BIOLOGY OF POSTHARVEST DISEASE AND THE ROLE OF PLANT CELL WALLS

Transcription

1 Postharvest Technology of Horticultural Crops 17 June 2010, UC Davis BIOLOGY OF POSTHARVEST DISEASE AND THE ROLE OF PLANT CELL WALLS Ann Powell, Plant Sciences Department, Univ. of California, Davis Dario Cantu, Barbara Blanco-Ulate, KaLai Lam, John Labavitch, Alan Bennett Tomato fruit provide inexpensive nutrients world-wide Grown on all continents in temperate to tropical conditions Utilized in the diets of all cultures Provide human nutrients (Vitamin C, antioxidants - lycopene, carotene, anthocyanin)

2 Tomato is a valuable crop Total US crop value ca. $1.5 billion California produces 35% of the world s processing tomatoes (value: $650 million) Value of the exported crop in US is about $300 million $39 million crop in Solano County in 2009 Most consumed fruit vegetable in the world Top Tomato Producers 2008 (in tons) China 33,811,702 United States 12,575,900 Turkey 10,985,400 India 10,260,600 Italy 5,976,912 World Total Ripening results in palatable, marketable fruit BREAKER TURNING PINK LIGHT RED

3 During fruit ripening Relative value Ethylene Cell wall disassembly Soluble sugars Lycopene Aroma volatiles Pathogen susceptibility Chlorophyll Firmness ph Starch Ripening time Ripening and susceptibility Ripening Infection

4 Botrytis cinerea Botrytis cinerea (Botryotinia fuckeliana, gray mold) Filamentous ascomycete Infects more than 200 host plants Model of polyphagous necrotrophic plant pathogen Genomes of two isolates have been sequenced and annotated Targeted mutagenesis possible Mutants are available ( known virulence factors) Botrytis cinerea infects and rots ripe fruit

5 Due to ripe fruit rotting Mature viable seeds released Important crop losses Plant is helped by breakdown of fruit during rotting to release mature seed >50% of crop in developing countries is lost and 85% of that is due to soft rot Seeds in green fruit are not viable and are, therefore, protected in non- decomposed fruit Treatments and packaging to reduce losses are expensive and sophisticated Provides an environment where human pathogens can grow To reduce rotting of ripe fruit, we would like to separate ripening and rotting Ripening processes Botrytis could utilize Softening Sugar accumulation Changes in ph etc. Developmental/ripening changes that facilitate infections Defense/pathogen response system Senescence

6 Is susceptibility separable from ripening? Infections of green and ripe fruit 1 dpi 2 dpi 3 dpi 3 dpi GFP-B. cinerea MG RR Cantu et al., Plant Phys. 150:

7 Fruit and fungal gene expression Fungal attack strategies vs fruit response tactics Role of development/ripening Simultaneous analysis of Botrytis and tomato genes Tomato transcriptome analysis Affymetrix Tomato microarrays MG ca. 9K genes RNA from 1 day post inoculation RR 3 Biological replicates Statistics with R in BioConductor SMA (Statistics for Microarray Analysis) for analysis ANOVA, P<0.001, > 1.5x change 9% 78% Cantu et al., Plant Phys. 150:

8 Transcriptional profiling of fruit responses Infection with Botrytis induces the expression of genes encoding defense or antimicrobial functions Cantu et al., Plant Phys. 150: Fruit and fungal proteomes Proteins released from intact infected fruit (overnight, 1.5 M NaCl) 3 days post inoculation LC-MS/MS of 5 x gel separated protein samples (CCRC, U. Ga) Peptides identified using B. cinerea and tomato sequence databases

