyl coa reductase 1 Ciclev m.g AT3G47860 CHL stress.abiotic chloroplastic lipocalin Ciclev m.g AT2G44480 BGLU17 misc.gluco-, galacto- and mannosidases beta glucosidase 17 Ciclev m.g AT5G51350 Leucine-rich repeat signalling.receptor kinases transmembrane protein kinase leucine rich repeat XIV family protein Ciclev m.g AT2G25790 signalling.receptor kinases. Leucine-rich receptor-like leucine rich repeat IV protein kinase family protein Ciclev m.g AT2G22570 NIC1 secondary metabolism.phenylpropanoids nicotinamidase 1 Ciclev m.g Ciclev m.g AT1G35190 AT1G06840 secondary metabolism. N misc.alkaloid-like signalling.receptor kinases. leucine rich repeat VIII oxoglutarate (2OG) and Fe(II)-dependent oxygenase superfamily protein Leucine-rich repeat protein kinase family protein 177

178 Table 4-8. Continued C. Clementina_ID Arabi ID Ciclev m.g AT4G31500 Gene Name CYP83B1, SUR2 Bin Name Log2 FC Arabidospsis_define secondary metabolism. sulfurcontaining.glucosinolates.synthesis. shared.cyp83b1 phenylacetaldoxime monooxygenase Ciclev m.g AT3G19000 redox.ascorbate and glutathione Ciclev m.g AT2G02100 LCR69, PDF2.2 stress.biotic Ciclev m.g AT4G25570 ACYB-2 redox.ascorbate and glutathione Ciclev m.g AT1G53710 misc.calcineurin-like phosphoesterase family protein cytochrome P450, family 83, subfamily B, polypeptide 1 2-oxoglutarate (2OG) and Fe(II)-dependent oxygenase superfamily protein low-molecular-weight cysteine-rich 69 Cytochrome b561/ferric reductase transmembrane protein family Calcineurin-like metallophosphoesterase superfamily protein * The negative sign in the column of Log2 FC indicates comparative downregulation of the gene expression level in VAL/CAN and upregulation in VAL/SW combination. Classification of the measured parameter into a set a functional category in the MapMan analysis tool. 178

179 Figure 4-1. Graphical presentation of DEGs involved in environmental biotic and abiotic signaling.biotic and abiotic signaling overview in the PageMan depicting DGE in leaves and roots of HLB-asymptomatic and -symptomatic treatment in VAL/CAN and VAL/SW combinations. Log2 fold changes are indicated as a gradient between blue (up-regulated in VAL/CAN combination) and red (up-regulated and VAL/SW combination. Asymp; Asymptomatic, Sympt; symptomatic 179

180 Figure 4-2. Graphical presentation of DEGs involved in biotic and abiotic stress. Stress overview in the PageMan depicting DGE in leaves and roots of the HLB-asymptomatic and -symptomatic treatments in VAL/CAN and VAL/SW combinations. Log2 fold changes are indicated as a gradient between blue (upregulated in VAL/CAN combination) and red (upregulated in VAL/SW combination). Asymp; Asymptomatic, Sympt; symptomatic Figure 4-3. Display of HLB-induced biotic stress responses in the leaves of the HLB asymptomatic VAL/CAN and VAL/SW leaves. The log2 fold change of DGE was analyzed using MapMan. Log2 fold changes are indicated as a gradient between blue (upregulated in the asymptomatic VAL/CAN) and red (upregulated in asymptomatic VAL/SW combination). 180

181 Figure 4-4 Display of HLB-induced biotic stress responses in leaves of the HLBsymptomatic VAL/CAN and VAL/SW leaves. The log2 fold change of DGE was analyzed using MapMan. Log2 fold changes are indicated as a gradient blue (upregulated in the symptomatic VAL/CAN combination) and red (upregulated in symptomatic VAL/SW combination). Figure 4-5. Display of HLB-induced biotic stress responses in roots of the HLBsymptomatic VAL/CAN and VAL/SW leaves. The log2 fold change of DGE was analyzed using MapMan. Log2 fold changes are indicated as a gradient between blue (upregulated in the symptomatic VAL/CAN combination) and red (upregulated in symptomatic VAL/SW combination). 181

182 CHAPTER 5 DIFFERENTIAL EXPRESSION OF PLANT GROWTH AND DEVELOPMENT- ASSOCIATED GENES IN LEAVES AND ROOTS OF TWO SCION/ROOTSTOCK COMBINATIONS AT ASYMPTOMATIC AND SYMPTOMATIC STAGES OF HLB DISEASE Introduction Physical growth and development are essential for the plant life cycle. Various components of plant growth are embryogenesis, development of seed and flower, fruit set and ripening, development of structural components and their expansion, growth of reproductive and vegetative organs, plant senescence and plant death, and interaction of plant with biotic and abiotic stress. Plant growth is a highly coordinated process which mostly depends on energy production (photosynthesis) and consumption (respiration), supply and demand of photosynthates flow between source-sink organs, regeneration of secondary plant growth, reproduction, and external and/or internal signaling mechanism. Plant growth may be compromised severely under biotic and abiotic stresses. When plants are impacted by biotic and abiotic challenges, defense is inevitable for survival, but plant sustainability is indeed dependent on continued photosynthesis and respiration for plant growth. In citrus, (huanglongbing) HLB-affected plants have shown the decline in plant production and sustainability. Interactions between HLB and citrus are still unclear. Anatomical and molecular analysis of HLB affected citrus trees suggests that a disparity between plant growth and defense affects the plant sustainability. Anatomical studies of Candidatus Liberibacter asiaticus (CaLas) -infected citrus cultivars have shown a severely affected plant photosynthesis machinery, interrupted phloem channels, reduced root growth, and disproportionate distribution of plant photosynthates between sink-source tissue, leading to the appearance of HLB symptoms on the leaves (Etxeberria et al., 2009; Folimonova and 182

183 Achor, 2010; Koh et al., 2012). Molecular analysis of the CaLas-infected leaves, roots and fruits samples supported the findings from the anatomical studies (Fan et al., 2012; Albrecht and Bowman, 2012b; Aritua et al., 2013). The anatomical, transcriptomic, proteomic, and microscopic -assisted studies of HLB-citrus interaction in various cultivars suggest that activation of many non-host specific defenses, inability to repair damaged phloem system, and limited available photosynthate resources hamper plant growth. Improved plant growth can, therefore, help to enhance the plant sustainability and performance of HLB-affected plants. This Chapter discusses the possible role of a putative HLB-tolerant candidate rootstock (CAN) to promote the plant growth in asymptomatic and symptomatic CaLas-infected Valencia scion. Plant tissues and organs are often injured because of insect feeding, wounding, disease attack and abiotic stresses. Damaged tissues are either repaired, regenerated or removed by selective cell death to keep essential plant biological functions working. Regeneration is a common strategy for plants to repair the damage caused by biotic and abiotic stress. The plant vascular system is a crucial system for plant survival and has a capacity to repair or regenerate damaged phloem, xylem and cambium tissues. Plant tissue culture techniques and microscopy have revealed a hierarchy of plant growth and development from totipotent meristematic tissue (Allan, 2015). Anatomical studies on plant regeneration of the model plant Arabidopsis thaliana showed that induction of the phloem regeneration is a three-step process: maintenance of stem cell microenvironment, proliferation and growth, and differentiation of dividing cells into tissues. Studies in Arabidopsis showed that Auxins (AU), gibberellins (GA), cytokinins (CT) and ethylene (ET) regulate vascular cambium activity and differentiation of 183

184 secondary vascular tissue (Ursache et al., 2013). The genes encoding vascular tissue regeneration transcription factors (TFs) such as KNOX1, AP-2, HD ZIPIII, KANADI (KAN), LATERAL ORGAN BOUNDARIES (LBD), and Vascular related NAC domain protein-1 (VND1) (Pang et al., 2008; Chen et al., 2014; Zhou et al., 2014). However, there are not enough studies on the genetic aspect of how the totipotent cells differentiate into different primary and secondary vascular structures. Microarray analysis of secondary vascular tissue regeneration in girdled Populus tissue showed changes in gene expression at each step of xylem, phloem and cambium regeneration (Zhang et al., 2011b). The study of girdled Populus reported differential changes in the expression level of genes encoding transcription factors (TFs) belong to R2R3-MYB, WRKY, DNA- binding with one zinc finger (DOFs), Knotted1-like homeobox (KNOX) and other phloem specific TFs. Also, the comparative gene expression analysis between xylem, phloem, and cambium regeneration stages showed effects of AU, CT and GA responsive genes in the vascular tissue development (Zhang et al., 2011b). A review of the genetic and hormonal regulation of cambial development reported that AU, CT, and GA act synergistically to regulate cambial development (Ursache et al., 2013). HLBaffected plants are a classic example of vascular tissue (phloem) damage caused by psyllid (Diaphrina citri Kuwayama) feeding (piercing) and callose/starch depositions in the phloem, which block the source-sink transport in the plants (Koh et al., 2012). Weakening strength of HLB-affected plants is the indirect effect of CaLas-infection and the direct impact of photosynthate supply shortage for plant growth, development, and defense (Cimò et al., 2013). Considering the significant impact of phloem damage in developing HLB symptoms and reducing plant sustainability, developing strategies that 184

185 can increase phloem regeneration ability in the CaLas-infected plants is a priority for citrus researchers. Plant secondary metabolites are involved in plant development and growth. The secondary metabolites are divided into 3 major categories: terpenes, phenolics, and nitrogen containing compounds. The groups of plant hormones such as GA and brassinosteroids (BR) are considered as diterpenes and tetraterpenes (Haubrick and Assmann, 2006; Urbanov et al., 2011). Abscisic acid (ABA) is a C15 terpene produced by degradation of a carotenoid precursor (Raghavendra et al., 2010). The carotenoids (red, orange, yellow colored) are tetraterpenes which are important pigments in the plant photosystem (Singh and Sharma, 2015). Phenylalanine (PHE) is a key intermediate in the secondary metabolite biosynthesis which belongs to the phenolics group. Many phenolic compounds in the plants are derived by PHE catabolic enzyme phenylalanine ammonia lyase (PAL). PAL is also a branching point which leads to different biosynthetic pathways (Hahlbrock and Scheel, 1989). PAL acts as a signal in response to nutrient level changes, light response, and potential plant danger signaling. Phenylpropanoid, a derivative of PHE, is involved in lignin biosynthesis (Besseau et al., 2007). Lignin is the component of plant cell wall cytoskeleton which supports cell integrity and strengthens the plant vascular system (Ruprecht and Persson, 2012). Phenolic compounds are divided into major 4 subgroups: flavonoids, flavones, isoflavonoids, and tannins. These phenolic subgroups are involved in developing plant pigmentation, cell wall modification, protection from the oxidative burst, and plant defense, (Gould, 2004; Besseau et al., 2007; Hussey et al., 2013). 185

186 Plants are autotrophs that produce their own energy using photosystems coupled with organelles that metabolize the energy into the form of adenosine triphosphate (ATP). Energy harvesting chloroplasts and energy production mitochondria are inevitable organelles in the plant cells that balance the energy demand and supply throughout the plant life cycle. Photosynthesis and plant respiration synthesized metabolites, and ATPs are the life driving force in plants. Plant metabolites are the complex molecules that are synthesized from simple sugar monomers or amino acids and other inorganic building blocks. Carbohydrates (CHOs) or sugars are invaluable for plant growth and defense (Sturm and Tang, 1999; Koch, 2004). Roles of monomers or polymers of CHOs are well known, and they are involved in the diverse plant biological functions. Polymers such as starch and fructans are the nocturnal energy reserve when photosynthesis is inactive (Orzechowski, 2008) whereas, sucrose is the primary provider of energy to the growing plant tissue. CHOs are not only the source of energy but also provide the plant cytoskeleton. The polysaccharides such as homogalacturonan (HGA), rhamnogalacturonan I (RGI), rhamnogalacturonan II (RGII) and xylogalacturonan (XGA) are components of the cell wall cytoskeleton. HGA is a component of pectins in the plant cell wall. Pectinmethylesterases (PMEs) are a carbohydrate esterase that catalyzes dimethyl esterification of HGA, and it remodels the plant cell wall during plant growth and defense (Pelloux et al., 2007). Nutrient availability controls plant development (Krouk et al., 2011). Plant minerals are essentials to produce plant energy and required metabolites. Plant minerals are divided into macronutrients: nitrogen (N), phosphorus (P), potassium (K) secondary nutrients: magnesium (Mg), calcium (Ca), Sulfur (S) and micronutrients: iron 186

187 (Fe), manganese (Mn), zinc (Zn), copper (Cu), and boron (B), nickel (Ni) and molybdenum (Mo). Macro, secondary and micronutrients are important plant regulatory components are essential for plant growth, energy production, maintenance plant structures, cellular redox environment, and resource supply to the source organs. Active plant growth, reproduction, and defense demand more energy and, therefore, nutrient requirement is more in the plant. Hence, nutrient homeostasis is critical to perform plant functions at optimum levels. Nutrient homeostasis is regulated through plant internal or cellular signaling system which creates hormonal changes, enhanced or reduced metal transporter activity, and expansion of the root system. The different components of nutrient homeostasis are highly interdependent (Krouk et al., 2011). Genomics studies in dicot and monocot plants revealed the differential expressional changes in the transporter genes to maintain the metal homeostasis in response to mineral deficiencies (Wintz et al., 2003; Grotz and Guerinot, 2006). Resource limitations or pathogen infection can lead to nutrient deficiencies or heavy metal toxicity in plants. It is not unusual that disease affected plants are deficient in various mineral nutrients. The depleted pool of nutrients in the disease affected plants can be enhanced by nutrient rich fertilizer applications. However, the time and space interaction is highly correlated to revive the plant s health under nutrient deficit and lack of immunity conditions. The similar scenario of space-time-disease complex induced nutrient deficiency is seen in the CaLas-infected citrus plants. HLB-affected citrus plants are found to exhibit Ca, Mg, Zn, Mn and B deficiencies in leaves, whereas K concentration is found to be increased (Gottwald et al., 2007). The same deficiencies were found to be even much greater in roots from both greenhouse and field trees 187

188 infected with CaLas (J.W. Grosser, personal communication). The dysfunctionality of vascular systems in HLB-affected plants not only imbalances the source-sink compartmentalization but also creates localized or systemic nutrient deficiencies which inhibit plant growth (Nwugo et al., 2013b). There is no comprehensive study on the nutrition challenges in the CaLas infected citrus plants. Another factor that strongly impacts the nutrient translocation and transport in the CaLas-infected citrus plants is the scion/rootstock combination. Effect of citrus rootstocks on mineral nutrition was discussed by Wutscher (1973). The purpose of studying differential transcriptomic changes between a putatively HLB-tolerant rootstock and the HLB-susceptible Swingle is to analyze the role of genes encoding nutrient transporters and transmembrane proteins to reprogram CaLas-infected scion growth and development. Materials and Methods Plant Material Two combinations of scion and rootstock were used in this experiment. Field grown seven-year-old experimental plants were planted in the Lee Family's Alligator Grove, east of St. Cloud, Florida. The first combination of trees was Valencia' (VAL) sweet orange (Citrus sinensis [L.] Osbeck) grafted onto a putative HLB-tolerant candidate (CAN) rootstock. The CAN rootstock is a hybrid of Hirado Buntan Pink' pummelo (HBP) (Citrus maxima Merr.) and Cleopatra' mandarin (Citrus reticulata Blanco.). The 2 nd combination was Valencia' (VAL) scion grafted onto commercially important Swingle citrumelo (SW) rootstock. Swingle is a hybrid of grapefruit (Citrus paradisi [Macf.]) and trifoliate orange (Poncirus trifoliata [L.] Raf). Each combination of VAL/CAN and VAL/SW, plants were divided into two treatments based on the visible presence of HLB-like symptoms (Table 5-2). Highly infected and symptomatic trees in 188

189 each combination were grouped into the symptomatic treatment. Whereas, trees with fewer or no symptoms were grouped into the asymptomatic treatment (Table 5-1). All biological replicates in each treatment and combination were tested using quantitative PCR (qpcr) based CaLas detection, and ELISA assisted citrus tristeza virus (CTV) detection. Sampling, RNA Extraction, and RNA Sequencing A detailed protocol of sampling, RNA extraction, and RNA sequencing is explained in chapter#2. In brief, differentially expressed genes (DEGs) in the pairwise comparison between asymptomatic VAL/CAN and VAL/SW combinations, and symptomatic VAL/CAN, and VAL/SW combinations were identified with RNA-Seq and the Tuxedo pipeline (Chapter 2). The significant differentially regulated genes in leaves and roots tissue were annotated to C. clementina genome database in the Phytozome V1.0 (Goodstein et al., 2012). Functional categories of the significantly expressed genes were identified using A. thaliana annotation in the Phytozome server and MapMan (Thimm et al., 2004) software (Chapter 2.). Blast2GO algorithm (Conesa et al., 2005) was also used to identify molecular, cellular and biological functional categories. Overview of DEG developed using PageMan analysis tool in the MapMan software (Usadel et al., 2009). Results HLB Detection and RNA Sequencing Output The results of qrt PCR-based HLB detection and RNA-Seq output in all combinations and treatments are discussed in Chapter

190 Differentially Expressed Plant Growth and Development-Associated Genes in Leaves and Roots of Asymptomatic VAL/CAN and VAL/SW combinations Leaf samples The pairwise comparison between the leaf samples of the asymptomatic treatment of VAL/CAN and VAL/SW combinations showed differential expression of genes encoding the phloem regeneration specific transcription factors (TFs). From this list, genes encoding TFs belonging to G2 like transcription factor family KANADI, LBD2, late elongation hypocotyl (LHY), and homeobox domain (HB2 and HB16) were upregulated in the VAL/CAN leaves as compared to VAL/SW leaves (Table 5-3). Growth regulating factor 7 (GRF7) is a positive regulator of growth that suppresses the growth inhibiting osmotic stress responsive genes, was also overexpressed in the leaves of the asymptomatic VAL/CAN combination. In asymptomatic treatment, VEIN PATTERNING 1 (VEP1) gene was upregulated in the asymptomatic VAL/CAN leaves as compared to VAL/SW leaves (Table 5-3). Leaves collected from asymptomatic VAL/CAN overexpressed genes encoding AUXIN RESPONSIVE FACTOR 8 and 16 (ARF8 and 16), CT- associated response regulator ARR9 and CT-two components responsive regulator APRR2. In asymptomatic treatment, DGE analysis showed overexpression of genes involved in the secondary metabolite pathways in VAL/CAN leaves as compared to VAL/SW leaves. These genes are encoding PMEs, XYLOGLUCAN ENDOTRANSGLYCOSYLASE/HYDROLASE 33 (XTH33), Expansin family protein (EXP), anthocyanin and carotenoid biosynthesis TRANSPARENT TESTA 4 and 7 (TT4 and TT7), FLAVANONE 3-DIOXYGENASE (F3H), PAL, Wax biosynthesis and 3-KETOACYL-COA SYNTHASE 6 (KCS6) (Figure 5-1 and 5-2). In the category of carbohydrate metabolism, genes encoding starch degrading Vacuolar invertases and 190

191 MYO-INOSITOL PHOSPHATE SYNTHASE 2 (MIPS2) were increased in their expression in the asymptomatic VAL/CAN leaves, whereas, starch biosynthesis, starch degradation and storage, synthesis genes were upregulated in the asymptomatic VAL/SW combination. Multiple transcripts encoding trehalose synthesis gene were strongly increased in their expression levels in the asymptomatic VAL/CAN leaves (Table 5-3). In the asymptomatic treatment, genes encoding nutrient transporters NODULIN- LIKE PROTEINS (NIP), MAJOR INTRINSIC PROTEIN (MIP), oligopeptide transporters and ATO BINDING CASSETTE (ABC) superfamily proteins, were also significantly upregulated in VAL/CAN leaves as compared to VAL/SW leaves (Figure 5-3). Nutrient transporter genes were AMMONIUM TRANSPORTER 1;2 (AMT1;2), and carbohydrate transmembrane INOSITOL TRANSPORTER (INT1) (Table 5-2). Nutrient transporters such as ZINC TRANSPORTER PRECURSOR 1 (ZIP1), potassium inward rectifier K TRANSPORTER 1(AKT), nitrate transporter NRT1.1, low affinity phosphate transporter PHT2;1, 2 oxoglutarate and malate valve transporter DECARBOXYLATE TRANSPORTER 1 (DiT1) and genes encoding water channels NOD26-like intrinsic protein NIP 4;2 and plasma membrane intrinsic protein PIP 2;4 and PIP 2;5 were also upregulated in the asymptomatic VAL/CAN leaves as compared to asymptomatic VAL/SW leaves (Table 5-3). VAL leaves collected from the asymptomatic VAL/SW combination were significantly upregulated in the ABA and ET hormone-induced responsive genes. In this category, gene encoding ABA induced GRAM domain containing protein, ET signal transduction ERF1 and ET forming enzyme (EFE) were overexpressed in the range of 191

192 2-4 log2 FC. Genes encoding TFs involved in plant development were also overexpressed in the asymptomatic VAL/SW combination. These include LOB domain containing protein LBD 41, homeobox domain containing HB7 and many NAC domain containing (NAC002, NAC084, NAC084, NAC090, and NAC061), and SCARECROWlike SCR13 and SCR14 gene (Table 5-4). Asymptomatic VAL/SW also showed significant upregulation of genes encoding proteins and enzymes involved in the secondary metabolite pathways (Figure 5-1). These genes are encoding cell wall metabolism and lignin biosynthesis associated; UDP-D-XYLOSE SYNTHASE 2, ARABINOGALACTAN-PROTEIN 3 (AGP3), XYLOGLUCAN ENDOTRANSGLYCOSYLASE 6 (XTR6), ARABINOGALACTAN PROTEIN 16 (AGP16) and CELLULOSE SYNTHASE- like D3 (CSLD3). In the asymptomatic treatment, genes encoding proteins those are involved in dehydration response: LATE EMBRYOGENESIS ABUNDANT 14 (LEA14), EARLY RESPONSE DEHYDRATION (EDR7), and RESPONSIVE to DESICCATION 26 (RD26) were significantly upregulated in VAL/SW leaves as compared to VAL/CAN leaves (Table 5-4 and Figure 5-6). In the category of CHO metabolism, genes encoding starch forming and starch catabolic invertases (neutral and cell wall) were significantly overexpressed in the asymptomatic VAL/SW leaves (Figure 5-5). The chloroplast GERANYLGERANYL REDUCTASE (GGR) gene was also upregulated in the asymptomatic VAL/SW leaves as compared to VAL/CAN leaves. In the category of transporter functional category, not many nutrient transporter genes were overexpressed in the asymptomatic VAL/SW leaves. However, phosphate transporters PHT1;4 and PHT 1;9 were significantly upregulated. DGE analysis of asymptomatic VAL/SW leaves showed significant overexpression of 192

193 PEPTIDE TRANSPORTER 3 (PTR3), GLUCOSE 6-PHOSPHATE TRANSPORTER (GPT2), METAL TOLERANCE PROTEIN B1 (MTPB1) and genes encoding heavy metal transport/detoxification superfamily proteins (Table 5-4). Root samples The DGE analysis between roots collected from the asymptomatic VAL/CAN, and VAL/SW combinations showed significantly upregulated CHO metabolism related genes in the VAL/SW roots. Differentially Expressed Plant Growth and Development-Associated Genes in Leaves and Roots of Symptomatic VAL/CAN and VAL/SW Combinations Leaf samples In the symptomatic treatment, leaves collected from VAL/CAN combination showed significant upregulation of genes encoding hormone-dependent plant growth regulators such as auxin responsive ARF8, REVEILLEA1 (REV1), subtilisin like protease known as AIR3, and LIKE-AUXIN RESISTANT 2 (LAX2). In the symptomatic treatment, leaves collected from VAL/CAN showed significant upregulation of genes encoding BR ENHANCED EXPRESSION 3 (BEE3), BR SIGNALING KINASE (BSK2), GA biosynthesis KAO2, and CT-induced response regulators ARR4 and chase domain containing histidine kinase WOL as compared to VAL/SW leaves (Table 5-3 and Table B-3). In the symptomatic VAL/CAN combination, many genes involved in the phenylpropanoid pathway induced lignin biosynthesis and flavonoids biosynthesis were also significantly upregulated (Figure 5-1 and 5-2). Among these, CAFFEOYL-COA 3- O-METHYLTRANSFERASE (CCoAOMT), 4-COUMARATE-COA LIGASE-3 (4CL3), CINNAMYL-ALCOHOL-DEHYDROGENASE CAD4 and CAD9, HYDROXYLCINNAMOYL-COA-SHIKIMATE TRANSFER (HCT), TRANSPARENT 193

194 TESTA (TT7, TT5, TT4), CINNAMOYL-COA REDUCTASE 1 (CCR1), and genes involved anthocyanin biosynthesis were also overexpressed (Table 5-5 and Table B-3). Genes encoding proteins involved in the chloroplast development and photosynthesis reactions: photosystem II polypeptide subunit, DEOXYLULOSE-5-PHOSPHATE SYNTHASE (DXS), NON-PHOTOCHEMICAL QUENCHING 1(NPQ1), MONOGALACTOSYL DIACYLGLYCEROL SYNTHASE1 (MGD1), CYCLOPHILIN 38, and ATP SYNTHASE DELTA SUBUNIT (ATPD) were significantly upregulated in the symptomatic VAL/CAN leaves as compared to symptomatic VAL/SW leaves (Table 5-5). In the CHO metabolism category, starch, sucrose, raffinose and Myo-inositol phosphate biosynthesis related genes were overexpressed in symptomatic VAL/CAN combination (Table 5-5). Leaves collected from the symptomatic VAL/CAN combination overexpressed genes encoding developing vessel and xylem differentiation related NAC domain protein1 VND1, BSK2, LBD11, MADS box SEPALLATA 4 (SEP4), VEP1, and NAC transcription factors-like (NTL9) (Table 5-4). Symptomatic VAL/CAN leaves showed significant upregulation of many nutrient transporter genes that generally respond to nutrient deficiency or increased nutrient transportation (Figure 5-3). These include high affinity nitrate transporter NRT2;7, phosphate transporter PHT2;1, potassium transporter AKT1 and AKT2, sulfate transmembrane transporter SULTR4;1 and SULTR4;2, CALCIUM ANION EXCHANGE CAX1 and CAX7, and CAX interacting protein CXIP4. Among water channel and sugar transporters, NIP4;2, PIP1;4, INT1, and inorganic phosphate transmembrane transporter encoding genes were upregulated in leaves of the symptomatic VAL/CAN 194

195 combination. In the symptomatic treatment, transcripts encoding chloroplast ATPase subunits and mitochondrial carrier A BOUT DE SOUFFLE (BOU) genes were also upregulated in VAL/CAN leaves as compared to VAL/SW leaves (Table 5-5). In the symptomatic VAL/SW combination, leaves showed significant upregulation of a number of genes involved in the starch biosynthesis. Among these STARCH BRANCHING ENZYME SBE2.1 and SBE2.2, ADP-GLUCOSE PYROPHOSPHORYLASE (AGPase), and ALPHA GLUCAN PHOSPHORYLASE 2 (PHS2) were upregulated (Figure 5-5). The transcriptomic data also showed the upregulation of starch biosynthesis regulator GLUCAN WATER DIKINASE or STARCH EXCESS 1 (SEX1) gene in the symptomatic VAL/SW leaves. In the symptomatic treatment, VAL/SW leaves showed significantly upregulated starch degrading neutral SUCROSE INVERTASE gene transcripts (Table 5-6). Transcripts encoding the photosystem I and II polypeptide subunits, RUBISCO ACTIVASE (RCA), ferredoxin oxidoreductase family HY2, CHLORORESPIRATORY REDUCTION 3 (CCR3) and ATP SYNTHASE were upregulated VAL/SW leaves as compared to VAL/CAN, in the symptomatic treatment. There was no significant overexpression of secondary vascular growth associated genes in leaves of the symptomatic VAL/SW combination, except a few genes involved in cell wall modification, lignin biosynthesis, and flavonoid metabolism pathway. Among transportation category, transcripts encoding phosphate transporter, zinc efflux, amino acid carriers, mitochondrial membrane carriers, phosphate transporter, and Zn transporter precursor 1; ZIP1 were significantly upregulated in leaves of the symptomatic VAL/SW combination. Transcripts encoding transition metal ion transporters such as NATURAL RESISTANCE ASSOCIATED 195

196 MACROPHAGE PROTEIN (NRAMP), HEAVY METAL ATPase (HMA1), a copper transporter COP1, CATION EXCHANGERS (CAXs), and sugar transporters were also significantly upregulated in leaves of the symptomatic VAL/SW combination (Table 5-6). Root samples RNA extracted from roots of the symptomatic VAL/CAN combination showed significant overexpression of the genes encoding auxin dependent Au efflux carrier family protein F14G6.12, Au responsive AIR12, ARF16 and ARF2, and BR-induced stem elongation promoting HERCULES RECEPTOR KINASE 2 (HERK2) (Table 5-7). In addition, PHYTOSULFOKININE precursor 3 (PSK3) growth factor transcripts upregulated by 3.34 log2 FC in CAN roots as compared to the SW roots in the symptomatic treatment. Roots collected from the symptomatic VAL/CAN combination showed significantly upregulated expression of genes encoding TFs of GARP family KAN4, and NAC domain containing NAC047, which are involved in the secondary vascular tissue development (Table 5-7 and Table B-5). In the symptomatic treatment, roots collected from VAL/CAN combination did not show significant upregulation of any starch synthesis precursor genes (Figure 5-5). However, glucogenesis pathway genes, trehalose metabolism, and sucrose synthesis genes were significantly overexpressed (Table 5-7). Roots collected from the symptomatic VAL/CAN combination showed an abundant number of transcripts encoding cell wall modification enzymes such as PMEs, BETA XYLOSIDASE 1 (BXL1) (Figure 5-2). Roots from symptomatic VAL/CAN combination showed upregulation of nitrate, iron and phosphate transporters: NRT1.1, NRT2.7, NRT1:2, Fe carriers, IRON-REGULATED 2 (IREG2), FER-like regulated iron uptake, FERRIC OXIDASE 7 (FRO7), and Phosphate transporters: PHT1;3, PHT1;7; PHT2;1 (Table 5-7). In symptomatic VAL/CAN, transition metal carriers NRAMP6, 196

197 HMA5, organic cation/carnitine transporter OCT1, METAL TOLERANCE PROTEIN (MTP), and CATION EFFLUX EXCHANGER (CAX) transcripts were also upregulated. Other transporter genes upregulated in the symptomatic VAL/CAN roots encoded heavy metal detoxification superfamily proteins, nodulin MtN21-like intrinsic protein, TONOPLAST INTRINSIC PROTEIN (TIP), oligopeptide sugar and mitochondrial substrate carriers (Table 5-7). Roots collected from the symptomatic treatment of VAL/SW showed upregulation of a few genes associated with root and shoot growth, including Au efflux carrier PIN4, Au response factor ARF19 and LAX1. Genes encoding homeobox like transcription factors that impact plant development and lateral root initiation such as HB13, HB7, HB8 and SHORT ROOTS (SHR) were significantly overexpressed in the symptomatic VAL/SW roots. In the symptomatic treatment, VAL/SW roots showed significant overexpression of transcripts encoding NAC domain containing XND1, NAC036, NAC042, NAC096, AINTGUMENTA-LIKE 5 (AIL5), SCR, and HAT22 TFs (Table 5-8). Roots collected from symptomatic VAL/SW combination, also, showed upregulation of amino acid and mitochondrial transmembrane carrier genes, and Nodlike 26 and plasma membrane major intrinsic protein (MIP) genes that are involved in water transport. Discussion Routine plant growth and defense trade-off is a matter of energy allocation. Plants generally do well in distributing energy under stress conditions. However, the burden of stress may disrupt the energy distribution balance between routine growth and defense. The complexity of CaLas-infection in citrus possibly leads to the imbalanced distribution of the energy resources in susceptible citrus scion/rootstock 197

198 combinations. In previous chapters, HLB-induced differentially regulated hormonal response genes and defense associated genes were discussed. This chapter discusses the qualitative and quantitative differential changes growth and development - associated genes in HLB asymptomatic and symptomatic VAL/CAN and VAL/SW combinations. HLB affected plants exhibit damaged vascular system which is caused by callose depositions and starch accumulation. The asymptomatic VAL/CAN leaves showed upregulated genes encoding TFs that are involved in phloem regeneration. They were KANADI and LBD2. Upregulation of transcripts encoding KANADI TF also reported in girdled Populus in the regenerating sieve elements (Zhang et al., 2011b; Hussey et al., 2013; Chen et al., 2014). Therefore, a significant increase in the KANADI encoded transcripts suggests that VAL/CAN plants possibly has increased phloem regeneration capacity in the asymptomatic CaLas-infected plants. Circadian rhythms maintain the plant biological clock. Transcription factors LHY, and REV1 are involved in regulating circadian clock regulation (Schaffer et al., 1998; Rawat et al., 2009). Transcriptomics studies of CaLas-infected citrus varieties reported a reduction in the LHY transcripts expression at the late stage of infection in sweet orange (Fu et al., 2016), Ponkan (Zhong et al., 2016), and Rough lemon (Fan et al., 2012). The growth regulating factor (GRF) and growth interacting factor (GIF) duo have a key role in plant organogenesis (Kim and Tsukaya, 2015). Of these, GRF7 is a negative regulator of osmotic stressresponsive genes that generally inhibit plant growth. The role of GRF7 has been studied in ABA-induced osmotic stress. ABA-induced drought stress upregulates dehydrationresponsive element binding protein 2A (DREB2A), which increases tolerance to osmotic 198

199 stress and retards plant growth. GRF7 binds to the DREB2A promoter region and therby, downregulates DREB2A induced growth and reproduction inhibition under normal environmental conditions (Kim et al., 2012). Upregulation of GRF7 transcripts in the leaves of asymptomatic VAL/CAN suggests the stimulation of growth in the asymptomatic VAL/CAN leaves. In asymptomatic treatment, VAL/SW leaves upregulated ABA biosynthesis genes, as discussed in the results presented in Chapter 4. This indicates the drought-like or abiotic stress conditions created in the CaLasinfected plants that increase expression of dehydration related and dehydration adaptation associated genes. Upregulation of transcripts encoding KANADI, LHY, GRF7 in the asymptomatic and REV1 in the asymptomatic leaves of VAL/CAN combinations highlights the CAN and SW rootstock induced growth changes in the HLB-affected VAL scion. In both symptomatic and asymptomatic treatments, VAL/CAN leaves showed significant upregulation of flavonoid biosynthesis, anthocyanin production, PMEs, and XTH33 transcripts. The role of PME (Pelloux et al., 2007) and XTH (Divol et al., 2007) has been well studied in plant-herbivore interactions. PMEs are, also, actively involved in the cell wall modification and expansions during plant growth (Pelletier et al., 2014). Vascular-related NAC domain 1 VND1 is involved in cell wall biosynthesis (Zhou et al., 2014), and this gene was upregulated only in the symptomatic VAL/CAN combination. Upregulation of cell wall modification related genes in both treatments of VAL/CAN leaves as compare to VAL/SW leaves, suggests the potential involvement of a cell wall modification mechanism to defend the CaLas-infection and Psyllid-induced wounding in the VAL/CAN combination. 199

200 Ethylene induced-ap2 TFs and NAC domain containing proteins are implicated in plant growth and stress response, and genes encoding these proteins were upregulated in the asymptomatic VAL/SW leaf combination. NAC domain proteins are implicated in plant senescence, stress, and cell wall biosynthesis (Wang et al., 2011; Nuruzzaman et al., 2013; Podzimska-Sroka et al., 2015). NAC090 transcripts were highly upregulated in the asymptomatic VAL/SW leaves. Although the NAC090 specific biological function has not yet been studied, overexpression of transcripts encoding ETactivated TFs and NAC genes supports the activation of the biotic stress response, leaf senescence and cell wall modifications in both the asymptomatic and symptomatic VAL/SW leaves. Hormone-induced, growth-associated genes were differentially expressed in VAL/CAN, and VAL/SW leaves. Plant growth hormones BR, CT and AU signaling pathways were strongly stimulated in the asymptomatic VAL by CAN rootstock in CaLas infected plants. AU and BR have been reported to work synergistically in plant developmental processes such as stem elongation and expansion (Nemhauser et al., 2004). Although AUs are involved in the plant susceptibility to the disease, they are indispensable plant growth hormones which promote plant development through their responsive genes. AU promotes plant growth through their ARF responsive genes (Liscum and Reed, 2002; Li et al., 2016). Also, interdependency of BR and AU signaling operates via ARFs, as reported in A. thaliana (Nemhauser et al., 2004). The upregulated ARF genes identified in this study, ARF8 and ARF16, are implied in plant parthenocarpy (Goetz et al., 2007) and control of seed germination (Liu et al., 2013). Upregulation of ARF8 was also reported in the regenerating phloem in girdled Populus 200

201 (Zhang et al., 2011b). Therefore, despite the involvement of ARF8 in the fruit set, a significant overexpression of the ARF8 in the symptomatic and asymptomatic VAL/CAN leaves suggests a possible role in phloem regeneration specific to the CAN rootstock. In HLB -asymptomatic and -symptomatic VAL/SW combinations, SW rootstock strongly upregulated ABA, AU, and ET-regulated genes expression. However, the overexpression of ET and ABA along with AU biosynthesis genes appeared to be the negative effect of CaLas infection interaction rather than a benefit to plant development. A key factor that severely reduces the sustainability of CaLas-infected plants is the deficiency of essential micronutrients (Schumann and Spann, 2009), and imbalanced sink-source partitioning (Fan et al., 2010). Leaves of HLB-affected citrus plants have shown an increase in the K while Ca, B, Fe, Zn, Mn, and Mg are reduced significantly (Schumann and Spann, 2009). The same deficiencies are even greater in roots of infected trees (J.W. Grosser, personal communication). Zn deficiency-like symptoms are typical of the HLB-affected plants. Plants generally increase the uptake and the transporter s activity of deficient nutrients. The overexpression of ZIP5 precursor was reported in the HLB-susceptible Cleopatra rootstock (Albrecht and Bowman, 2012b). The increased expression of Zn efflux transporter ZIP1 and ZIP4 genes in the symptomatic VAL/SW combination suggests that plant experienced Zn deficiency. Therefore, to obtain Zn either from soil or storage organelles, ZIP transporter genes expression was enhanced. The role of ZIP family proteins in the Zn homeostasis is explained in many plant nutrients studies (Sinclair and Krämer, 2012; Assunção et al., 2013; Bashir et al., 2016). Cu transporter genes such as HMA1 and COPT1 were also overexpressed in the leaves collected from the symptomatic VAL/SW. The Cu 201

202 transporters are present on the chloroplast envelope and play a significant role in photosynthetic electron transport (Grotz and Guerinot, 2006). Increased expression of chloroplast localized metal transporter transcripts suggests enhanced chloroplast activity in the symptomatic VAL/SW leaves. Phosphorus deficiency is also prominent in HLB-affected plants, and occurs because of the interference of small RNAs (Zhao et al., 2013). Phosphorus starvation-induced P transporters PHT 2;1 (asymptomatic and symptomatic leaves) and PHT1;3 and PHT1;7 (symptomatic roots) transcripts were upregulated in the VAL/CAN as compared to VAL/SW in the respective treatments and tissues. Whereas, VAL/SW combination showed upregulation of PHT1;4 and PHT1;9 in asymptomatic leaves as compared to asymptomatic VAL/CAN leaves. Excessive starch biosynthesis is a signature of HLB-affected plants (Etxeberria et al., 2009). Enhanced production of transcripts encoding starch biosynthesis AGPase, starch branching SBE2.1 and SBE2.2, starch excess 1 (SEX1) was seen in the symptomatic VAL/SW leaves as compared to symptomatic VAL/CAN leaves. However, starch degrading enzyme and invertase genes upregulation was observed only in the asymptomatic VAL/SW leaves. The pattern of starch biosynthesis and catabolism gene transcripts in the VAL/SW combination supports the typical behavior of the HLBsusceptible cultivars. Trehalose is a common sugar in lower organisms such as bacteria, fungi, and invertebrates, but also accumulates in plants when they are infected with micro-organisms. The role of trehalose in the plant is still uncertain. However, it is predicted that trehalose may interfere with sugar sensing and plant development (Müller et al., 1999). Overexpression of trehalose synthesis genes in the asymptomatic VAL/SW combination suggests a higher CaLas activity in the infected rootstock. While 202