9 Proteins in the microenvironment of infected fruit Infected MG Infected RR Infected rin Total MS/MS spectra 1,623 7,560 5,583 Total tomato spectra 1,196 (74%) 6,711 (89%) 4,847 (87%) Total Botrytis spectra 427 (26%) 849 (11%) 736 (13%) Total proteins (<1% FDR) Total tomato proteins 119 (73%) 456 (92%) 374 (89%) Total Botrytis proteins 44 (27%) 41 (8%) 47 (11%) Could distinguish between tomato and Botrytis proteins MG (119) RR (456) MG (44) RR (41) rin (374) 12 rin (47) Botrytis and tomato proteins Tomato Botrytis

10 Defenses activated in green fruit Frequency of defense-like HR responses decreases with ripening MG BR LR RR At 18 hpi: Oxidative burst H 2 O 2 Cantu et al., Plant Phys. 150: Cell wall reinforcements Lignin Suberin Defenses activated but outcome depends on fruit development Oxidative burst and cell wall reinforcements after MG infection Microarray and proteomics results show that defenses activated in MG fruit in response to Botrytis. But ripening of uninfected fruit increases expression of pathogen response genes SA deficient MG nahg fruit fail to develop the HR-like response and are more susceptible than WT fruit ABA deficient sitiens fruit show HR-like responses at RR stage, but eventually are as susceptible as WT fruit

11 Botrytis induces susceptibility functions in host TRANSCRIPTIONAL CHANGES ELICITED BY BOTRYTIS ALSO INDUCED/SUPPRESSED BY NORMAL RIPENING Cantu et al., Plant Phys. 150: Botrytis induces ripening functions Microarray results q RT-PCR results Botrytis induces susceptibility in the host Cantu et al., Plant Phys. 150:

12 Botrytis induces both resistance and susceptibility in fruit CNR What if ripening does not occur? DEVELOPMENTAL CONTROL Transcription Factors??? TAGL1 CNR NOR RIN Non Ethylene Mediated Pathway Ethylene Biosynthesis Competence to Respond to Ethylene CLIMACTERIC RESPIRATION Ethylene Sensing and Signal Transduction GR NR Expression of Ripening Related Genes RIPENING

13 Non-ripening tomato mutants Perturbed ripening program: Fail to ripen in response to ethylene Do not turn red Cnr Do not soften MG (31 dpa) RR (42 dpa) Ac wild type MG (31 dpa) RR (42 dpa) rin nor dpa = days post-anthesis Ripening mutant susceptibility to Botrytis MG (31 dpa) RR (42 dpa) 1 dpi 2 dpi 3 dpi 1 dpi 2 dpi 3 dpi

14 What fruit ripening events does Botrytis need to infect? Separation of susceptibility pathways and ripening pathways (RIN, CNR, Ethylene) Identify ripening functions that Botrytis requires (NOR) Plant cell walls The cell wall polysaccharide matrix - Cellulose microfibrils - Hemicelluloses (xyloglucans) Expansin - Pectin (HGA, RGI, RGII) Polygalacturonases (PG)

15 Breakdown and remodeling plant cell walls Plants disassemble their cell walls (e.g., cell growth, fruit ripening) Brummell and Harpster, 2001 Botrytis degrades plant cell walls Has at least 6 PGs (BcPGs) that are differentially expressed in vitro and in planta (Wubben et al., 1999) BcPG1 and BcPG2 are required for full virulence (ten Have et al., 1998) Most BcPGs show distinct product profiles when assayed in vitro (Kars et al., 2005) Plant susceptibility to Botrytis correlates with expression of proteins that inhibit BcPGs (PGIPs - PG-inhibiting protein, Powell et al., 2000) Plant cell wall disassembly in infected fruit Uninfected Infected or rotted

16 Tomato PG induced in only some infections 31 dpa 42 dpa H W I H W I H W I H W I H W I H W I H W I H W I 31 dpa 42 dpa H I-1 I-3 H I H I-1 I-3 H I Cell wall polysaccharide degrading enzymes Pathogenesis Botrytis cinerea Ripening Tomato fruit PG PL PME etc. Plant Cell wall PG PL PME etc. -PG 35S-antisense LePG (0.85% PG) Smith et al., Exp 35S-suppressed LeEXP1 (~3% Exp1) Brummell et al., 1999 Host tissue maceration and colonization Fruit softening -PG-Exp Powell et al., 2003