203 the asymptomatic VAL/CAN combination showed upregulation of genes encoding starch degrading enzymes, vacuolar invertases, and raffinose synthesis and MIPS biosynthesis proteins highlighted the genetic response of CAN rootstock to avoid starch accumulations and utilization of other sugar resources. Roots obtained from the asymptomatic treatment of VAL/CAN and VAL/SW showed very few differentially expressed genes. Among these, strong upregulation of genes associated with the light reaction system of chloroplasts was observed in the asymptomatic VAL/SW roots. In contrast, many genes were differentially expressed in the roots collected from the symptomatic treatment of the VAL/CAN and VAL/SW. At the advanced stage of infection, CAN roots showed significant upregulation of BR and AU response genes. Whereas, SW roots were found to enhance the expression of genes that increase lateral root spread. These genes were AU efflux carrier PIN4, SHORT ROOTS (SHR), SCR, and WUSCHEL related WOX4. HLB-affected SW rootstock show decreased root growth. Increased expression of lateral root growth associated genes suggests the SW is promoting root growth to reach out to the nutrient resources available in the soil. Higher expression of the HD-zip II family HAT22 gene in the SW roots supports the theory of reduced root growth and the ABA-dependent drought response in the HLB affected plants (Huang et al., 2008). Roots collected from the symptomatic treatment of VAL/CAN combination showed upregulated genes encoding phloem regenerating KANADI TF that was also upregulated in the leaves of the VAL/CAN. Upregulation of KANADI in the roots and leaves of the VAL/CAN combination indicates phloem regeneration ability is specific to the putative HLB-tolerant CAN rootstock. Phytosulfokine-α is a signaling peptide and 203

204 acts as a root growth factor in Arabidopsis suspension cell culture (Kutschmar et al., 2009). In the absence of any other significant root promoting factors, phytosulfokines precursor 3 and 4 may support enhanced root growth in the CaLas-infected CAN rootstock. Nitrate and sulfur transporters, major intrinsic protein, and nodulin-like NIP transporter transcripts were downregulated in the CaLas-infected roots of SW. However, the transcripts encoding nitrate transporters, sulfate transmembrane transporters, calcium exchanger (CAX), water and sugar transporter activities were upregulated in roots of the symptomatic VAL/CAN combination. In addition, transcripts for a mitochondrial carrier BOU that is involved in plant meristem growth and a mitochondrial substrate carrier were also upregulated in the roots of the symptomatic VAL/CAN combination, suggesting upregulated mitochondrial activity in the CaLasinfected VAL/CAN plants. The upregulation of N, S, Ca, sugar, and water transporter transcripts in the CAN rootstock as compared to the SW rootstock suggests a possible increased root growth of the CAN rootstock under CaLas-infection. Upregulation of transition metal transporter genes in the symptomatic VAL/CAN roots suggests that the infected plant is trying to achieve homeostasis by effluxing nutrients from the storage organelles (Hall and Williams, 2003; Krämer et al., 2007). Plants under stress often exhibit compromised growth. Optimizing the balance and allocation of energy between routine growth and defense is critical when the plant is consistently under stress. CaLas-infected citrus plants exhibit a similar scenario, and it appears that the susceptible SW rootstock partitions too much energy to defense at the expense of energy needed for adequate nutrition management. Use of HLB-tolerant rootstock cultivars and optimal nutrition management may be the key to prolonging the 204

205 economic viability of CaLas-infected plants. The study of comparative transcriptomic analysis between VAL/CAN and VAL/SW in symptomatic and asymptomatic treatments showed that rootstock has potential to reprogram the scion transcriptome at different stages of HLB disease development. The changes in the scion are not only manipulated based on rootstock specific genetics, but also the stage of HLB disease development in the scion. The results of this study suggest that enhanced VAL performance in the infected plants is probably due to a combination of phloem regeneration ability, nutrient homeostasis, and BR-AU dependent plant development induced by the putative HLBtolerant CAN rootstock. 205

206 Table 5-1. Experimental treatments and scion/rootstock combinations Treatments based on visual Rootstock Rootstock Parents Scion observations of HLB symptoms Swingle; 2n (SW) putative HLBtolerant candidate; 2n (CAN) Grapefruit X Trifoliate orange HBP Pummelo X Cleopatra Mandarin Valencia sweet orange (VAL) Valencia sweet orange (VAL) Symptomatic VAL/SW Slightly Symptomatic VAL/CAN Asymptomatic VAL/SW Asymptomatic VAL/CAN Approximately 7- year old trees planted in the Lee Family s Alligator Grove east of St. Cloud, FL. Table 5-2. Comparison pairs used for differential gene expression analysis in leaves and roots of the experimental scion/rootstock combinations Leaves Roots Asymptomatic VAL/SW vs. Asymptomatic Asymptomatic VAL/SW vs. Asymptomatic VAL/CAN VAL/CAN Symptomatic VAL/SW vs. Symptomatic Symptomatic VAL/SW vs. Symptomatic VAL/CAN VAL/CAN 206

207 Table 5-3. Differentially expressed growth and development -associated genes significantly upregulated in leaves of asymptomatic VAL/CAN combination as compared to asymptomatic VAL/SW leaves C. Clementina_ID Arabi ID Gene name Bin Name Log2 FC Arabidopsis_define Ciclev m.g AT2G13610 T10F5.15 transport.abc transporters and multidrugresistance systems ABC-2 type transporter family protein Ciclev m.g AT2G40460 T2P4.19 transport.peptides and oligopeptides Major facilitator superfamily protein Ciclev m.g AT1G01060 LHY RNA. regulation of transcription. MYBrelated transcription factor family Homeodomain-like superfamily protein Ciclev m.g AT5G23730 MRO11.23 development.unspecified Transducin/WD40 repeat-like superfamily protein Ciclev m.g AT5G06710 HAT14 development. unspecified homeobox from Arabidopsis thaliana Ciclev m.g AT1G64780 AMT1;2 transport. ammonium ammonium transporter 1;2 Ciclev m.g AT3G14770 AT3G14770 development. unspecified Nodulin MtN3 family protein Ciclev m.g AT5G13930 TT4 Ciclev m.g AT2G22240 MIPS2 Ciclev m.g AT1G32240 KAN2 secondary metabolism. flavonoids. chalcones.naringenin-chalcone synthase minor CHO metabolism.myoinositol.insp Synthases RNA. regulation of transcription. G2- like transcription factor family, GARP Chalcone and stilbene synthase family protein myo-inositol-1-phosphate synthase Homeodomain-like superfamily protein Ciclev m.g AT2G43330 INT1 transporter. sugars inositol transporter 1 Ciclev m.g AT1G21460 F24J8.9 development. unspecified Nodulin MtN3 family protein Ciclev m.g AT2G45190 AFO development. unspecified Plant-specific transcription factor YABBY family protein Ciclev m.g AT1G59740 F23H11.6 transport. peptides and oligopeptides Major facilitator superfamily protein Ciclev m.g AT2G26650 AKT1 transport. potassium K+ transporter 1 secondary metabolism.flavonoids. Ciclev m.g AT3G29670 AT3G29670 anthocyanins. HXXXD-type acyl-transferase family anthocyanin 5-aromatic protein acyltransferase Ciclev m.g AT1G24100 UGT74B1 secondary metabolism.sulfurcontaining.glucosinolates.synthesis UDP-glucosyl transferase 74B1 Ciclev m.g AT1G61680 TPS14 secondary metabolism terpene synthase 14 isoprenoids.terpenoids Ciclev m.g AT1G02630 T14P4.9 transport.unspecified cations Nucleoside transporter family protein Ciclev m.g AT4G36710 AP22.56 development. unspecified GRAS family transcription factor Ciclev m.g AT1G78020 F28K19.24 development Protein of unknown function (DUF581) 207

208 Table 5-3. Continued C. Clementina_ID Arabi ID Gene Log2 Bin Name name FC Arabidopsis_definition Ciclev m.g AT3G12750 ZIP1 transport.metal zinc transporter 1 precursor Ciclev m.g AT3G02380 COL2 development.unspecified CONSTANS-like 2 Ciclev m.g AT5G15410 DND1 transport.cyclic nucleotide or Cyclic nucleotide-regulated ion calcium regulated channels channel family protein Ciclev m.g AT2G02450 NAC035 development.unspecified NAC domain containing protein 35 Ciclev m.g AT1G27940 PGP13 transport.abc transporters and multidrugresistance systems P-glycoprotein 13 Ciclev m.g AT4G36920 AP2 development.unspecified Integrase-type DNA-binding superfamily protein Ciclev m.g AT1G12240 BETA- major CHO metabolism. Glycosyl hydrolases family FRUCT4 degradation.sucrose.invertases.vacuolar protein Ciclev m.g AT2G30300 T9D9.11 development.unspecified Major facilitator superfamily protein Ciclev m.g AT4G01840 KCO5 transport.potassium Ca2+ activated outward rectifying K+ channel 5 Ciclev m.g AT1G12110 NRT1.1 transport.nitrate nitrate transporter 1.1 Ciclev m.g AT2G28260 CNGC15 transport.cyclic nucleotide or calcium regulated channels cyclic nucleotide-gated channel 15 Ciclev m.g AT1G18590 SOT17 secondary metabolism. sulfur-containing. glucosinolates sulfotransferase 17 Ciclev m.g AT5G37820 NIP4;2 transport.major Intrinsic Proteins.unspecified NOD26-like intrinsic protein 4;2 Ciclev m.g AT2G28500 LBD11 RNA.regulation of transcription. AS2, Lateral Organ Boundaries Gene Family LOB domain-containing protein 11 Ciclev m.g AT1G12480 OZS1 transport. metabolite transporters at the C4-dicarboxylate transporter/malic mitochondrial membrane acid transport protein Ciclev m.g AT1G79360 OCT transport.misc organic cation/carnitine transporter 2 Ciclev m.g AT2G37360 F3G5.15 transport. ABC transporters and ABC-2 type transporter family multidrug resistance systems protein Ciclev m.g AT5G55180 MCO15.13 misc.beta 1,3 glucan hydrolases O-Glycosyl hydrolases family 17 protein Ciclev m.g AT2G37040 PAL1 secondary metabolism. phenylpropanoids. lignin biosynthesis.pal PHE ammonia lyase 1 Ciclev m.g AT1G10960 FD1 PS.lightreaction.other electron carrier (ox/red).ferredoxin ferredoxin 1 Ciclev m.g AT2G40610 EXPA8 cell wall. modification expansin A8 208

209 Table 5-3. Continued C. Clementina_ID Arabi ID Gene name Bin Name Log2 FC Arabidopsis_definition Ciclev m.g AT5G12860 DiT1 transport.metabolite transporters at the dicarboxylate mitochondrial membrane transporter 1 Ciclev m.g AT3G29590 AT5MAT secondary metabolism.flavonoids.anthocyanins. anthocyanin 5-aromatic acyltransferase HXXXD-type acyltransferase family protein Ciclev m.g AT3G58060 AT3G58060 transport.metal Cation efflux family protein Ciclev m.g AT3G13620 AT3G13620 transport.amino acids Amino acid permease Ciclev m.g AT5G60660 PIP2;4 transport.major Intrinsic Proteins.PIP Ciclev m.g AT3G51240 F3H secondary metabolism.flavonoids. dihydroflavonols. flavanone 3-dioxygenase Ciclev m.g AT4G01070 GT72B1 secondary metabolism.flavonoids.dihydroflavonols family protein plasma membrane intrinsic protein 2;4 flavanone 3- hydroxylase UDP- Glycosyltransferase superfamily protein Ciclev m.g AT1G10550 XTH33 cell wall.modification xyloglucan:xyloglucosyl transferase 33 Ciclev m.g AT2G39210 T16B24.15 development. unspecified Major facilitator superfamily protein Ciclev m.g AT3G54820 PIP2;5 transport.major Intrinsic Proteins.PIP plasma membrane intrinsic protein 2;5 Ciclev m.g AT4G40060 HB16 RNA.regulation of transcription.hb,homeobox transcription factor family Ciclev m.g AT4G24220 VEP1 development.unspecified Ciclev m.g AT1G68530 KCS6 secondary metabolism.wax Ciclev m.g AT4G33220 PME44 cell wall.pectin*esterases.misc Ciclev m.g AT5G54860 MBG8.12 transport. misc Classification of the measured parameter into a set a functional category in the MapMan analysis tool homeobox protein 16 NAD(P)-binding Rossmann-fold superfamily protein 3-ketoacyl-CoA synthase 6 pectin methylesterase 44 Major facilitator superfamily protein 209

210 Table 5-4. Differentially expressed growth and development-associated genes significantly upregulated in leaves of asymptomatic VAL/SW combination as compared to asymptomatic VAL/CAN leaves C. Clementina_ID Arabi ID Gene name Bin Name Log2 FC* Arabidopsis_define Ciclev m.g AT2G26560 PLA2A development. storage proteins phospholipase A 2A Ciclev m.g AT5G51990 CBF4 RNA. regulation of transcription. AP2/EREBP/Ethylene-responsive element C-repeat-binding factor 4 Ciclev m.g AT1G12060 BAG5 development.unspecified BCL-2-associated athanogene 5 Ciclev m.g AT4G31500 CYP83B1 secondary metabolism. glucosinolates. cytochrome P450, family 83, synthesis. shared. CYP83B1 subfamily B, polypeptide 1 Ciclev m.g AT4G38460 GGR secondary metabolism. geranylgeranyl pyrophosphate synthase geranylgeranyl reductase Ciclev m.g AT1G61800 GPT2 transport. metabolite transporters glucose-6-phosphate/phosphate at the envelope membrane translocator 2 Ciclev m.g AT5G22380 NAC090 development. unspecified NAC domain containing protein 90 Ciclev m.g AT1G53885 AT1G53903 development. unspecified Protein of unknown function (DUF581) Ciclev m.g AT3G06500 F5E6.17 major CHO metabolism Plant neutral invertase family degradation.sucrose.invertases.neutral protein Ciclev m.g AT5G41330 MYC6.4 transport. potassium BTB/POZ domain with WD40/YVTN repeat-like protein Ciclev m.g AT2G46330 AGP16 cell wall.cell wall proteins.agps arabinogalactan protein 16 Ciclev m.g AT3G13790 ATBFRUCT1 major CHO metabolism.degradation. Glycosyl hydrolases family sucrose.invertases.cell wall protein Ciclev m.g AT1G69490 NAP development. unspecified NAC-like, activated by AP3/PI Ciclev m.g AT1G07050 F10K1.24 development. unspecified CCT motif family protein Ciclev m.g AT1G61340 T1F9.17 development. unspecified F-box family protein Ciclev m.g AT5G14000 NAC084 Development. unspecified NAC domain containing protein 84 Ciclev m.g AT5G19120 T24G5.20 development. storage proteins Eukaryotic aspartyl protease family protein Ciclev m.g AT5G06760 LEA4-5 development. late embryogenesis Late Embryogenesis Abundant abundant 4-5 Ciclev m.g AT3G28960 AT3G28960 transport. amino acids Transmembrane amino acid transporter family protein Ciclev m.g AT1G23870 TPS9 minor CHO metabolism. trehalose. trehalose-phosphatase/synthase potential TPS/TPP 9 210

211 Table 5-4. Continued C. Clementina_ID Arabi ID Gene name Bin Name Log2 FC Arabidopsis_definition Ciclev m.g AT1G01720 ATAF1 development.unspecified Ciclev m.g AT3G02550 LBD41 RNA.regulation of transcription.as2,lateral Organ Boundaries Gene Family NAC (No Apical Meristem) domain transcriptional regulator superfamily protein LOB domain-containing protein 41 Ciclev m.g AT4G25810 XTR6 cell wall.modification xyloglucan endotransglycosylase 6 Ciclev m.g AT4G27410 RD26 development.unspecified NAC (No Apical Meristem) domain transcriptional regulator superfamily protein Ciclev m.g AT5G67150 K21H1.11 secondary metabolism HXXXD-type acyl-transferase family protein Ciclev m.g AT2G47180 GolS1 minor CHO metabolism.raffinose family.galactinol synthases.known galactinol synthase 1 Ciclev m.g AT4G40090 AGP3 cell wall.cell wall proteins.agps arabinogalactan protein 3 Ciclev m.g AT1G80300 NTT1 transport.misc nucleotide transporter 1 Ciclev m.g AT5G24520 TTG1 development.unspecified Transducin/WD40 repeat-like protein Ciclev m.g AT5G40780 LHT1 transport.amino acids lysine histidine transporter 1 Ciclev m.g AT2G36380 PDR6 transport.abc transporters and multidrugresistance systems pleiotropic drug resistance 6 Ciclev m.g AT4G15560 CLA1 secondary metabolism.isoprenoids. non-mevalonate Pathway.DXS Deoxyxylulose-5-phosphate synthase Ciclev m.g AT1G35910 F10O5.8 minor CHO metabolism.trehalose Ciclev m.g AT1G68840 RAV2 RNA.regulation of transcription.ap2/erebp, APETALA2/Ethylene-responsive element binding protein family Haloacid dehalogenase-like hydrolase (HAD) superfamily protein related to ABI3/VP1 2 Ciclev m.g AT4G31290 F8F transport.unspecified cations ChaC-like family protein Ciclev m.g AT1G20510 OPCL1 secondary metabolism.phenylpropanoids OPC-8:0 CoA ligase1 Ciclev m.g AT2G35980 YLS9 development.unspecified Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family Ciclev m.g AT3G03050 CSLD3 cell wall.cellulose synthesis.cellulose synthase cellulose synthase-like D3 Ciclev m.g AT4G37990 ELI3-2 secondary metabolism.phenylpropanoids.lignin biosynthesis.cad elicitor-activated gene

212 Table 5-4. Continued C. Clementina_ID Arabi ID Gene name Bin Name Log2 FC Arabidopsis_define Ciclev m.g AT2G18950 HPT1 secondary metabolism.isoprenoids. tocopherol biosynthesis homogentisate phytyltransferase 1 Ciclev m.g AT4G07960 CSLC12 cell wall.cellulose synthesis Cellulose-synthase-like C12 Ciclev m.g AT5G18840 F17K4.90 transporter.sugars Major facilitator superfamily protein Ciclev m.g AT1G02850 BGLU11 misc.gluco-, galacto- and mannosidases beta glucosidase 11 Ciclev m.g AT1G35190 T32G9.27 secondary metabolism.n misc.alkaloid-like oxoglutarate (2OG) and Fe(II)- dependent oxygenase superfamily protein Ciclev m.g AT3G05290 PNC1 transport.metabolite transporters at the peroxisomal adenine nucleotide mitochondrial membrane carrier 1 Ciclev m.g AT2G36950 T1J8.13 transport.misc Heavy metal transport/detoxification superfamily protein Ciclev m.g AT1G07530 SCL14 RNA.regulation of transcription. GRAS transcription factor family SCARECROW-like 14 Ciclev m.g AT5G13790 AGL15 development.unspecified AGAMOUS-like 15 Ciclev m.g AT2G27500 F10A12.18 misc.beta 1,3 glucan hydrolases.glucan Glycosyl hydrolase superfamily endo-1,3-beta-glucosidase protein Ciclev m.g AT5G46050 PTR3 transport.peptides and oligopeptides peptide transporter 3 Ciclev m.g AT4G34950 AT4G34950 development.unspecified Major facilitator superfamily protein Ciclev m.g AT2G22500 UCP5 transport.metabolite transporters at the mitochondrial membrane uncoupling protein 5 Ciclev m.g AT1G06410 TPS7 minor CHO metabolism.trehalose.potential TPS/TPP trehalose-phosphatase/synthase 7 Ciclev m.g AT1G01470 LEA14 development.late embryogenesis abundant Late embryogenesis abundant protein Ciclev m.g AT1G76430 PHT1;9 transport.phosphate phosphate transporter 1;9 Ciclev m.g AT1G77380 AAP3 transport.amino acids amino acid permease 3 Ciclev m.g AT2G23810 TET8 development. unspecified tetraspanin8 Ciclev m.g AT1G64760 F13O11.7 Misc. beta 1,3 glucan hydrolases. O-Glycosyl hydrolases family glucan endo-1,3-beta-glucosidase protein Ciclev m.g AT5G61430 NAC100 development. unspecified NAC domain containing protein 100 Ciclev m.g AT4G08250 T12G13.90 development. unspecified GRAS family transcription factor Ciclev m.g AT1G13980 GN development.unspecified sec7 domain-containing protein Ciclev m.g AT1G03940 F21M11.13 secondary metabolism.flavonoids. anthocyanins HXXXD-type acyl-transferase family protein 212

213 Table 5-4. Continued C. Clementina_ID Arabi ID Name Bin Name Log2 FC Arabidopsis_definition Ciclev m.g AT5G36160 MAB16.11 secondary metabolism.isoprenoids.tocopherol Tyrosine transaminase biosynthesis family protein Ciclev m.g AT4G17230 SCL13 development.unspecified SCARECROW-like 13 Ciclev m.g AT5G54160 OMT1 secondary metabolism.phenylpropanoids.lignin biosynthesis.comt O-methyltransferase 1 Ciclev m.g AT3G49530 NAC062 development.unspecified NAC domain containing protein 62 Ciclev m.g AT5G26340 MSS1 transporter.sugars Major facilitator superfamily protein Ciclev m.g AT3G18080 BGLU44 misc.gluco-, galacto- and mannosidases B-S glucosidase 44 Ciclev m.g AT1G78950 AT1G78950 secondary metabolism.isoprenoids.terpenoids Terpenoid cyclases family protein Ciclev m.g AT1G59870 PEN3 ABC-2 and Plant PDR transport.abc transporters and ABC-type transporter multidrugresistance systems family protein Ciclev m.g AT4G26140 BGAL12 misc.gluco-, galacto- and mannosidases.betagalactosidase beta-galactosidase 12 Ciclev m.g AT1G29690 CAD1 development.unspecified MAC/Perforin domain- Ciclev m.g AT1G10760 SEX1 Ciclev m.g AT3G21250 MRP6 Ciclev m.g AT1G07640 OBP2 major CHO metabolism.degradation.starch.glucan water dikinase transport.abc transporters and multidrug resistance systems secondary metabolism. sulfurcontaining.glucosinolates.regulation.indole Ciclev m.g AT1G08200 AXS2 cell wall. precursor synthesis.axs Ciclev m.g AT2G18700 TPS11 minor CHO metabolism. trehalose.potential TPS/TPP containing protein Pyruvate phosphate dikinase, PEP/pyruvate binding domain multidrug resistanceassociated protein 6 Dof-type zinc finger DNAbinding family protein UDP-D-apiose/UDP-Dxylose synthase 2 trehalose phosphatase/synthase 11 * The negative sign in the column of Log2 FC indicates comparative downregulation of the gene expression level in VAL/CAN and upregulation in VAL/SW combination. Classification of the measured parameter into a set a functional category in the MapMan analysis tool. 213

214 Table 5-5. Differentially expressed growth and development -associated genes significantly upregulated in leaves of the symptomatic VAL/CAN combination as compared to symptomatic VAL/SW leaves C. Clementina_ID Arabi ID Name Bin Name Log2 FC Arabidopsis_definition Ciclev m.g AT1G01060 LHY RNA. regulation of transcription. MYBrelated transcription factor family protein Homeodomain-like superfamily Ciclev m.g AT2G22240 MIPS2 minor CHO metabolism.myo-inositol.insp myo-inositol-1-phosphate synthase Synthases 2 Ciclev m.g AT2G13610 T10F5.15 transport.abc transporters and ABC-2 type transporter family multidrugresistance systems protein Ciclev m.g AT2G03710 SEP-4 RNA.regulation of transcription.mads K-box region and MADS-box box transcription factor family transcription factor family protein Ciclev m.g AT1G73830 BEE3 RNA.regulation of transcription. bhlh,basic Helix-Loop-Helix family BR enhanced expression 3 Ciclev m.g AT4G35580 NTL9 development NAC transcription factor-like 9 Ciclev m.g AT3G02380 COL2 development CONSTANS-like 2 Ciclev m.g AT2G37040 PAL1 secondary metabolism.phenylpropanoids.lignin biosynthesis.pal PHE ammonia lyase 1 Ciclev m.g AT4G11080 T22B4.60 RNA.regulation of transcription.high HMG (high mobility group) box mobility Group (HMG) family protein Ciclev m.g AT1G24625 ZFP7 RNA.regulation of transcription.c2h2 zinc finger family zinc finger protein 7 Ciclev m.g AT2G40460 T2P4.19 transport.peptides and oligopeptides Major facilitator superfamily protein Ciclev m.g AT2G22800 HAT9 RNA.regulation of Homeobox-leucine zipper protein transcription.hb,homeobox transcription family factor family Ciclev m.g AT1G04770 F13M7.24 development Tetratricopeptide repeat (TPR)-like superfamily protein Ciclev m.g AT1G23200 F26F24.2 cell wall.pectin*esterases.misc Plant invertase/pectin methylesterase inhibitor superfamily Ciclev m.g AT5G46800 BOU transport.metabolite transporters AT the Mitochondrial substrate carrier mitochondrial membrane family protein Ciclev m.g AT1G61820 BGLU46 misc.gluco-, Galacto- and mannosidases beta glucosidase 46 Ciclev m.g AT2G18060 VND1 development vascular related NAC-domain protein 1 Ciclev m.g AT2G44940 T13E Integrase-type DNA-binding superfamily protein 214

215 Table 5-5. Continued C. Clementina_ID Arabi ID Name Bin Name Ciclev m.g AT3G60160 MRP9 Ciclev m.g AT4G34050 CCoAOMT1 Ciclev m.g AT5G13080 WRKY75 Ciclev m.g AT1G16070 TLP8 Ciclev m.g AT1G CL3 transport. ABC transporters and multidrug resistance systems secondary metabolism.phenylpropanoids lignin biosynthesis. CCoAOMT RNA.regulation of transcription. WRKY domain transcription factorfamily RNA.regulation of transcription.tub transcription factor family secondary metabolism.lignin biosynthesis.4cl Log2 FC Ciclev m.g AT5G13870 XTH5 cell wall.modification Arabidopsis_definition multidrug resistance-associated protein 9 S-adenosyl-L-methionine-dependent methyltransferases superfamily WRKY DNA-binding protein tubby like protein coumarate:CoA ligase 3 xyloglucan endotransglucosylase/hydrolase 5 Ciclev m.g AT2G26690 F18A8.6 transport. nitrate Major facilitator superfamily protein Ciclev m.g AT1G17140 ICR1 development interactor of constitutive active rops 1 Ciclev m.g AT1G02730 CSLD5 cell wall.cellulose synthesis cellulose synthase-like D5 Ciclev m.g AT4G19420 T5K cell wall.pectin*esterases.acetyl esterase Pectinacetylesterase family protein Ciclev m.g AT2G26650 AKT1 transport.potassium K+ transporter 1 Ciclev m.g AT2G28500 LBD11 RNA.regulation of transcription. AS2,Lateral Organ Boundaries LOB domain-containing protein 11 Gene Family Ciclev m.g AT5G53660 GRF growth-regulating factor 7 Ciclev m.g AT2G21650 MEE3 RNA.regulation of transcription. MYB-related transcription factor family Homeodomain-like superfamily protein Ciclev m.g AT3G19450 CAD4 secondary metabolism. phenylpropanoids. GroES-like zinc-binding alcohol lignin biosynthesis.cad dehydrogenase family protein secondary metabolism Ciclev m.g AT3G CL2.phenylpropanoids coumarate: CoA ligase 2 lignin biosynthesis.4cl Ciclev m.g AT5G23420 HMGB6 RNA.regulation of transcription Nucleosome/chromatin assembly group high-mobility group box 6 Ciclev m.g AT2G26910 PDR4 transport.abc transporters and multidrugresistance systems pleiotropic drug resistance 4 Ciclev m.g AT3G61430 PIP1A transport.major Intrinsic Proteins plasma membrane intrinsic protein 1A Ciclev m.g AT3G15540 IAA19 RNA.regulation Aux/IAA family indole-3-acetic acid inducible

216 Table 5-5. Continued C. Clementina_ID Arabi ID Name Bin Name Ciclev m.g AT5G07050 MOJ9.22 development Log2 FC Arabidopsis_definition nodulin MtN21 /EamA-like transporter family protein Ciclev m.g AT4G09650 ATPD PS.lightreaction. ATP synthase. delta chain ATP synthase delta-subunit gene Ciclev m.g AT2G43330 INT1 transporter. sugars inositol transporter 1 Ciclev m.g AT2G05160 F5G3.6 RNA.regulation of transcription CCCH-type zinc finger family protein with RNA-binding domain Ciclev m.g AT2G28470 BGAL8 misc.gluco-, Galacto- and mannosidases. beta-galactosidase beta-galactosidase 8 Ciclev m.g AT5G65730 XTH6 cell wall.modification xyloglucan endotransglucosylase/hydrolase 6 Ciclev m.g AT5G03760 CSLA09 cell wall. cellulose synthesis Nucleotide-diphospho-sugar transferases superfamily protein Ciclev m.g AT4G00370 ANTR2 transporter.sugars Major facilitator superfamily protein Ciclev m.g AT3G19620 AT3G19620 cell wall. degradation. mannan-xylose-arabinose-fucose Glycosyl hydrolase family protein Ciclev m.g AT1G60950 FED A PS.lightreaction. other electron carrier (ox/red).ferredoxin Fe-2S ferredoxin-like superfamily protein Ciclev m.g AT4G14550 IAA14 RNA.regulation of transcription. Aux/IAA family indole-3-acetic acid inducible 14 Ciclev m.g AT2G21050 LAX2 transport. amino acids like AUXIN RESISTANT 2 Ciclev m.g AT2G32540 CSLB04 cell wall. cellulose synthesis. cellulose synthase cellulose synthase-like B4 Ciclev m.g AT1G58370 RXF12 cell wall. degradation. glycosyl hydrolase family10 protein / mannan-xylose-arabinose-fucose carbohydrate-binding domain-containing protein secondary metabolism. Ciclev m.g AT4G39330 CAD9 phenylpropanoids. lignin biosynthesis. CAD cinnamyl alcohol dehydrogenase 9 Ciclev m.g AT1G30690 T5I8.14 transport. misc Ciclev m.g HCT hydroxycinnamoyl-coa shikimate/quinate hydroxycinnamoyl transferase Sec14p-like phosphatidylinositol transfer family protein lignin bisynthesis, flavonoid accumulation/auxin tranport Ciclev m.g AT5G14570 NRT2.7 transport. nitrate high affinity nitrate transporter 2.7 Ciclev m.g AT5G43700 AtAUX

217 Table 5-5. Continued C. Clementina_ID Arabi ID Name Bin Name Log2 FC Arabidopsis_definition Ciclev m.g AT5G53370 PME cell wall.pectin*esterases.misc pectin methylesterase PCR fragment F Ciclev m.g AT1G51400 F5D21.10 PS. Light reaction. photosystem II.PSII polypeptide subunits Photosystem II 5 kd protein Ciclev m.g AT2G30570 PSBW PS. Light reaction. photosystem II.PSII polypeptide subunits photosystem II reaction center W Ciclev m.g AT3G09600 AT3G09610 RNA.regulation of transcription.mybrelated transcription factor family Homeodomain-like superfamily protein Ciclev m.g AT3G18080 BGLU44 misc.gluco-, Galacto- and mannosidases B-S glucosidase 44 Ciclev m.g AT3G61950 AT3G61950 RNA.regulation of basic helix-loop-helix (bhlh) DNA-binding transcription.bhlh,basic Helix-Loop- superfamily protein Ciclev m.g AT1G21460 F24J8.9 development Nodulin MtN3 family protein Ciclev m.g AT1G11580 PMEPCRA cell wall.pectin*esterases.pme methylesterase PCR A Ciclev m.g AT1G75880 T4O12.12 development SGNH hydrolase-type esterase superfamily Ciclev m.g AT3G18660 PGSIP1 major CHO plant glycogenin-like starch initiation protein metabolism.synthesis.starch 1 Ciclev m.g AT1G78060 F28K19.27 cell wall.degradation. mannan-xylose-arabinose-fucose Glycosyl hydrolase family protein Ciclev m.g AT2G26500 T9J22.17 PS.lightreaction.cytochrome b6/f cytochrome b6f complex subunit (petm), putative Ciclev m.g AT5G65380 MNA5.11 development MATE efflux family protein Ciclev m.g AT4G38960 F19H22.60 RNA. regulation of transcription.c2c2(zn) CO-like, Constans-like zinc finger family Ciclev m.g AT5G23730 MRO11.23 development B-box type zinc finger family protein Transducin/WD40 repeat-like superfamily protein Ciclev m.g AT1G66330 T27F4.8 development senescence-associated family protein Ciclev m.g AT5G37820 NIP4;2 transport. Major Intrinsic Proteins NOD26-like intrinsic protein 4;2 Ciclev m.g AT4G28500 NAC073 development NAC domain containing protein 73 Ciclev m.g AT3G53720 CHX20 transport. metal cation/h+ exchanger 20 Ciclev m.g AT1G69580 F24J1.30 RNA. regulation of transcription. G2- like transcription factor family, GARP Homeodomain-like superfamily protein Ciclev m.g AT5G49330 MYB111 RNA.regulation of transcription.myb domain transcription factor family myb domain protein

218 Table 5-5. Continued C. Clementina_ID Arabi ID Name Bin Name Log2 FC Arabidopsis_definition Ciclev m.g AT5G10770 T30N20.40 RNA.regulation of transcription Eukaryotic aspartyl protease family protein Ciclev m.g AT3G14770 AT3G14770 development Nodulin MtN3 family protein Ciclev m.g AT4G28660 PSB28 PS.liGhtreaction.photosystem photosystem II reaction center II.PSII polypeptide subunits PSB28 protein Ciclev m.g AT5G14880 T9L3.180 transport.potassium Potassium transporter family protein Ciclev m.g AT4G15920 DL4000C development Nodulin MtN3 family protein Ciclev m.g AT1G10470 ARR4 RNA. regulation of transcription. ARR response regulator 4 Ciclev m.g AT2G37630 AS1 RNA.regulation of myb-like HTH transcriptional transcription.myb domain regulator family protein transcription factor family. Classification of the measured parameter into a set a functional category in the MapMan analysis tool. 218

219 Table 5-6. Differentially expressed growth and development-associated genes significantly upregulated in leaves of the symptomatic VAL/SW combination as compared to symptomatic VAL/CAN leaves C. Clementina_ID Arabi ID Gene name Bin Name Log2 FC* Arabidopsis_define Ciclev m.g AT1G02850 BGLU11 misc.gluco-, Galacto- and mannosidases beta glucosidase 11 Ciclev m.g AT3G12750 ZIP1 transport. metal zinc transporter 1 precursor Ciclev m.g AT3G13080 MRP3 transport. ABC transporters and multidrug resistance-associated multidrug resistance systems protein 3 Ciclev m.g AT5G53130 CNGC1 transport. cyclic nucleotide or calcium regulated channels cyclic nucleotide gated channel 1 Ciclev m.g AT4G35160 T12J5.30 secondary metabolism. phenylpropanoids O-methyltransferase family protein Ciclev m.g AT1G69530 EXPA1 cell wall.modification expansin A1 Ciclev m.g AT1G61800 GPT2 transport.metabolite transporters at the glucose-6-phosphate/phosphate envelope membrane translocator 2 Ciclev m.g AT4G34950 AT4G34950 development Major facilitator superfamily protein Ciclev m.g AT3G52450 PUB22 RNA. regulation of transcription. PHOR plant U-box 22 Ciclev m.g AT4G02620 VHA -F transport.p- and v-atpases. vacuolar ATPase subunit F family H+-transportinG two-sector ATPase protein Ciclev m.g AT5G37490 MPA22.3 RNA. regulation of transcription. PHOR ARM repeat superfamily protein Ciclev m.g AT1G53885 AT1G53903 development Protein of unknown function (DUF581) Ciclev m.g AT1G80840 WRKY40 RNA.regulation of transcription. WRKY domain transcription factor family WRKY DNA-binding protein 40 Ciclev m.g AT5G54160 OMT1 secondary metabolism.phenylpropanoids. lignin biosynthesis.comt O-methyltransferase 1 Ciclev m.g AT1G07050 F10K1.24 development CCT motif family protein Ciclev m.g AT2G37060 NF-YB8 RNA.regulation of transcription. CCAAT box binding factor family, HAP nuclear factor Y, subunit B8 Ciclev m.g AT5G59820 RHL41 RNA.regulation C2H2 zinc finger family C2H2-type zinc finger family protein Ciclev m.g AT4G23990 CSLG3 cell wall.cellulose synthesis. cellulose synthase cellulose synthase like G3 Ciclev m.g AT4G39210 APL3 major CHO metabolism. Glucose-1-phosphate synthesis.starch.agpase adenylyltransferase family protein Ciclev m.g AT3G18830 PMT5 transporter.sugars polyol/monosaccharide transporter 5 Ciclev m.g AT5G19500 T20D1.20 transport.amino acids Tryptophan/tyrosine permease Ciclev m.g AT1G07030 F10K1.26 transport.metabolite transporters Mitochondrial substrate carrier family at the mitochondrial membrane protein 219

220 Table 5-6. Continued C. Clementina_ID Arabi ID Name Bin Name Log2 FC Arabidopsis_definition Ciclev m.g AT4G04450 WRKY42 RNA. regulation of transcription. WRKY domain transcription factor family WRKY family transcription factor Ciclev m.g AT1G76430 PHT1;9 transport. phosphate phosphate transporter 1;9 Ciclev m.g AT4G27410 RD26 development NAC (No Apical Meristem) domain transcriptional regulator superfamily protein Ciclev m.g AT5G62680 MRG21.10 transport. peptides and oligopeptides Major facilitator superfamily protein Ciclev m.g AT4G18780 IRX1 cell wall.cellulose synthesis. cellulose synthase cellulose synthase family protein Ciclev m.g AT2G40340 DREB2C RNA. regulation of Integrase-type DNA-binding transcription.ap2/erebp, superfamily protein APETALA2/Ethylene-responsive Ciclev m.g AT5G45380 DUR3 transport. cations solute:sodium symporters;urea transmembrane transporters Ciclev m.g AT5G26340 MSS1 transporter. sugars Major facilitator superfamily protein Ciclev m.g AT3G07650 COL9 RNA. regulation.c2c2(zn) CO-like, Constans-like zinc finger family Ciclev m.g AT3G16640 TCTP development CONSTANS-like 9 translationally controlled tumor protein Ciclev m.g AT2G39730 RCA PS.calvin cycle.rubisco interacting rubisco activase Ciclev m.g AT5G55930 OPT1 transport. peptides and oligopeptides oligopeptide transporter 1 Ciclev m.g AT2G31180 MYB14 RNA.regulation of transcription. MYB domain transcription factor family myb domain protein 14 Ciclev m.g AT2G38940 PHT1;4 transport. phosphate phosphate transporter 1;4 Ciclev m.g AT1G10970 ZIP4 transport. metal zinc transporter 4 precursor Ciclev m.g AT3G23250 MYB15 RNA. regulation of transcription. MYB domain transcription factor family myb domain protein 15 Ciclev m.g AT1G77380 AAP3 transport. amino acids amino acid permease 3 Ciclev m.g AT1G20510 OPCL1 secondary metabolism. phenylpropanoids OPC-8:0 CoA ligase1 Ciclev m.g AT3G28960 AT3G28960 transport. amino acids Transmembrane amino acid transporter family protein Ciclev m.g AT5G54860 MBG8.12 transport.misc Major facilitator superfamily protein Ciclev m.g AT5G13330 Rap2.6L RNA.regulation of transcription. AP2/EREBP, APETALA2/Ethylene related to AP2 6l 220

221 Table 5-6. Continued C. Clementina_ID Arabi ID Name Bin Name Log2 FC Arabidopsis_definition Ciclev m.g AT2G35980 YLS9 development Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family Ciclev m.g AT1G80300 NTT1 transport.misc nucleotide transporter 1 Ciclev m.g AT3G54700 PHT1;7 transport. phosphate phosphate transporter 1;7 Ciclev m.g AT2G01590 CRR3 PS.lightreaction.NADH DH chlororespiratory reduction 3 Ciclev m.g AT4G32551 LUG development LisH dimerisation motif;wd40/yvtn repeat-like-containing domain Ciclev m.g AT3G04070 NAC047 development NAC domain containing protein 47 Ciclev m.g AT3G20810 AT3G oxoglutarate (2OG) and Fe(II)- RNA.regulation of transcription dependent oxygenase superfamily JUMONJI family protein Ciclev m.g AT2G20880 F5H14.15 RNA.regulation of transcription.ap2/erebp, APETALA2/Ethylene-responsive Integrase-type DNA-binding superfamily protein Ciclev m.g AT5G66770 MUD21.1 development GRAS family transcription factor Ciclev m.g AT1G01720 ATAF1 development NAC (No Apical Meristem) domain transcriptional regulator superfamily protein Ciclev m.g AT4G10770 OPT7 transport.peptides and oligopeptides oligopeptide transporter 7 Ciclev m.g AT1G19210 T29M8.8 RNA.regulation of Integrase-type DNA-binding transcription.ap2/erebp, superfamily protein APETALA2/Ethylene-responsive Ciclev m.g AT3G21250 MRP6 transport.abc transporters and multidrug resistance-associated multidrugresistance systems protein 6 Ciclev m.g AT1G61340 T1F9.17 development F-box family protein Ciclev m.g AT5G03650 SBE2.2 major CHO metabolism.synthesis. starch.starch branching starch branching enzyme 2.2 Ciclev m.g AT5G23810 AAP7 transport.amino acids amino acid permease 7 Ciclev m.g AT4G40090 AGP3 cell wall.cell wall proteins.agps arabinogalactan protein 3 Ciclev m.g AT1G52190 F9I5.4 transport.peptides and oligopeptides Major facilitator superfamily protein Ciclev m.g AT3G06500 F5E6.17 major CHO metabolism.degradation.sucrose. invertases.neutral Plant neutral invertase family protein 221