17 Botrytis and reduced fruit PG and expansin 80 LR Fruit showing disease symptoms [%] RR Botrytis biomass [SI] LR AC -Exp -PG -PG-Exp a b ab c a b b 36 hpi 72 hpi c RR a b a b b c c d 36 hpi 72 hpi dpi Cantu et al PNAS 105: Role for cell wall architecture and composition? Botrytis biomass [SI] 20 a a a a MG LR RR a a aa aaaa a a a a a a a a a a b a aa a a a b AC -Exp -PG -PG-Exp BOTRYTIS GROWS IN VITRO ON UNINFECTED FRUIT CELL WALLS IF PG AND EXP ARE EXPRESSED MG LR AC AC -Exp -PG -PG-Exp 10 0 b aaa b a 1 dpi 3 dpi 6 dpi RR Cantu et al., PNAS 105:

18 Tissue maceration and rotting AC 9 dpi -PG-Exp 9 dpi AC -PG-Exp 3 dpi 3 dpi Uninfected fruit soften during ripening Infected fruit softening is accelerated Maceration and decomposition Uninfected fruit softening reduced 15% Infected fruit softening is eliminated No maceration and decomposition Plant cell wall architecture WHAT IS IT S INFLUENCE ON THE POPULATION OF APOPLASTIC PROTEINS? Apoplastic proteins: PR (pathogen response) proteins Cell wall remodeling proteins Defense proteins Receptors AC -PG-Exp IM MG BR LR RR IM MG BR LR RR Wild-type +ppgip LePGIP DOES CELL WALL POLYSACCHARIDE DISASSEMBLY SUPPRESSION RETAIN PGIPS AND OTHER ANTI-FUNGAL PROTEINS AT STRATEGIC POSITIONS?

19 Cell wall polysaccharide degrading enzymes Pathogenesis Botrytis cinerea PG PL PME etc. Plant Cell wall Ripening Tomato fruit PG PL PME etc. Host tissue maceration and colonization Fruit softening Fruit cell wall targets of B. cinerea MG (31 dpa) RR (42 dpa)

20 Pectin targets of B. cinerea Botrytis Artillery (PGs) deconstruct pectins

21 31 dpa 42 dpa 31 dpa 42 dpa 31 dpa 42 dpa 31 dpa 42 dpa Tomato Actin 31 dpa 42 dpa 31 dpa 42 dpa 31 dpa 42 dpa 31 dpa 42 dpa

22 Tomato Actin 31 dpa 42 dpa 31 dpa 42 dpa 31 dpa 42 dpa 31 dpa 42 dpa Tomato Actin 31 dpa 42 dpa 31 dpa 42 dpa 31 dpa 42 dpa 31 dpa 42 dpa

23 Fruit rotting or decomposition Relies on some, but not all, fruit ripening events Botrytis induces ripening processes in green fruit initiates the induction of susceptibility by exploiting endogenous developmental programs Anti-pathogen defense responses are induced in green fruit Pectin polysaccharides in the fruit cell wall are target Specific BcPGs used in ripening and non-ripening susceptible fruit But significant plasticity of the plant responses to Botyrtis that changes during ripening/development Thank you NSF IOB NSF IOS Plant Sciences Department GSR-ships Dario Cantu Zac Chestnut Barbara Blanco Institute of Technology, Costa Rica Fulbright Fellowship (JL) Hong Wang Hong Wang Dario Cantu Javier Lopez Zac Chestnut Ariel Vicente John Labavitch Carl Greve Alan Bennett Jan Van Kan, U. Wageningen Carl Bergmann, U. Georgia KaLai Lam Anthony Zhou Barbara Blanco