222 Table 5-6. Continued C. Clementina_ID Arabi ID Name Bin Name Log2 FC Arabidopsis_definition Ciclev m.g AT5G51990 CBF4 RNA.regulation.AP2/EREBP, APETALA2/Ethylene-responsive C-repeat-binding factor 4 Ciclev m.g AT1G15520 PDR12 transport. ABC transporters and multidrug resistance systems pleiotropic drug resistance 12 Ciclev m.g AT5G46590 NAC096 development NAC domain containing protein 96 Ciclev m.g AT1G68710 F24J5.6 ATPase E1-E2 type family protein / RNA. regulation of transcription haloacid dehalogenase-like bzip transcription factor family hydrolase family protein Ciclev m.g AT2G39890 PROT1 transport.amino acids proline transporter 1 Ciclev m.g AT2G38470 WRKY33 RNA.regulation of transcription. WRKY domain transcription factor family WRKY DNA-binding protein 33 Ciclev m.g AT5G26170 WRKY50 RNA.regulation of transcription. WRKY domain transcription factor family WRKY DNA-binding protein 50 Ciclev m.g AT2G26150 HSFA2 RNA.regulation of transcription.hsf,heat -shock transcription factor family heat shock transcription factor A2 Ciclev m.g AT1G07640 OBP2 RNA.regulation of transcription. Dof-type zinc finger DNA-binding C2C2(Zn) DOF zinc finger family family protein Ciclev m.g AT5G24110 WRKY30 RNA. regulation of transcription. WRKY domain transcription factor family WRKY DNA-binding protein 30 Ciclev m.g AT5G59030 COPT1 transport. metal copper transporter 1 Ciclev m.g AT2G46330 AGP16 cell wall.cell wall proteins.agps arabinogalactan protein 16 Ciclev m.g AT3G17611 RBL14 RNA. regulation of transcription RHOMBOID-like protein 14 Ciclev m.g AT3G28345 AT3G28345 transport. ABC transporters and multidrug resistance systems ABC transporter family protein Ciclev m.g AT5G14940 F2G14.60 transport. peptides and oligopeptides Major facilitator superfamily protein Ciclev m.g AT1G78600 LZF1 RNA.regulation of transcription. C2C2(Zn) CO-like, light-regulated zinc finger protein 1 Constans-like zinc finger family Ciclev m.g AT3G21690 AT3G21690 transport.misc MATE efflux family protein Ciclev m.g AT1G15960 NRAMP6 transport. metal NRAMP metal ion transporter 6 Ciclev m.g AT4G37270 HMA1 transport.metal heavy metal atpase 1 Ciclev m.g AT3G46970 PHS2 major CHO metabolism. degradtion. starch.starch phosphorylase alpha-glucan phosphorylase 2 Ciclev m.g AT4G37990 ELI3-2 secondary metabolism. phenylpropanoids. lignin biosynthesis. CAD elicitor-activated gene

223 Table 5-6. Continued C. Clementina_ID Arabi ID Name Bin Name Log2 FC Arabidopsis_definition Ciclev m.g AT5G62020 HSFB2A RNA.regulation of transcription.hsf,heatshock transcription factor family factor B2A heat shock transcription Ciclev m.g AT3G54090 FLN1 major CHO metabolism.degradation.sucrose.fructokinase fructokinase-like 1 Ciclev m.g AT5G58490 MQJ2.6 secondary metabolism.phenylpropanoids.lignin biosynthesis.ccr NAD(P)-binding Rossmann-fold superfamily protein Ciclev m.g AT2G41710 T11A7.19 Ciclev m.g AT1G60470 GolS4 RNA.regulation of transcription.ap2/erebp, APETALA2/Ethylene-responsive element binding protein family minor CHO metabolism.raffinose family.galactinol synthases.putative Integrase-type DNA-binding superfamily protein galactinol synthase 4 PLATZ transcription factor Ciclev m.g AT4G17900 T6K21.80 RNA.regulation of transcription.unclassified family protein PS.lightreaction.cyclic electron flowchlororespiration fluorescence increase post-illumination chlorophyll Ciclev m.g AT3G15840 PIFI Ciclev m.g AT3G51860 CAX3 transport.metal cation exchanger 3 transport.metabolite transporters AT the Mitochondrial substrate Ciclev m.g AT1G14140 F7A mitochondrial membrane carrier family protein nodulin MtN21 /EamA-like Ciclev m.g AT5G40240 MSN9.140 development transporter family protein * The negative sign in the column of Log2 FC indicates comparative downregulation of the gene expression level in VAL/CAN and upregulation in VAL/SW combination. Classification of the measured parameter into a set a functional category in the MapMan analysis tool. 223

224 Table 5-7. Differentially expressed growth and development -associated genes significantly upregulated in roots of the symptomatic VAL/CAN combination as compared to symptomatic VAL/SW roots C. Clementina_ID Arabi ID Gene name Bin Name log2 FC Arabidopsis_define Ciclev m.g AT2G46320 transport. metabolite transporters at the Mitochondrial substrate mitochondrial membrane carrier family protein Ciclev m.g AT5G53130 CNGC1 transport. cyclic nucleotide or cyclic nucleotide gated calcium regulated channel 1 channels Ciclev m.g AT1G74840 RNA. regulation of transcription. MYBrelated transcription factor family superfamily protein Homeodomain-like Ciclev m.g AT5G49360 BXL1 cell wall.degradation. mannan-xylose-arabinose-fucose beta-xylosidase 1 Ciclev m.g Ciclev m.g AT2G22780 PMDH1 Gluconeogenesis. Malate DH peroxisomal NAD-malate dehydrogenase 1 Ciclev m.g AT1G45474 LHCA5 PS.lightreaction. photosystem I.LHC-I photosystem I light Ciclev m.g AT1G59960 Ciclev m.g AT5G54160 OMT1 secondary metabolism.flavonoids.chalcones secondary metabolism.phenylpropanoids. lignin biosynthesis.comt harvesting complex gene 5 NAD(P)-linked oxidoreductase superfamily protein O-methyltransferase 1 Ciclev m.g AT4G19010 secondary metabolism.phenylpropanoids AMP-dependent synthetase and ligase family protein Ciclev m.g AT1G09230 RNA.RNA binding RNA-binding (RRM/RBD/RNP motifs) family protein Ciclev m.g AT2G26560 PLA2A,PLP2 development. storage proteins phospholipase A 2A Ciclev m.g AT3G44735 AtPSK3,PSK1 development PHYTOSULFOKINE 3 PRECURSOR Ciclev m.g AT5G oxoglutarate (2OG) and secondary metabolism. flavonoids Fe(II)-dependent oxygenase anthocyanins superfamily protein Ciclev m.g AT4G31500 ATR4,CYP83B1 secondary metabolism. sulfur-containing. Glucosinolates. synthesis. shared. CYP83B cytochrome P450, family 83, subfamily B, polypeptide 1 Ciclev m.g AT5G65640 bhlh093 RNA. regulation of transcription. bhlh, Basic Helix-Loop-Helix family beta HLH protein 93 Ciclev m.g AT4G24040 AtTRE1, TRE1 minor CHO metabolism.trehalose.trehalase trehalase 1 224

225 Table 5-7. Continued C. Clementina_ID Arabi ID Gene name Bin Name Log2 FC Arabidopsis_definition Ciclev m.g AT3G29590 At5MAT secondary metabolism.flavonoids. HXXXD-type acyl-transferase anthocyanins. anthocyanin 5-aromatic family protein acyltransferase Ciclev m.g RNA.regulation of transcription. Ciclev m.g AT1G68840 RAV2,TEM2 AP2/EREBP, APETALA related to ABI3/VP1 2 /Ethylene-responsive Ciclev m.g Ciclev m.g AT5G10830 development S-adenosyl-L-methioninedependent methyltransferases superfamily protein Ciclev m.g AT4G18372 RNA.processing Small nuclear ribonucleoprotein family protein Ciclev m.g AT5G14570 NRT2.7 transport.nitrate high affinity nitrate transporter 2.7 Ciclev m.g Ciclev m.g AT1G68010 HPR PS.photorespiration. hydroxypyruvate reductase hydroxypyruvate reductase Ciclev m.g Ciclev m.g AT2G39510 development nodulin MtN21 /EamA-like transporter family protein Ciclev m.g AT4G35160 secondary metabolism. O-methyltransferase family phenylpropanoids protein Ciclev m.g Ciclev m.g AT1G12110 CHL11, NRT1, NRT1.1 transport.nitrate nitrate transporter 1.1 Ciclev m.g AT2G36870 XTH32 cell wall.modification xyloglucan endotransglucosylase/hydrolase 32 Ciclev m.g AT4G00050 UNE10 RNA.regulation of transcription. basic helix-loop-helix (bhlh) bhlh,basic Helix-Loop-Helix family DNA-binding superfamily protein Ciclev m.g AT2G39510 development nodulin MtN21 /EamA-like transporter family protein Ciclev m.g AT2G39510 development nodulin MtN21 /EamA-like Ciclev m.g AT2G29290 secondary metabolism.n misc.alkaloid-like transporter family protein NAD(P)-binding Rossmann-fold superfamily protein 225

226 Table 5-7. Continued C. Clementina_ID Arabi ID Gene name Bin Name Log2 FC Arabidopsis_definition Ciclev m.g AT2G39510 development nodulin MtN21 /EamA-like transporter family protein Ciclev m.g AT2G29290 secondary metabolism.n NAD(P)-binding Rossmannfold superfamily protein misc.alkaloid-like Ciclev m.g AT4G16370 OPT3 transport.peptides and oligopeptides oligopeptide transporter Ciclev m.g AT5G54160 OMT1 secondary metabolism.phenylpropanoids. lignin biosynthesis. COMT O-methyltransferase 1 RNA.regulation of Ciclev m.g AT4G25470 CBF2, transcription.ap2/erebp, DREB1C APETALA2/Ethylene-responsive C-repeat/DRE binding factor 2 element binding protein family Ciclev m.g AT1G51340 transport.misc MATE efflux family protein Ciclev m.g AT3G53720 CHX20 transport.metal cation/h+ exchanger 20 Ciclev m.g AT3G21090 transport.abc transporters and ABC-2 type transporter family multidrug resistance systems protein Ciclev m.g Ciclev m.g AT4G23730 minor CHO metabolism. others Galactose mutarotase-like superfamily protein Ciclev m.g Ciclev m.g AT1G67940 AtNAP3,AtSTAR1 transport. ABC transporters and non-intrinsic ABC protein 3 multidrugresistance systems Ciclev m.g AT5G55930 OPT1 transport.peptides and oligopeptides Ciclev m.g AT1G51310 RNA.processing Ciclev m.g AT1G61110 NAC025 development oligopeptide transporter 1 transferases;trna (5- methylaminomethyl-2- thiouridylate)- methyltransferases NAC domain containing protein 25 Ciclev m.g AT1G58030 CAT2 transport.amino acids cationic amino acid transporter 2 Ciclev m.g AT2G37460 development nodulin MtN21 /EamA-like transporter family protein Ciclev m.g AT3G16910 AAE7, ACN1 Gluconeogenese/ Glyoxylate cycle acyl-activating enzyme 7 226

227 Table 5-7. Continued C. Clementina_ID Arabi ID Gene name Bin Name Log2 FC Arabidopsis_definition Ciclev m.g AT3G47420,PS3 transporter.membrane system phosphate starvation-induced unknown gene 3 Ciclev m.g AT3G10960 AZG1 transport.misc AZA-guanine resistant1 Ciclev m.g AT2G40100 LHCB4.3 PS.lightreaction. light harvesting complex photosystem II photosystem II LHC-II Ciclev m.g AT4G17030 EXLB1,EXPR1 cell wall. modification expansin-like B1 Ciclev m.g AT5G58460 CHX25 transport.metal cation/h+ exchanger 25 Ciclev m.g AT4G08250 development Ciclev m.g AT5G54160 OMT1 secondary metabolism. phenylpropanoids. lignin biosynthesis.comt Ciclev m.g AT5G41330 transport.potassium Ciclev m.g AT2G22500 PUMP5, UCP5 Ciclev m.g AT4G01470 TIP1.3, GAMMA-TIP3 Ciclev m.g AT1G08650 PPCK1 Ciclev m.g AT1G68570 Ciclev m.g AT1G62280 SLAH1 transport. metabolite transporters at the mitochondrial membrane transport.major Intrinsic Proteins.TIP Glycolysis.cytosolic branch.phospho-enolpyruvate carboxylase kinase (PPCK) transport.peptides and oligopeptides transport.metabolite transporters AT the mitochondrial membrane secondary metabolism.phenylpropanoids GRAS family transcription factor O-methyltransferase 1 BTB/POZ domain with WD40/YVTN repeat-like protein uncoupling protein tonoplast intrinsic protein 1; phosphoenolpyruvate carboxylase kinase 1 Major facilitator superfamily protein SLAC1 homologue 1 Ciclev m.g AT1G AMP-dependent synthetase and ligase family protein Ciclev m.g AT5G49740 FRO7 metal handling.acquisition ferric reduction oxidase 7 Ciclev m.g AT4G01470 ATTIP1.3,GAMMA- TIP3 transport.major Intrinsic Proteins.TIP tonoplast intrinsic protein 1;3 227

228 Table 5-7. Continued C. Clementina_ID Arabi ID Gene name Bin Name Log2 FC Arabidopsis_definition Ciclev m.g AT2G38100 transport. peptides and proton-dependent oligopeptide oligopeptides transport (POT) family protein Ciclev m.g AT1G58030 CAT2 transport. amino acids cationic amino acid transporter 2 Ciclev m.g AT4G30380 cell wall. modification Barwin-related endoglucanase Ciclev m.g AT2G28260 CNGC15, CNGC15 transport. cyclic nucleotide or calcium regulated channels cyclic nucleotide-gated channel 15 Ciclev m.g AT5G54160 ATOMT1, secondary metabolism O-methyltransferase 1 OMT1 phenylpropanoids.lignin biosynthesis.comt Ciclev m.g AT3G54700 PHT1;7 transport. phosphate phosphate transporter 1;7 Ciclev m.g AT1G17840 ABCG11, WBC11, transport. ABC transporters and multidrug resistance systems white-brown complex homolog protein 11 Ciclev m.g AT3G25882 NIMIN-2 RNA. regulation of transcription NIM1-interacting 2 unclassified Ciclev m.g AT5G53130 CNGC1, transport. cyclic nucleotide or cyclic nucleotide gated channel 1 CNGC1 calcium regulated channels Ciclev m.g AT5G15140 minor CHO metabolism. others Galactose mutarotase-like superfamily protein Ciclev m.g AT3G58060 transport. metal Cation efflux family protein Classification of the measured parameter into a set a functional category in the MapMan analysis tool. 228

229 Table 5-8. Differentially expressed growth and development -associated genes significantly upregulated in roots of the symptomatic VAL/SW combination as compared to symptomatic VAL/CAN roots C. Clementina_ID Arabi ID Gene name Bin Name Log2 FC* Arabidopsis_definition Ciclev m.g AT4G12240 RNA. regulation of zinc finger (C2H2 type) family transcription.c2h2 zinc finger family protein Ciclev m.g Ciclev m.g Ciclev m.g AT3G51420 SSL4 secondary metabolism. N misc.alkaloid-like strictosidine synthase-like 4 Ciclev m.g AT1G59960 secondary NAD(P)-linked oxidoreductase metabolism.flavonoids.chalcones superfamily protein Ciclev m.g AT5G15090 VDAC3 transport. porins voltage dependent anion channel 3 Ciclev m.g AT5G54160 OMT1 secondary metabolism. phenylpropanoids. lignin biosynthesis. COMT O-methyltransferase 1 Ciclev m.g AT3G15270 SPL5 development. squamosa promoter squamosa promoter binding binding like (SPL) protein-like 5 Ciclev m.g Ciclev m.g AT1G17020 SRG1 secondary metabolism. flavonoids.flavonols senescence-related gene 1 Ciclev m.g AT5G06760 LEA4-5 development.late embryogenesis abundant Late Embryogenesis Abundant 4-5 Ciclev m.g AT1G68570 transport.peptides and oligopeptides Major facilitator superfamily protein Ciclev m.g AT3G24450 Heavy metal metal handling.binding, chelation, transport/detoxification superfamily and storage protein Ciclev m.g AT3G53480 ABCG37, transport.abc transporters and ATPDR9, multidrugresistance systems pleiotropic drug resistance 9 Ciclev m.g AT1G33440 transport. peptides and oligopeptides Major facilitator superfamily protein Ciclev m.g Ciclev m.g AT2G28660 metal handling. binding, chelation, Chloroplast-targeted copper and storage chaperone protein Ciclev m.g AT4G24000 ATCSLG2, cell wall. cellulose cellulose synthase like G2 CSLG2 Ciclev m.g AT1G60470 AtGolS4, GolS4 synthesis.cellulose synthase minor CHO metabolism raffinose family.galactinol synthases.putative galactinol synthase 4 229

230 Table 5-8. Continued C. Clementina_ID Arabi ID Gene name Bin Name Log2 FC Arabidopsis_definition Ciclev m.g AT4G15010 transport. metabolite transporters at the mitochondrial membrane Mitochondrial substrate carrier family protein Ciclev m.g AT4G15010 transport. metabolite transporters at the mitochondrial membrane Mitochondrial substrate carrier family protein Ciclev m.g AT5G59800 MBD7 RNA.regulation of transcription. Methyl binding methyl-cpg-binding domain proteins domain 7 Ciclev m.g AT3G14420 PS. photorespiration.glycolate oxidase Aldolase-type TIM barrel family protein Ciclev m.g AT3G54340 AP3, ATAP3 development K-box region and MADSbox transcription factor family protein Ciclev m.g AT2G17840 ERD7 development Senescence/dehydrationassociated protein-related Ciclev m.g AT4G37990 ATCAD8, ELI3 secondary metabolism.phenylpropanoids.lignin biosynthesis.cad elicitor-activated gene 3-2 Ciclev m.g AT2G18950 TPT1, VTE2 secondary metabolism.isoprenoids.tocopherol homogentisate biosynthesis.homogentisate phytyltransferase phytyltransferase 1 Ciclev m.g AT2G45050 GATA2 RNA. regulation of transcription.c2c2(zn) gata transcription factor family GATA transcription factor 2 Ciclev m.g AT1G02460 cell wall. degradation. pectate lyases and Pectin lyase-like polygalacturonases superfamily protein Ciclev m.g Ciclev m.g AT2G46440 CNGC11 transport.cyclic nucleotide or calcium cyclic nucleotide-gated regulated channels channels Ciclev m.g AT1G22710 SUC2, SUT1 transporter. sugars.sucrose sucrose-proton symporter 2 Ciclev m.g AT5G11450 Mog1/PsbP/DUF1795-like PS.lightreaction.photosystem II.PSII photosystem II reaction polypeptide subunits center PsbP family protein Ciclev m.g AT5G23870 cell wall.pectin*esterases.acetyl esterase Pectinacetylesterase family Ciclev m.g Ciclev m.g AT2G38300 AT5G15410 CNGC2, DND1 RNA.regulation of transcription. G2-like transcription factor family, GARP transport. cyclic nucleotide or calcium regulated channels protein myb-like HTH transcriptional regulator family protein Cyclic nucleotide-regulated ion channel family protein 230

231 Table 5-8. Continued C. Clementina_ID Arabi ID Gene name Bin Name Log2 FC Arabidopsis_definition Ciclev m.g AT3G53480 ATPDR9, PDR9 transport. ABC transporters and multidrug resistance systems pleiotropic drug resistance 9 Ciclev m.g AT3G57670 NTT,WIP2 RNA. regulation of transcription. C2H2-type zinc finger family C2H2 zinc finger family protein Ciclev m.g AT4G05180 PSBQ-2,PSII-Q PS. lightreaction. photosystem II. PSII polypeptide subunits photosystem II subunit Q-2 Ciclev m.g AT1G69850 NRT1:2, NTL1 transport. nitrate nitrate transporter 1:2 Ciclev m.g AT5G49630 AAP6 transport. amino acids amino acid permease 6 Ciclev m.g AT1G52190 transport. peptides and Major facilitator superfamily oligopeptides protein Ciclev m.g AT1G63680 acid-amino acid ATMURE, MURE, development ligases;ligases;atp PDE316 binding;atp binding;ligases Ciclev m.g AT3G13080 ATMRP3, MRP3 transport. ABC transporters and multidrug resistanceassociated protein multidrug resistance systems Ciclev m.g Ciclev m.g AT5G40390 SIP1 minor CHO metabolism. raffinose Raffinose synthase family family.raffinose synthases.known protein Ciclev m.g AT3G19330 RNA.processing Protein of unknown function (DUF677) Ciclev m.g Ciclev m.g AT1G59740 transport. peptides and oligopeptides Major facilitator superfamily protein Ciclev m.g Ciclev m.g AT5G26170 WRKY50 RNA.regulation of transcription.wrky WRKY DNA-binding protein domain transcription factor family 50 Ciclev m.g AT3G28180 ATCSLC04, cell wall.cellulose synthesis Cellulose-synthase-like C4 ATCSLC4, Ciclev m.g AT3G50740 UGT72E1 secondary metabolism. phenylpropanoids.lignin biosynthesis UDP-glucosyl transferase 72E1 Ciclev m.g AT3G54140 ATPTR1,PTR1 transport. peptides and oligopeptides peptide transporter 1 Ciclev m.g AT4G00430 PIP1;4, PIP1E,TMP- C transport.major Intrinsic Proteins.PIP plasma membrane intrinsic protein 1;4 Ciclev m.g AT3G24310 ATMYB71,MYB305 RNA.regulation of transcription.myb myb domain protein 305 domain transcription factor family Ciclev m.g AT2G44745 RNA.regulation of transcription WRKY family 231

232 Table 5-8. Continued C. Clementina_ID Arabi ID Gene name Bin Name Log2 FC Arabidopsis_definition secondary UDP-Glycosyltransferase Ciclev m.g AT5G65550 metabolism.flavonoids.anthocyanins superfamily protein Ciclev m.g AT3G07650 COL9 RNA.regulation of transcription. C2C2(Zn) CO-like, Constans-like zinc finger family CONSTANS-like 9 Ciclev m.g AT1G03650 RNA.regulation of transcription.histone Acyl-CoA N-acyltransferases acetyltransferases (NAT) superfamily protein Ciclev m.g AT5G65380 development MATE efflux family protein Ciclev m.g AT2G45660 AGL20, RNA.regulation of transcription.mads box SOC1 transcription factor family AGAMOUS-like 20 Ciclev m.g AT4G36810 GGPS1 secondary metabolism.isoprenoids.nonmevalonate Pathway.geranylgeranyl geranylgeranyl pyrophosphate synthase 1 pyrophosphate synthase Ciclev m.g AT5G10970 RNA.regulation of transcription. C2H2 and C2HC zinc fingers C2H2 zinc finger family superfamily protein Ciclev m.g AT1G30650 ATWRKY14, RNA. regulation of transcription. WRKY14 WRKY domain transcription factor family WRKY DNA-binding protein 14 Ciclev m.g AT5G46590 anac096, NAC domain containing protein development NAC Ciclev m.g AT3G43660 development Vacuolar iron transporter (VIT) family protein Ciclev m.g AT5G65060 AGL70, FCL3, RNA.regulation of transcription. K-box region and MADS-box MAF3 MADS box transcription factor family transcription factor family protein Ciclev m.g AT2G05760 transport.misc Xanthine/uracil permease family protein Ciclev m.g AT1G75900 development GDSL-like Lipase/Acylhydrolase superfamily protein Ciclev m.g AT5G41610 CHX18 transport.metal cation/h+ exchanger 18 Ciclev m.g AT3G50410 OBP1 RNA.regulation of transcription. C2C2(Zn) DOF zinc finger family OBF binding protein 1 Ciclev m.g AT5G07050 development Ciclev m.g AT5G22300 AtNIT4,NIT4 secondary metabolism. sulfur-containing. glucosinolates.degradation.nitrilase Ciclev m.g AT5G49120 development nitrilase 4 nodulin MtN21 /EamA-like transporter family protein Protein of unknown function (DUF581) 232

233 Table 5-8. Continued C. Clementina_ID Arabi ID Gene name Bin Name Log2 FC Arabidopsis_definition Ciclev m.g Ciclev m.g AT1G08290 WIP3 RNA. regulation of transcription.c2h2 zinc finger family WIP domain protein 3 Ciclev m.g AT4G14210 PDS, PDS3 secondary metabolism. isoprenoids. carotenoids. phytoene phytoene desaturase 3 dehydrogenase Ciclev m.g AT5G20420 CHR42 RNA.regulation of transcription. Chromatin Remodeling Factors chromatin remodeling 42 Ciclev m.g AT5G53130 CNGC1 transport.cyclic nucleotide or cyclic nucleotide gated channel calcium regulated channels 1 Ciclev m.g AT1G08230 GAT1 transport.amino acids Transmembrane amino acid transporter family protein Ciclev m.g Ciclev m.g AT2G37630 AS1, MYB91 RNA.regulation of transcription.myb myb-like HTH transcriptional domain transcription factor family regulator family protein RNA.regulation of transcription. Ciclev m.g AT4G32730 MYB3R-1, MYB3R1 MYB domain transcription factor Homeodomain-like protein family Ciclev m.g AT1G04250 AXR3, IAA17 RNA.regulation of transcription.aux/iaa family AUX/IAA transcriptional regulator family protein Ciclev m.g Ciclev m.g AT4G00950 MEE47 RNA.regulation of transcription.c2c2(zn) DOF zinc finger family Ciclev m.g AT5G53130 CNGC1 transport.cyclic nucleotide or calcium regulated channels Ciclev m.g AT5G50740 metal handling.binding, chelation, and storage Ciclev m.g AT4G34530 CIB1 RNA.regulation of transcription.bhlh,basic Helix- Loop-Helix family Ciclev m.g AT4G21440 ATM4, RNA.regulation of transcription.myb Protein of unknown function (DUF688) cyclic nucleotide gated channel Heavy metal transport/detoxification superfamily protein cryptochrome-interacting basic-helix-loop-helix MYB-like 102 MYB102 domain transcription factor family Ciclev m.g AT2G32950 COP1 development Transducin/WD40 repeat-like superfamily protein 233

234 Table 5-8. Continued C. Clementina_ID Arabi ID Gene name Bin Name Log2 FC Arabidopsis_definition Ciclev m.g AT5G53130 CNGC1 transport.cyclic nucleotide or calcium regulated channels cyclic nucleotide gated channel 1 Ciclev m.g AT5G53130 CNGC1 transport.cyclic nucleotide or calcium regulated channels cyclic nucleotide gated channel 1 Ciclev m.g AT3G27400 cell wall. degradation.pectate lyases Pectin lyase-like and polygalacturonases superfamily protein Ciclev m.g AT5G56840 RNA.regulation of transcription. MYBrelated transcription factor family factor family protein myb-like transcription Ciclev m.g AT2G26910 ATPDR4, PDR4 transport.abc transporters and pleiotropic drug multidrug resistance systems resistance 4 Ciclev m.g AT1G27940 PGP13 transport.abc transporters and multidrug resistance systems P-glycoprotein 13 Ciclev m.g AT3G10600 CAT7 transport.amino acids cationic amino acid transporter 7 Ciclev m.g AT5G66460 cell wall.degradation.mannan-xylosearabinose-fucose superfamily protein Glycosyl hydrolase Ciclev m.g AT5G64530 ANAC104, XND1 development xylem NAC domain 1 * The negative sign in the column of Log2 FC indicates comparative downregulation of the gene expression level in VAL/CAN and upregulation in VAL/SW combination. Classification of the measured parameter into a set a functional category in the MapMan analysis tool. 234

235 Figure 5-1. Graphical presentation of secondary metabolite biosynthesis pathway involved genes found in DGE analysis of asymptomatic and symptomatic VAL/CAN and VAL/SW -leaves and -roots. Log2 fold changes are indicated as a gradient between blue (up-regulated in VAL/CAN combination) and red (up-regulated VAL/SW combination). Asymp; Asymptomatic, Sympt; symptomatic 235

236 Figure 5-2. Graphical presentation of cell wall modification-associated pathways involved genes found in DGE analysis of asymptomatic and symptomatic VAL/CAN and VAL/SW -leaves and -roots. Log2 fold changes are indicated as a gradient between blue (up-regulated in VAL/CAN combination) and red (up-regulated VAL/SW combination). Asymp; Asymptomatic, Sympt; symptomatic 236

237 Figure 5-3. Graphical presentation of nutrient transportation-associated Genes found in DGE analysis of asymptomatic and symptomatic VAL/CAN VAL/CAN and VAL/SW -leaves and -roots. Log2 fold changes are indicated as a gradient between blue (up-regulated in VAL/CAN combination) and red (up-regulated VAL/SW combination). Asymp; Asymptomatic, Sympt; symptomatic Figure 5-4. Graphical presentation of Nitrogen metabolism-associated genes found DGE analysis of asymptomatic and symptomatic VAL/CAN VAL/CAN and VAL/SW -leaves and -roots. Log2 fold changes are indicated as a gradient between blue (up-regulated in VAL/CAN combination) and red (up-regulated VAL/SW combination). Asymp; Asymptomatic, Sympt; symptomatic 237

238 Figure 5-5. Graphical presentation of carbohydrate metabolism-associate genes found in DGE analysis of asymptomatic and symptomatic VAL/CAN and VAL/SW -leaves and -roots. Log2 fold changes are indicated as a gradient between blue (up-regulated in VAL/CAN combination) and red (up-regulated VAL/SW combination). Asymp; Asymptomatic, Sympt; symptomatic 238

239 Figure 5-6. Graphical presentation of genes encoding transcription regulators involved in plant growth and development, and found in DGE analysis of asymptomatic and symptomatic VAL/CAN and VAL/SW leaves and roots. Log2 fold changes are indicated as a gradient between blue (up-regulated in VAL/CAN combination) and red (up-regulated and VAL/SW combination). Asymp; Asymptomatic, Sympt; symptomatic 239

240 CHAPTER 6 EFFECTS OF IMPROVED CITRUS ROOTSTOCK AND ENHANCED NUTRIENT FORMULATION ON HLB-DISEASE SEVERITY IN VALENCIA SWEET ORANGE SCION Introduction Plants are autotrophic organisms which synthesis their own food by the process of photosynthesis. The products of photosynthesis are called photosynthates, and they perform various plant functions. Plant photosynthates are translocated from area of supply called sources, to areas of metabolism, storage, and growth, called sinks. Biotic or abiotic stress may cause imbalance between source and sink tissue photosynthates allocation/distribution. Plants face environmental challenges in their natural habitat. Hence, plant s surveillance and defense systems are activated to defend the environmental adversaries. Pathogen detection and resistance are energy consuming processes that can create an imbalance of resource distribution between plant growth and defense. Plant defense is generally beneficial, but not always. A model discussed by Ellen et al. (1987) and other studies (Simms and Rausher, 1987) showed that energy invested in the defense against herbivores was rather non-beneficial and increased plant resource consumption that could have been used for other biological functions. Whereas, benefits of herbivory-induced defense (Heil, 2004), pathogen-activated defense (van Hulten et al., 2006) and cost/benefit of defense responses (Mauricio and Rausher, 1997) were also reported in many plant-pathogen and plant-herbivore interactions. The benefit or cost of defense is dependent on the environmental conditions and resource availability (Purrington and Bergelson, 1997; Heil et al., 2000). Cost and benefits of induced and constitutively expressed defenses are explained by the optimal defense theory (ODT) (Moreira et al., 2012). The theory of ODT explains the 240

241 differential allocation of induced and constitutive defense based on the importance of the organs or tissues in the plant s survival. According to the ODT, valuable reproductive organs exhibit constitutive defense, whereas, leaves and roots, which are the plant s expandable organs, show the induced defense. It is hypothesized that induced resistance costs fewer resources and more benefits as compared to the constitutive defense. However, the calculations of defense-induced costs and benefits in the plant are not easy and simple to understand. It is an intricate networking between threat sensing signaling components, defense activation, and nutrient status of the plant. Plant growth hormones (PGRs) and nutrients status are the critical components to optimize the balance of energy supply between growth and defense. PGRs, whether defense-associated salicylic acid (SA), jasmonic acid (JA), ethylene (ET) or growth related auxins (AU), cytokinins (CT), gibberellic acid (GA), brassinosteroids (BR) and abscisic acid (ABA), directly or indirectly regulate the plant defense (Huot et al., 2014). PGRs have an antagonistic or synergistic effect on each other that regulates growth and defense responses (Nemhauser et al., 2004; Robert-Seilaniantz et al., 2011; Naseem et al., 2015). Nutrient homeostasis in the plants is also regulated by plant hormones (Rubio et al., 2009; Krouk et al., 2011). Effect of cultivars and nutrient interactions in the plant defense was discussed in the Hyaloperonospora parasitica infection in A. thaliana (Heidel and Dong, 2006). The study reported that systemic acquired resistance (SAR) is beneficial to the wild phenotype plants exhibiting normal SAR, under a normal or lownutrient condition in H. parasitica infected plants. Whereas, the high-nutrient condition did not show any increased benefit to wild phenotype plants the under H. parasitica 241

242 infection. Also, the study showed that in the overexpressed NPR1 genotype, constitutive expression of SAR is beneficial under a strong damage caused by pathogen attack. JA and ET induced resistance-associated defenses are studied widely in plants attacked by herbivores and the necrotrophs (Simms and Rausher, 1987; Agrawal, 1999; Elle et al., 1999). Although JA induced chemical defense is reported to be cost-effective in the defense against herbivores, it is found that constitutive expression of JA-inducible responses delay plant phenology (Cipollini, 2010). Pathogen infection can have a deleterious effect on the plant health due to its direct or indirect damage. Citrus plants infected with HLB disease, caused by putative bacteria Candidatus Liberibacter asiaticus (CaLas) infection, suffer severely compromised growth, immature fruit drop, and leaf senescence. CaLas-infected plant phloem tissues are found to have callose deposition that barriers the spread of CaLas. However, the overaccumulation of callose in the phloem also creates blockages for nutrient and photosynthate flow. There are no reports on CaLas specific immunity or effector triggered immunity (ETI) exhibited by any infected plants. Therefore, in the absence of resistance, plants induce different non-host specific defenses to fight against the CaLas infection. CaLas-infected susceptible citrus scions and rootstocks showed upregulation of genes involved in different plant defenses (Martinelli et al., 2012; Martinelli et al., 2013; Zhong et al., 2016). In such a scenario, improved HLB-tolerant scions and rootstocks are important to increase plant sustainability. The economically enduring and biological HLB tolerance can be achieved either by developing HLB tolerant citrus cultivars, and/or modifying cultural practices that can increase plant sustainability under HLB pressure. Different levels of HLB tolerance are observed in the 242

243 hybrids of trifoliate orange (Poncirus trifoliata [L.] Raf), rough lemon (Citrus jambhiri Lush.), pummelos (Citrus maxima Merr.) and lemons (Citrus limon L. [Burm.] f.) (Folimonova et al., 2009; Fan et al., 2012; Albrecht and Bowman, 2012b; Aritua et al., 2013; Martinelli et al., 2016). The citrus breeding program at the University of Florida, Citrus Research and Education Center, Lake Alfred (UF-CREC) has developed putative HLB-tolerant hybrid rootstocks in response to the need of improved plant sustainability under HLB disease pressure. HLB disease development in citrus trees is not only attributed to the lack of immunity but also the inadequate supply of nutrients (Cimò et al., 2013). The role of proper nutrition management is vital to reducing the disease severity (Dordas, 2008), whereas improper nutrition management may not achieve the desirable results (Gottwald et al., 2012). An understanding of nutrient interactions with disease development and cultivars is vital to implement effective nutrition management. CaLasinfected plants are found to have a similarity to those showing zinc (Zn) deficiency symptoms. Moreover, boron (B), calcium (Ca), magnesium (Mg) and phosphorus (P) deficiencies are also prominent in the CaLas-infected plants (Spann and Schumann, 2009). Enhanced nutrient foliar application programs (ENPs) have been implemented to address nutrient deficiencies in the CaLas-infected plants. However, ENPs did not show a significant role in reducing HLB disease severity (Gottwald et al., 2012). Foliar application of nutrients does not address the deficiencies in the roots. Soil application of nutrients in combination with insecticide and hormones was found to be effective in reducing HLB disease severity (Shen et al., 2013). Therefore, implementation of 243

244 efficient nutrient management focusing more on root health could be a key to achieving desirable results in CaLas-infected plants. The interaction between nutrition and citrus rootstocks plays a vital role in HLB disease development in Citrus. Therefore, to address the issue of nutrient deficiency and symptoms severity in the CaLas-infected plants, combinations of a putatively HLBtolerant tetraploid (4x) rootstock and an improved nutrient formulation were tested in CaLas-infected plants under greenhouse conditions. The improved nutrient formulation is a controlled release fertilizer delivering elevated levels of secondary and micronutrients impacted by HLB In this study, the role of the improved nutrient formulation was also tested in reducing CaLas infection in the Valencia sweet orange (Citrus sinensis [L.] Osbeck) scion grafted onto HLB-susceptible Swingle citrumelo rootstock combination (a standard in the Florida industry). The results obtained from the greenhouse study will be helpful to understand the potential role of nutrition and citrus rootstocks to relieve the HLB disease severity and enhance the economic tolerance in the susceptible Citrus plants. Materials and Methods Plant Material and Nutrition Treatment The greenhouse-based experiment was designed to analyze the effect of interaction between citrus rootstocks and nutrient formulations on CaLas-infected Valencia (VAL) scion. In the rootstock treatment, two different rootstocks were tested (Table 6-1). The putatively HLB-tolerant 4x hybrid used in this study is a complex candidate rootstock hybrid that contains a combination of more than four different citrus species and/or cultivars. The 4x hybrid is a cross of a cybrid autotetraploid [Amblycarpa (Citrus amblycarpa) + Volkmer lemon (Citrus Volkmariana)] and tetrazyg Orange

245 (released as UFR-4). The somatic cybrid between Amblycarpa mandarin + Volkmer lemon is an autotetraploid which was created using the protoplast fusion technique. This unusual combination retained two copies of the Volkmariana nuclear genotype in combination with Amblycarpa cytoplasm. Orange 19 is called a tetrazyg, which is a zygotic allotetraploid produced from crossing two tetraploids that can be somatic hybrids or tetrazygs. Orange 19 (commercially released as UFR-4) is a hybrid of two somatic hybrids: [ Nova mandarin + Hirado Butan Pink Pummelo] x [ Cleopatra mandarin + Argentine Trifoliate orange]. The 4x hybrid rootstock used in this study will be named Tetr here onward. The commercially important and HLB-susceptible Swingle (Sw) rootstock used in this study is a hybrid between grapefruit (Citrus paradisi [Macf.]) and Trifoliate orange. In the nutrition treatment, two formulations of Harrell s POLYON coated controlled-release fertilizer (CRF) were used. One of the formulations, the nursery mix (NM), is designed to fertilize greenhouse plants; whereas, the other nutrient formulation contained increased concentration of micronutrients and 4% Ca compared to the NM. It will be referred to as Enhanced-CRF (ECRF) (Table 6-2). The ECRF formulation was designed to compensate for the secondary and micronutrient deficiencies observed in roots of CaLas infected trees. Multiple plant biological replicates (4-6) and randomized experimental design were used to test the effects of the interaction between nutrient formulations and rootstock treatments on VAL scion in the downstream analyses of this experiment. 245

246 CaLas-Inoculation and Detection CaLas-infected VAL sticks were grafted onto the rootstocks grown using the nutrient formulation combinations in 4 x 4-inch pots to test the effects of the interaction between rootstock and nutrient formulation (Figure 6-1). In addition, healthy VAL sticks were also grafted onto Sw and provided with NM formulation, which served as the control in this experiment. Two methods of CaLas infection were performed in the ECRF-applied scion/rootstock combination. In the first type of inoculation, plants were infected by grafting a 6- to 7-inch-long CaLas-infected VAL bud-sticks onto the healthy rootstock (stick-grafting infection). In the second type of inoculation, healthy scion/rootstock grafts were infected by grafting a CaLas-infected VAL blind-bud onto the rootstock (blind-bud infection) using the standard T-grafting technique. Plants under NM formulation were infected by only the stick-grafting infection method. Stick grafting ensures CaLas inoculum availability for the infection and enough plant material for the sampling. CaLas-infected VAL sticks were collected from the UF-CREC, Block 2B from commercial VAL trees grown under a conventional nutrition program. These plants were tested for the detection of CaLas and citrus tristeza virus (CTV) strains. Healthy VAL sticks were procured from a certified citrus nursery. CaLas-infected VAL sticks and buds collected from the field were sterilized thoroughly using 10% bleach and 70% alcohol treatment to avoid any contamination other than HLB, and then immediately used for the grafting. Grafting of all plants was conducted in the months of September and October, Quantitative real-time PCR (qrt-pcr) based CaLas detection was conducted to test the cycle threshold (Ct) values and bacterial titer of the VAL sticks and post-inoculated grafts. The GenElute Plant Genomic DNA Miniprep Kit (Sigma 246

247 Aldrich, Woodlands, TX) was used to extract DNA from leaves, and the PowerMax Soil DNA Isolation Kit (MOBIO Laboratories, Inc., Carlsbad, CA) was used to isolate root DNA. The extracted DNA was quantified in a NanoDrop ND-100 spectrophotometer (Thermo Scientific, Wilmington, DE). CaLas specific CQULA04F and CQULA04R primer pair and CQULAP10 TaqMan-probe (FAM fluorophore dye) were used for the amplification of CaLas 16sRNA (Wang et al., 2006). The TaqMan Gene Expression Master Mix Kit (Applied Biosystems, Foster City, CA) was used to perform qpcr assay. All reactions were carried out in 25 µl reaction volume containing 50 ng DNA and 0.3 µm probe and primer concentrations. Amplification was conducted over 40 cycles of qpcr in StepOnePlus PCR system (Applied Biosystems). Sampling Three time-dependent leaf samplings were conducted in this experiment. Roots were collected at the end of the experiment to avoid changes in the leaf gene expression analysis that may arise because of root damage. The first sampling of leaves was performed based on the sprouting and availability of enough plant material for the gene expression and CaLas detection. At the end of 13 weeks after bud sprout (WAB), enough sampling material was available on the grafted VAL scion in all rootstock and nutrient combinations. The rest of the time-dependent samplings were done at 25 and 37 WAB. Roots were collected at the end of 37 WAB to analyze gene expression and CaLas detection. All three time-dependent leaf samples were used for gene expression and CaLas detection. At each time point, the same leaves were collected for CaLas detection to monitor the effect of nutrient and rootstock treatments to the Ct value changes. Sampling for CaLas detection was performed by collecting leaf 247

248 discs from the leaf midrib portion using a paper punch; whereas, for gene expression analysis, fully expanded leaves were collected from each biological replicate. RNA Extraction and Gene Expression Quantification Total RNA was extracted from the collected leaves or roots, using Qiagen RNeasy Mini Plant RNA Extraction kit (Qiagen Inc., Valencia, CA). The manufacturer s protocol was followed using 100 milligram starting material and on-column DNase treatment performed with RNase free DNase Free kit (Qiagen). Quantity and quality of the extracted RNA were analyzed in a NanoDrop spectrophotometer. Quantitative realtime reverse transcriptase (qrt-rt) PCR technique was used to amplify the selected genes. A total of six genes were selected to analyze gene expression pattern in response to nutrient formulation and rootstock treatments. These genes were nutrient transporters, defense and HLB-response associated genes (Table 6-3). Gene specific primers and probes were designed using Integrated DNA Technologies (IDT) Primer Quest Tool (IDT, Coralville, IA). For normalization, GLYCERADEHYDE-3-PHOSPHATE DEHYDROGENASE C2 (GAPC) forward and reverse primer pairs and GAPC specific probe were used as the reference gene control. TaqMan and Power SYBR green RNA-to-Ct 1-Step RT PCR (Applied Biosystems) master mix was used to convert total RNA into cdna and then amplify it with the primers and probe combination. A total of 50 ng of DNase free RNA and 500 nm of each primer and probe were used to amplify and quantify the real-time gene expression. Amplification was performed over 40 cycles in a StepOnePlus real-time PCR machine (Applied Biosystems). Relative gene expression quantification was calculated using ΔΔCt method (Livak and Schmittgen, 2001). For each sample, the Ct value of GAPC was subtracted from the Ct value for the gene of interest to get ΔCt value. ΔCt values obtained from 248

249 leaves of healthy VAL grafted onto Sw_NM interaction were used as the control. The relative gene expression was calculated as Log2 fold change (Log2 FC) scale. Data analysis of differential gene expression (DGE) in response to rootstock and nutrient interactions was conducted using the generalized mixed model in the JMP13 statistical software (SAS Inc., Cary, CA). The significant differences were estimated using HSD Tukey test at p The visual presentation of the data was created, using Microsoft Office Excel 10. Plant Phenotype Analysis Plant phenotypic analyses were conducted at 37 WAB time point. These analyses include the presence of HLB symptoms in leaves, plant height, and the number of branches and diameters of the plants (5, 10 and 15 centimeters above and below the graft union). Significant differences in the phenotypic observations were analyzed, using JMP13 (SAS Inc.) and tested using HSD Tukey test at p The visual presentation of the data was created with Microsoft Office Excel 10. Results CaLas-Detection The Ct value of the tree that used for budsticks collection was in the range of 25-27, and selected budsticks tested negative for CTV. In the ECRF treatment, VAL/Sw, and VAL/Tetr plants that were infected by two different HLB inoculation methods, stickgraft infection and blind-bud infection, did not show a significant difference in their Ct values. Therefore, stick-graft infected and blind-bud infected plants in each rootstock treatment were pooled into their respective rootstock and nutrient formulation combinations, and named as Sw_ECRF and Tetr_ECRF. Real-time qrt- PCR based CaLas detection results showed that in all combinations, the 13 WAB time point had the 249

250 lowest Ct values compared to the 25 WAB and 37 WAB time point Ct values (Figure 6-2A). At 13 WAB, VAL grafted onto the Sw_NM combination had the lowest mean Ct value as compared to 13 WAB Cts in other combinations. At 13 WAB time point, the mean of Ct values of CaLas-infected VAL grafted onto Sw_NM, Sw_ECRF, Tetr_NM and Tetr_ECRF were 24.3, 30.4, 26.7 and 31.1, respectively (Figure 6-2A). At the 25 WAB time point, the mean Ct value of VAL grafted onto Sw_NM interaction increased non-significantly compared to its 13 WAB. However, the mean Ct of Sw_NM combination at 25 WAB was significantly low compared to the Ct values of the rest of three combinations at the 25 WAB (Figure 6-2A). At the 25 WAB, mean Ct of VAL grafted onto Sw_ECRF, Tetr_NM and Tetr_ECRF combinations increased significantly compared to their 13 WAB (Figure 6-2A). At the 37 WAB time point, the mean Ct value of VAL grafted on Sw_NM, Sw_ECRF, Tetr_NM and Tetr_ECRF combinations changed compared to their Ct at 25 WAB, but indifferently (Figure 6-2A). The mean Ct value of Sw_NM interaction at 37 WAB time point was 25 which was the lowest as compared to the other three combinations (Figure 62-A). Overall results of CaLas detection showed that the mean Ct value of VAL grafted onto Sw_NM remained the lowest at all three-time points compared to VAL grafted onto the Sw_ECRF, Tetr_NM and Tetr_ECRF combinations. Roots collected from three biological replicates of Sw_NM combination detected Ct value in the range of In Sw_ECRF, Tetr_NM, and Sw_ECRF combinations, only one biological replicate in each combination detected CaLas in the roots. (Figure 6-2B). 250

251 DGE Analysis of The Defense and Transporter Genes in Leaves Differential expression analysis of the NPR1 gene Comparative gene expression analysis of NPR1 in leaves of CaLas-infected VAL grafted onto two rootstocks under two different nutrient formulations showed significantly altered NPR1 transcripts expression levels at three different time points. The combination of Sw and NM upregulated NPR1 expression level in the CaLasinfected VAL at the 13 WAB, but the NPR1 expression level decreased significantly in 25 WAB and 37 WAB in the same combination (Figure 6-3A). CaLas-infected VAL grafted onto Sw_ECRF combination had downregulated NPR1 expression at 13 WAB and 25 WAB. However, at the 37 WAB time point, NPR1 expression level upregulated significantly in the same combination (Figure 6-3A). The pattern of NPR1 regulation in the CaLas-infected VAL grafted onto Tetr_NM and Tetr_ECRF was similar to that of the Sw_ECRF-NPR1 expression pattern at all three-time points (Figure 6-3A). The expression level of NPR1 at 13 WAB and 25 WAB was significantly lowered in Tetr_ECRF as compared to the respective time points in Tetr_NM. There were no significant differences in the NPR1 expression levels in VAL grafted onto Tetr_NM and Tetr_ECRF combinations at 37 WAB. Overall, the pattern of Tetr_NM and Tetr_ECRFinduced NPR1 expression in the CaLas-infected VAL was significantly different from the VAL/Sw_NM combination. Differential expression analysis of the NPR3 gene Differential expression analysis of NPR3 at three-time points showed fluctuations in the expression levels in all nutrient and rootstock combinations. In all nutrition and rootstock interaction combinations, CaLas-infected VAL showed either suppression or negligible expression of the gene at three different time points. However, at 25 WAB, 251

252 the expression level of NPR3 was highly increased in the Sw_ECRF, and slightly upregulated in the Tetr_NM combinations (Figure 6-3B). Differential expression analysis of the PP2B gene Relative expression quantification of PP2B in CaLas-infected VAL under the influence of different rootstocks and nutrient formulations did not change significantly. Sw_NM, Sw_ECRF and Tetr_ECRF combinations had higher PP2B expression levels at the 13 WAB among all three-time points in the CaLas-infected VAL, which was then downregulated significantly in the later two time points (Figure 6-3C). In the Tetr_NM combination, the mean of PP2B expression level was close to 0 log2 fold change (log2 FC), which was then increased significantly at 25 WAB and again decreased significantly at 37 WAB (Figure 6-3C). Overall, regardless of differences in the nutrient formulations and rootstock, PP2B followed the similar expression pattern over three different time points in VAL for all rootstock and nutrition combinations. Differential expression analysis of the ZRT2 gene ZRT2 expression pattern in VAL scion was different in response to all combinations of rootstocks and nutrient formulations. At 13 WAB, all combinations showed downregulated ZRT2 expression levels (Figure 6-3D). In the Sw_NM combination, the expression levels of ZRT2 in VAL upregulated significantly in 25 WAB and 37 WAB as compared to 13 WAB, and in Sw_ECRF combination, ZRT2 expression levels always remained lower compared to Sw_NM at all three-time points (Figure 6-3D). In the Sw_ECRF, ZRT2 expression level was significantly downregulated at 13 WAB as compared to the 25 WAB and 37 WAB, but the differences between 25 WAB and 37 WAB time points were non-significant (Figure 6-3D). CaLas-infected VAL grafted onto 4x Tetr rootstock followed the similar pattern of ZRT2 expression in both nutrition 252

253 formulations. VAL- ZRT2 gene expression always remained downregulated in all three times points in the Tetr_NM and Tetr_ECRF combinations (Figure 6-3D). Differential expression analysis of the NRAMP2 gene Expression analysis of NRAMP2 encoding metal co-transporter in the leaves of CaLas-infected VAL grafted onto the Sw and Tetr rootstocks under NM nutrient formulation followed a different pattern as compared to the ECRF nutrient formulation (Figure 6-3E). The interaction between Sw and NM downregulated NRAMP2 expression level in VAL at 13 and 25 WAB and upregulated NRAMP2 expression significantly at the 37 WAB time point (Figure 6-4E). Whereas, the effect of Tetr_NM combination suppressed NRAMP2 expression in VAL at 13 WAB, and then increased significantly at 25 WAB, and again downregulated non-significantly at 37 WAB (Figure 6-3E). ECRF nutrient formulation had a similar effect on the expression of NRAMP2 in the CaLasinfected VAL regardless of rootstocks. The expression level of NRAMP2 was remarkably high at 13 WAB time point in the CaLas-infected VAL under ECRF formulation, which later decreased significantly in 25 WAB and 37 WAB time points in the both rootstocks (Figure 6-3E). Differential expression analysis of the NIP6 gene CaLas-infected VAL exhibited differential expression of boron transporter NIP6 pattern in the different rootstocks and nutrition formulation interactions. NIP6 expression level remained upregulated in CaLas-infected VAL under the influence of Sw_NM combination. In the same combination, NIP6 expression levels were upregulated at all time points. Among these, the significantly highest expression level of NIP6 was observed at 13 WAB. (Figure 6-3F). NIP6 expression in the CaLas-infected VAL followed the zigzag pattern among all three-time points in Tetr_NM, which showed NIP6 253

254 expression upregulated at 13 and 37 WAB, and downregulated at 25 WAB (Figure 6-3F). ECRF supplemented Sw, and the Tetr rootstock showed the similar pattern of NIP6 expression in the CaLas-infected VAL in which NIP6 expression downregulated at 13 and 25 WAB, but increased significantly at 37 WAB in VAL scion (Figure 6-3F). DGE Analysis of Defense and Transporter Genes in Roots Comparative analysis of the selected genes in roots of all the combinations showed downregulation in their expression levels except NPR3, FRO2 and PP2B genes in the NM_Tetr combination (Table 6-4). NPR1 expression level downregulated nonsignificantly in roots collected from all the combinations except Sw_NM combination. Root-NPR3 expression level was significantly downregulated in ECRF applied formulation as compared to the NM formulation in both the rootstocks. Roots collected from Tetr_NM combination showed increased expression of FRO2, but this increased expression was insignificant compared to Tetr_ECRF combination. The expression level of NRAMP2 and NIP5 remained downregulated in roots of all the combinations with no significant differences. The expression level of PP2B was significantly low in Sw roots as compared to Tetr roots in both the nutrient formulations. Plant Phenotype Analysis Rootstock and nutrient formulation interactions had a significant impact on the total plant growth and appearance of the HLB-like symptoms at the end of the experiment. CaLas-infected VAL grafted onto the Sw rootstock showed significant differences in plant phenotypes under the two different nutrient formulations. CaLasinfected VAL/Sw plants fertilized with ECRF were significantly taller as compared to the NM formulation (Figure 6-5A). The average height of VAL/Sw plants under ECRF nutrition was 96 cm with the significantly lower percentage of HLB-like symptomatic 254

255 leaves at the end of 37 WAB. Whereas, the average height of the VAL/Sw combination plants grown under NM formulation was 64 cm, and about 33% of a total number of leaves were showing HLB-like symptoms at the end of 37 WAB (Figure 6-4). Phenotypic analysis of CaLas-infected VAL grown onto Tetr candidate rootstock exhibited notably improved plant phenotype as compared to the VAL/Sw under ECRF and NM nutrition (Figure 6-5B). VAL/Tetr plants were significantly taller; 108 cm and 122 cm in NM and ECRF nutrient formulations, respectively, and only 16% and 10% of a total number of leaves were showing HLB-like symptoms, respectively, in NM and ECRF fertilization formulations (Figure 6-4). VAL/Tetr_ECRF combination also showed significantly fewer branches among all combinations (Table 6-5). Discussion Defense is important for plant survival, but growth and development are essential for plant sustainability. Hence, plants optimize the distribution of energy resources towards defense and growth. However, some of the biotic and abiotic circumstances pose a great challenge to plants in maintaining a balance between defense and growth. Efficient nutrition management is a key component for increasing plant sustainability under disease and pest pressure (Dordas, 2008). In HLB, it is reported that CaLasinfected susceptible citrus cultivars have very low economic viability and sustainability (Folimonova et al., 2009). In addition, conventional nutrition management is inefficient to reduce the disease pressure and improve plant performance in the CaLas-infected plants (Xia et al., 2011; Gottwald et al., 2012). Also, DGE analysis of the nutrient transporters and plant growth associated genes discussed in Chapter 5, which proposes a hypothesis that a lack of adequate nutrition may increase HLB disease severity in VAL/SW. Therefore, learning from past experiences and referring to the 255

256 contemporaray work to understand HLB-citrus interaction, an important aim of this greenhouse study was to investigate the importance of utilizing a 4x putatively HLBtolerant rootstock and efficient nutrition management to enhance the plant performance of CaLas-infected scion and improve the overall HLB tolerance in the grafted trees. The relative quantification analysis of the micronutrient transporter encoding genes and defense associated NPR1 gene in this study showed that modifying conventional NM fertilizer formulation in combination with a newly developed putative HLB-tolerant rootstock can show differential molecular changes in the CaLas-infected VAL scion. Moreover, plant phenotypes can also be improved. The changes in the Ct values over three different time points showed that VAL grafted onto Sw and provided with ECRF nutrient formula could decrease CaLas population. These results highlight the effect of improved micronutrient management in the CaLas-infected plants. Deficiency symptoms of Zn, B, Fe and Cu nutrients in the CaLas-infected plants have been previously studied (Schumann and Spann, 2009). The results of this greenhouse experiment suggest that optimal increase of micronutrients content in the fertilizer formulation is helpful to improve plant phenotypes of HLB-susceptible citrus varieties such as the VAL/Sw combination. qrt-pcr based Ct values generated in CaLasinfected VAL/Tetr combination under NM formulation highlight the rootstock inherent capacity to lower the CaLas bacterial population at three different time points that were selected in this study. Although there were no significant differences observed between the Ct values of VAL/Tetr combination provided either with ECRF and NM, the phenotype of CaLas-infected VAL/Tetr was superior when provided with ECRF formulation. The improved phenotype of VAL/Tetr, provided with either of nutrient 256

257 formulations, shows the potential of the Tetr rootstock in defending CaLas infection under field conditions. NPR1 is a key protein in activating SAR induced plant defense (Kinkema et al., 2000; Wu et al., 2012). Also, a study on the cloning and transfer of Arabidopsis NPR1 gene into transgenic citrus reported that overexpression of Arabidopsis NPR1 in transgenic citrus could increase tolerance to HLB (Dutt et al., 2015). The differential expression patterns of NPR1 in response to the rootstock and nutrient formulations in this study underline the possibility of manipulation of defense responses in the CaLasinfected citrus by adopting improved rootstock(s) and nutrition practices. The NPR1 like- 3 (NPR3) is a negative regulator of NPR1 (Shi et al., 2013; Seyfferth and Tsuda, 2014). The role of NPR3 is to switch off the NPR1 expression under healthy plant conditions. However, it can affect the NPR1 expression under stressed conditions also. Hence, there is a negative correlation between NPR1 and NPR3 expression. In the Sw_NM combination, increased level of NPR1 in the CaLas-infected VAL at the 13 WAB time point and significant downregulation at later time points suggest that under conventional fertilizer formulation, Sw can activate the defense by upregulating NPR1 at an early stage of CaLas spread; but, NPR1-dependent defense lowers gradually as CaLas infection progresses in the plant. When susceptible Sw rootstock was fertilized with ECRF, the NPR1 expression pattern reversed in VAL scion. The opposite pattern of NPR1 expression in VAL grafted onto Sw_ECRF and Sw_NM combinations emphasizes an important role of micronutrients in altering NPR1 expression in CaLasinfected and susceptible plants. VAL/Tetr under NM and ECRF application showed lower expression of NPR1 at 13 WAB, and at later time points, it increased significantly. 257

258 The lower percentage of HLB-like symptoms, higher Ct values and reduced expression of NPR3 is consistent with the NPR1 induced possible defense at the 25 WAB and 37 WAB time points in VAL/Tetr_NM and VAL/Tetr_ECRF plants.both greenhouse and field CaLas-infected plants are found to be deficient in micronutrients such as B, Zn, Mg, Mn, Fe and Ca, with deficiencies in roots much greater than in leaves (J.W. Grosser, personal communication). Hence, genes encoding micronutrient transport ZRT2, NRAMP2, NIP6 were selected to analyze the effect of the interaction of the enhanced nutrient formulation and improved rootstock on the cellular alterations of micronutrients in the CaLas-infected VAL. Citrus is a strategy I plant which responds to Fe deficiency by acidifying rhizosphere Fe 3+ ions to Fe 2+ ions to make it available for the plant uptake (Martínez-Cuenca et al., 2013). There are many Fe 2+ regulators found in plants. Iron deficiency or toxicity regulates the expression of Fe 2+ regulators (Walker and Connolly, 2008). The Fe regulators are transcription factors, transporter genes, enzymes, etc. Fe specific transporters also compete for Zn and Mn mobility in the plants (Bashir et al., 2016). Especially Fe transporters belong to the ZIP family also regulate Zn and Mn uptake based on the availability of Fe, Mn and Zn (Guerinot, 2000). Another group of metal transporters is known as transition metal transporters such as NRAMP. The NRAMPs are present on the vacuolar membranes and their expression level changes according to the demand and supply of metals in the plant cytoplasm (Hall and Williams, 2003). The activity of ZRT2 gene is studied in Saccharomyces cerevisiae (Zhao and Eide, 1996) suggesting its role in Zn uptake in Zn limiting conditions. The results of ZRT2 expression changes in Sw_NM did not match the results obtained in S. cerevisiae which showed that ZRT2 expression increased in Zn replete condition and 258

259 downregulated under Zn deficiency (Bird et al., 2004). The whole scenario of ZRT2 and NRAMP2 regulation in the case of Sw_NM interaction suggests that under a traditional nutrition supply, and increasing CaLas infection, VAL/Sw plants experience the Zn deficiency which was exhibited by a higher percentage of HLB-like symptoms in VAL/Sw_NM plants; while, the suppression of ZRT2 and fluctuating NRAMP2 expression level in the Tetr_NM interaction suggests the efforts of Tetr rootstock to maintain Zn homeostasis in the CaLas-infected VAL. The interaction between ECRF with Sw and Tetr rootstocks influenced the ZRT2 and NRAMP2 expression levels in the CaLas-infected VAL in a similar fashion (Figure 6-3D and E). In VAL/Sw_ECRF and VAL/Tetr_ECRF plants, the expression level of ZRT2 was strongly downregulated, and NRAMP2 was significantly upregulated at 13 WAB, and in later two-time points, ZRT2 and NRAMP2 levels remained downregulated. Strong upregulation of NRAMP2 gene expression at 13 WAB time point indicates that the micronutrient deficiency may compensate by releasing the deficient nutrients from the storage organelles. The expression level patterns of ZRT2 and NRAMP2 in the VAL grafted onto two different rootstocks and grown under ECRF nutrition underscore the positive effect of enhanced micronutrient content on balancing the Zn status in CaLasinfected plants. CaLas-infected plants are also lacking adequate B content (Spann and Schumann, 2009). The deficiency of B is generally exhibited as corky veins in the plants (Yang et al., 2013). In Arabidopsis, B transporters were identified. These are Nod-like intrinsic protein (NIP) and boric acid channels. Expression of NIP6 is shoot-specific (Tanaka et al., 2008), whereas NIP5 is a root-specific transporter (Takano et al., 2006; 259

260 Tanaka et al., 2011). The study of Arabidopsis-NIP6 gene reported that NIP6 facilitates rapid permeation of boric acid across the membrane. Also, NIP6 mrna accumulated about 1.4 FC under B-deprived condition compared to the high B condition (Tanaka et al., 2008). The increased levels of NIP6 transcripts throughout all three-time points in the Sw_NM may indicate B deficiency in the CaLas-infected VAL. The pattern of NIP6 expression was quite similar in CaLas-infected VAL grafted onto Sw_ECRF and Tetr_ECRF at all three-time points. It showed downregulation of NIP6 at 13 WAB and 25 WAB and increased expression of NIP6 at 37 WAB (Figure 6-3F). The pattern of NIP6 expression in the ECRF provided rootstocks suggests a potential role of improved micronutrient nutrition practices in lowering the B deficiency. The fluctuations in the NIP6 expression levels in Tetr_NM combination support the rootstock specific response to maintain B homeostasis in the CaLas-scion. Gene expression analysis of roots did not show upregulation of the selected genes. However, there were significant differences in the expression of NPR1, NPR3, FRO2 and PP2B in both the rootstocks and nutrient formulation combinations. The NPR1 expression levels in roots of the Sw_NM combination were significantly suppressed as compared to the other combinations. A significant decrease of NPR1 in roots the Sw_NM supports the downregulated expression level of NPR1 in leaves of the same combination at 37 WAB. Significantly suppressed NPR3 expression in the ECRF formulation at the end of 37 WAB suggests that ECRF formulation may help to downregulate NPR3 expression. Suppression of NPR3 expression also supports the less suppression of NPR1 expression in roots of Sw_ECRF and Tetr_ECRF as compared to Sw_NM. Iron starvation indicator FRO2 expression changes were studied 260

261 in citrus (Martínez-Cuenca et al., 2013). According to the study, the higher levels of Zn 2+ and Mn 2+ disturb the balance between Fe, Zn and Mn in the root growth culture media of citrus. Also, FRO2 overexpression indicates an Fe 2+ starved condition (Walker and Connolly, 2008). FRO2, encoding ferric chelate reductase (FC-R) enzyme, has showed increased activity in the Fe deficient condition (Robinson et al., 1999). According to Martínez-Cuenca et al. (2013), the overexpression of FRO2 was a result of Zn 2+ and Mn 2+ competatition with Fe 2+ that could affected Fe 2+ compartmentalization, thereby induced to Fe 2+ starvation. Similarly, FRO2 upregulation in Tetr_NM and in Tetr_ECRF may be a result of competition between Zn, Mn and Fe uptake that led to possible Fe 2+ starved condition in the plants. In our study, there is not adequate evidence to explain the expression pattern of NRAMP2. However, from the results it can be speculated that Fe 2+ starvation would have increased the expression level of NRAMP2 in roots, but there was not a significant increase in the NRAMP2 expression level. Therefore, the expression pattern of NRAMP2 in roots observed in this study is not conclusive with the data available. NIP5 is a plasma membrane protein and required for B uptake under the B depleted condition (Takano et al., 2006). The downregulated NIP5 expression level in roots of all combinations suggest the plants were not experiencing B deficiency. Plants involve many other nutrient transporters that may influence the expression pattern of the selected genes in this study, directly or indirectly. However, the primarily results of the selected genes in this study underscore the importance of efficient micronutrient management to increase plant sustainability when plants are CaLas-infected. Further field evaluations and molecular analyses of the combinations are required to confirm the greenhouse results. 261

262 Overall, the greenhouse study underscores the potential role of root-applied improved micronutrient formulations to lessen the HLB disease symptoms that are caused by micronutrient deficiencies and other disease damage. Also, DGE analysis of the selected genes suggests that HLB-induced nutrient deficiency or defense development responses can be identified at the molecular level in the early stage of HLB infection. Identifying the plant's stress at an early stage of infection may help to implement preventive disease strategies needed to inhibit severe disease damage. The positive response of the Valencia/SW_ECRF trees in this study may help explain the positive turnaround of CaLas-impacted commercial sweet orange/sw trees in various groves that have been converted to new, evolving nutritional programs. The positive phenotypic and gene expression analysis results obtained in VAL grafted onto the putative HLB-tolerant Tetr rootstock opens new avenues for growers and researchers to test new scion/rootstock combinations utilizing improved HLB-tolerant citrus rootstocks along with enhanced nutrition programs in the field. 262

263 Table 6-1. Rootstock treatments Rootstock Rootstock parents Scion Swingle; 2n Valencia; Grapefruit X Trifoliate orange (Sw) (VAL) Putative HLB-tolerant tetraploid;4x (Tetr) Amblycarpa + Volkmeriana X Orange 19 (UFR-4) Valencia; (VAL) Table 6-2. Controlled release fertilizer formulations Harrell s Nursery Mix Nutrients (NM) % Harrell s enhanced controlled release fertilizer (ECRF) % N-P-K Calcium absent 4.5 Boron absent 0.07 Zinc Manganese Iron Magnesium Potassium Copper Nutrient s content is expressed in the percentage concentration. 263

264 Table 6-3. Primer sequences Gene Identification Abbr. Primer sequence* LOC / C. sinensis_nonexpressor of PR1 Ciclev m.g/LOC /NPR1 like-3 Ciclev m.g/Phloem protein 2B Ciclev m/Zinc-iron regulated 2 Ciclev m.g/Natural resistanceassociated macrophage 2 Ciclev m/Nod-like intrinsic protein;6 Ciclev m/ Nod-like intrinsic protein;5 Ciclev m/Ferric oxide reducatse-2 Glyceradehyde-3-phosphate dehydrogenase C2 NPR1 NPR3 PP2B ZRT2 NRAMP2 NIP6 NIP5 FRO2 GAPC F- 5 -CCAGAGTTGGTGGCTCTTTAT-3 R- 5 CTGAGTCTAGTGCTCGATGTATTC-3 F- 5 -AGGTTCTCAGCCTCCGGATTA-3 R- 5 -CCATCGGATTCCTCATTTC-3 F- 5 -AAGCAGATGGTGAAGTCAAGAG-3 R- 5 -CTCTCCCAATTCAACCTCCATC-3 P- 5 -/56-FAM/ACTGAAGGT/ZEN/GGTGGTGATGAAAGGT/3IABkFQ/-3 F- 5 -AAGTGGGAACCGATGGTAATC-3 R- 5 -AAACAGAGGGCGAGAATGAG-3 P- 5 -/56- FAM/CAGGACAAAG/ZEN/TTCCATTGGAGACACCA/3IABkFQ/-3 F- 5 -TGGAGTTGTGGGCTGTATTATC-3 R- 5 -CTTGGACACGGCCTTTCTTA-3 P- 5 -/56-FAM/CTTGATTGC/ZEN/ACAAGAGCGGAGTGC/3IABkFQ/-3 F- 5 -GCTGTCATGGTCGTCATCATCTTAT-3 R- 5 -TCCATGGAAAGTGCTTTAGGG-3 P- 5 -/56-FAM/CTGCTGTCA/ZEN/CCATTGCCTTTGCTG/3IABkFQ/-3 F- 5 -CTCTCAACAGGACAATCTCTG-3 R- 5 -TATGTAAGCCGGAACCTGAAC-3 P- 5 -/56-FAM/TCCGTCCCT/ZEN/TACCATAGCATTTGCG/3IABkFQ/-3 F- 5 - GGGCAGTTACAAACAACATCTC -3 R- 5 -CCACATGGCAAGACCAGATA-3 P- 5 -/56-FAM/AATATCAAA/ZEN/CGTGGCCGGAGAGCT/3IABkFQ/-3 F- 5 - GACATCAACGGTAGGAACTCG -3 R- 5 -CAATGAAGGACTGGAGAGGTG-3 P- 5 -/56-FAM/CTTCAACAT/ZEN/CATTCCCAGCAGCACC/3IABkFQ/-3 *F; forward primer sequence, R; reverse primer sequence; P; probe; /56- FAM/, 5 6 FAM FAM dye; /3IABkFQ/, ZEN- 3 Iowa Black FQ quencher. abbreviations used in the text and figures. 264

265 Table 6-4. Gene expression analyses in the roots Combinations Gene expression analysis changes (Log2 FC) NPR1 NPR3 FRO2 NRAMP2 NIP5 PP2B Sw_NM a a b a a c Sw_ECRF b c b a a c Tetr_NM b 1.25 b 2.10 a a a 0.70 a Tetr_ECRF b c ab a a b Significant differences were calculated at P < 0.05 using HD Tukey test. Different letters indicate the significant differences in the Log2 FC value for the same gene among the different combination at 37 WAB. 265

266 Table 6-5. Phenotypic measurements including tree diameters below and above the graft union, and no.of branches/tree Combinations Below graft union Above graft union No. of Dia_5cm Dia_10 cm Dia_15 cm Dia_5 cm Dia_10 cm Dia_15 cm branches Sw_NM Sw_ECRF Tetr_NM Tetr_ECRF * *Significant difference between combinations at p < 0.05 using HD Tukey s test 266

267 A B Figure 6-1. CaLas-infected Valencia sweet orange (VAL) stick grafts. A) VAL grafted onto SW rootstock. B) VAL grafted onto Tetr rootstock. 267

268 A B Figure 6-2. CaLas detection from leaves and roots of different combinations. A) Leaf samples B) Root samples at 37 WAB. In leaves, different letters indicate significant differences in the Ct values among the 3 time points for each combination. In roots, only the Sw_NM combination had all 3 biological replicates CaLas-infected, whereas only one plant was CaLas-infected in each of the remaining combinations at 37 WAB 268

269 A B C Figure 6-3. Gene expression analysis of the selected genes in leaves. A) NPR1, B) NPR3, C) PP2B, D) ZRT2, E) NRAMP2, F) NIP6. All values are expressed in Log2 fold change. Different letters indicate significant differences in the Log2 FC value for the same gene among the three different time points 269

270 D E F Figure 6-3. Continued 270

271 Figure 6-4. Phenotypic differences in rootstock-nutrient formulation combinations. Different letters indicate the significant differences in the height and % HLB symptoms within the four different scion/rootstock combinations. A B Figure 6-5. Plant phenotype at 37 WAB. A) VAL grafted onto Sw rootstock. B) VAL grafted onto Tetr rootstock. NM: nursery mix; ECRF: enhanced controlled release fertilizer 271

272 CHAPTER 7 SUMMARY AND CONCLUSIONS A good understanding of plant-pathogen interactions is important in plant breeding and plant pathology studies. Plant genetics, environmental conditions, and pathogenicity play key roles in plant-pathogen interactions. In Florida, prevalent huanglongbing (HLB) disease has been causing overwhelming economic losses due to reduced plant sustainability. Koch s postulates are not proven in case of HLB-causing putative Candidatus Liberibacter asiaticus (CaLas). Therefore, inadequate knowledge of CaLas limits researchers to identify the bacteria pathogenicity mechanism, and therefore find an efficient solution. Therefore, the overall goal of this study is to analyze the role of improved-rootstock/scion interactions to fight against HLB and to improve plant sustainability in HLB-affected plants. This dissertation includes a field study, and a greenhouse study that also includes a nutrition component. The citrus plant is a two-component system in which rootstock and scion are crucial to commercial citrus production. Citrus rootstocks and scions have a significant effect on important horticultural traits and fruit production. Therefore, the aim of the field study was to understand the differential effect of citrus rootstocks in regulating Valencia (VAL) sweet orange scion performance at different stages of HLB disease development. RNA-sequencing based comparative differential gene expression (DGE) analysis of two citrus scion/rootstock combinations was conducted. These combinations were Valencia /Swingle (VAL/SW) and Valencia /improved candidate rootstock (VAL/CAN). The commercially used SW rootstock is HLB susceptible under conventional nutrition programs. Whereas, the CAN is a putatively HLB-tolerant 272

273 rootstock developed by the citrus breeding program at the University of Florida, Citrus Research and Education Centre (UF-CREC). Fruit juice quality results obtained in the field study showed that irrespective of the stages of HLB development, the VAL fruit juice Brix and BRIX-acid ratio remained higher in VAL/CAN -asymptomatic and -symptomatic treatments as compared to the symptomatic VAL/SW combination. A lower BAR value of symptomatic VAL/SW fruit juice was possibly the result of a significant higher acidity percentage. The two years data of fruit juice quality from symptomatic and asymptomatic VAL/CAN suggest that the CAN rootstocks can produce standard VAL fruit juice quality, and therefore, this combination has potential to be commercially accepted in the citrus industry. Comparative DGE analysis of the VAL/CAN and VAL/SW combinations showed that asymptomatic VAL/SW had many strongly upregulated defense-associated genes and the defense was stronger in the symptomatic stage; whereas, CAN rootstock showed more tolerance to HLB by upregulating a smaller number of defense-related genes. Upregulation of a greater number of defense and immunity-associated genes in the VAL/SW combination as compared to VAL/CAN suggest the overreaction of commercial rootstock such as SW to HLB infection. The higher sensitivity of VAL/SW combination to HLB suggests that these plants may be investing more energy for defense and create an energy distribution imbalance between defense and routine metabolism/growth. Studies on the plant growth-defense tradeoff suggested that more energy investment in the defense comes at the cost of compromised plant growth and development (Denancé et al., 2013; Huot et al., 2014). The similar theory may support the gradually deteriorating health of HLB-affected commercial scion/rootstock 273

274 combinations. CaLas-infected VAL/CAN phenotypes showed that the plants were healthy and less symptomatic as compared CaLas-infected VAL/SW combination. DGE analysis of the field grown scion/rootstock combinations in this study showed distinct differences in the expression level of growth and development associated genes. VAL/CAN combination showed upregulation of growth factors GRF7 in asymptomatic leaves, cell wall modifying pectin methyl esterase in asymptomatic leaves, phloem regenerating transcription factor KANADI in symptomatic leaves and roots, and root promoting phytosulfokines in the symptomatic roots as compared to respective HLB stages and plant tissue of VAL/SW suggesting that CaLas-infected VAL/CAN plants may be improving tolerance to HLB through promoting phloem regeneration, cell wall modifications and root growth. Differential expression changes in the hormonal metabolism related genes between VAL/CAN and VAL/SW highlight the possible reasons of HLB-induced damage in the VAL/SW combination. Commercial rootstock such as SW has found to have severe root loss under HLB infection. Therefore, a significant overrepresentation of water deprivation and salt stress biological functional categories in the asymptomatic and symptomatic VAL/SW is apparent. Also, upregulation of abscisic acid (ABA) metabolism genes supports the significant presence of abiotic stress biological category identified by MapMan, Blast2go and Pathway studio functional analysis in VAL/SW. Auxin (AU) and ethylene (ET) -metabolism and -response genes were strongly upregulated in the VAL/SW combination; whereas, brassinosteroid (BR) and AU response genes were overexpressed in VAL/CAN combination suggesting that AU-ET crosstalk may favor the CaLas spread and HLB development in the Citrus, and AU-BR 274

275 interaction is potentially beneficial in balancing defense and growth in VAL/CAN. Jasmonic acid (JA)-activated MYC gene was strongly upregulated in the symptomatic VAL/CAN roots indicating the CAN rootstock specific hormonal genes reprogramming in the advanced stage of HLB development in plants. The activation of MYC branch also supports the suppression of ET and salicylic acid (SA) signaling pathway in the symptomatic VAL/CAN. The research findings of the transcriptome comparison of field grown VAL/CAN and VAL/SW suggest that CAN and SW rootstock are differentially acting on the VAL scion by reprogramming hormonal metabolism, defense, and growth-associated genes. The significant differential expression changes were also observed in the nutrient transporter and cell wall modification genes. Altogether, the transcriptomic analysis of CaLas-infected VAL/CAN, and VAL/SW supports that rootstock can differentially change CaLas-infected scion transcriptome. The significant upregulation of genes involved in AU-BR interactions, enhanced phloem regeneration ability, cell wall modifications, transporters regulations, and phytosulfokines signaling suggests possible role these mechanisms to improve CaLas-infected VAL/CAN sustainability and are useful to develop testable models for future HLB-citrus interactions studies. The greenhouse study included a nutrition component, as nutrition is another crucial aspect of HLB disease management. Nutrition and rootstock interactions are crucial to deciding the fate of CaLas-infected plants. Therefore, to address the issue of nutrient deficiency and symptoms severity in CaLas-infected plants, interactions of a UF-CREC developed putatively HLB-tolerant 4x (Tetr) rootstock, and an enhanced controlled release fertilizer (ECRF) formulation were studied in CaLas-infected plants 275

276 under greenhouse conditions. The two scion/rootstock combinations; VAL/Tetr and VAL/SW, and two nutrient formulations; nursery mix (NM) and enhanced controlled release fertilizer (ECRF) were tested to analyze the effect of rootstock and nutrient formulations in mitigating HLB disease severity in the VAL scion. The differential expression of nutrient transporter genes, improved plant phenotypes and a lower bacterial titer in the VAL/Tetr_ECRF, VAL/Ter_NM, and VAL/SW_ECRF support the hypothesis that rootstock and nutrient interactions can differentially modify the response of VAL scion to CaLas infection. The comparison between VAL/SW_NM and VAL/SW_ECRF plants showed that ECRF supplied VAL/SW plants had improved plant phenotype and reduced CaLas titer in the leaves, at the end of the experiment indicating that root applied ECRF formulation can improve plant phenotype and reduce CaLas spread in the infected VAL/SW commercial combination under greenhouse conditions. An improved understanding of rootstock/nutrition interactions should result in the development of improved production systems (efficient, affordable delivery of optimal nutrition) that should maximize the performance of improved rootstocks in an HLB-endemic Florida. In the situation of resource restrictions, plants must balance the energy resources spending towards growth and defense. In plants, while the defense is imperative for survival, it comes at the expense of energy that could use for routine plant growth/metabolism. As we observed that in the VAL/SW field plants, SA, JA, and ET hormonal dependent defense and plant immunity genes were strongly upregulated which suggest that VAL/SW is defending itself against the disease by deploying different defense mechanisms (Figure7-1). However, this defense may not be long 276

277 lasting, and resources required for plant growth and development is compromised. Whereas, in the VAL/CAN combination, overall in both the HLB stages SA, JA, and ETactivated defense gene responses were not strongly upregulated, but, BR and AUresponsive genes were significantly upregulated. Therefore, CAN rootstock may have been operating the defense against HLB at a lower level, but growth/metabolism is not compromised as to severely impact overall plant health (Figure7-2). Results obtained in the greenhouse study were supporting the conclusions of the transcriptomic study of field grown VAL/CAN and VAL/SW combinations. The greenhouse study results showed that improved root applied nutrition practices may help commercial rootstocks such as SW to enhance plant phenotype of CaLas-infected VAL scion. The conclusions of field and greenhouse studies showed that UF-CREC developed CAN and Tetr rootstocks can be part of the equation to combat the devastating HLB disease in Florida. Moreover, the combination of evolving, improved nutrition practices and HLB tolerant rootstocks should play a significant role in obtaining adequate HLB tolerance in new citrus plantings as necessary to ensure a sustainable and profitable future citrus industry. 277

278 Figure 7-1. Graphic presenting summary of CaLas-infected VAL/SW combination. Boxes present possible mechanism (Testable models) those are responsible for each growth and defense regulations in CaLas-infected combination. Figure 7-2. Graphic presenting summary of CaLas-infected VAL/CAN combination. Boxes present possible mechanism (Testable models) those are responsible for each growth and defense regulations in CaLas-infected combination. 278