High coverage in planta RNA sequencing identifies Fusarium oxysporum effectors and Medicago truncatularesistancemechanisms

Transcription

1 High coverage in planta RNA sequencing identifies Fusarium oxysporum effectors and Medicago truncatularesistancemechanisms Louise Thatcher Gagan Garg, Angela Williams, Judith Lichtenzveig and Karam Singh CSIRO AGRICULTURE 2015 Australasian Plant Pathology Conference, 14 th September 2015

2 Fusarium oxysporum: causal agent of Fusarium wilt disease globally ubiquitous soil-borne fungus root-infecting causes disease > 100 plant species pathogenic strains assigned to formae speciales based on host specificity Tomato Chickpea Cotton Banana Arabidopsis Medicago f.sp. lycopersici ciceris vasinfectum cubense conglutinans medicaginis Australasian Plant Pathology Conference, September 2015 Louise Thatcher

3 The cost of disease to legume industries High protein food source and livestock feed Agricultural benefits - improve soil, break crop reduce disease, pest and weed levels Fusariumwilts - losses upwards of 10% pa, but can lead to complete loss, reduce yield and quality No effective curative treatment Identify plants harbouring resistance R genes are static: new races may develop and overcome resistance C. Davies, CSIRO H. Casarotto, CSIRO Murray and Brennan (2012) Australasian Plant Pathology Conference, September 2015 Louise Thatcher

4 The potential cost of legume Fusariumwilt in Australia Significant biosecurity threat to Australian grain-legume industry Fusarium wilt of Potential Entry Establishment Spread Econ. impact Overall risk Chickpea medium high high high high ICRISAT Lentil medium high high medium medium ICARDA Lupin low high high high medium oregonstate.edu Source: Biosecurity Plan, Plant Health Australia Australasian Plant Pathology Conference, September 2015 Louise Thatcher

5 The need for a model legume pathosystem Cannot import the legume-infecting F. oxysporum f.sp. isolates Cannot predict entry of Fusarium races into Australia might introgress inappropriate R-genes R-genes don t offer long-term resistance how can we boost plant immunity or limit pathogen spread/virulence? Generated a model legume pathosystem Medicago truncatula - F. oxysporum f.sp. medicaginis (Fom) Generated robust infection assay Identified high level resistance Australasian Plant Pathology Conference, September 2015 Louise Thatcher

6 High level resistance identified Core 100 Medicago ecotype collection screened Major resistance loci identified (6.7Mb) Resistance to Fusarium oxysporum1 (RFO1) mock Relative fungal abundance (18s) Mock Fusarium 5E-10 resistant susceptible R S R S Relative fungal abundance 4E-10 3E-10 2E-10 1E Days post inoculation Thatcher and Singh unpublished Australasian Plant Pathology Conference, September 2015 Louise Thatcher

7 Tackling Fusarium wilt from host and pathogen perspectives Host Identify strong genetic resistance Decipher mechanism(s) of host resistance and susceptibility Pathogen Identify effectors non-core sequences Effector-like properties Highly in-planta inducible Australasian Plant Pathology Conference, September 2015 Louise Thatcher

8 Samples verified prior to RNAseq Verified plant defense gene response response Verified Fusariumresponse for detection of known effectors by RT-PCR estimated [fungal RNA] % Sequence at > 50 million reads per sample (10Gb Illuminia HiSeq) Verified infection completed as expected 2 days selected for RNAseq RNAseq Stranded, Illumina TruSeq Medicago reads mapped to reference Fusarium reads mapped Australasian Plant Pathology Conference, September 2015 Louise Thatcher

9 Resistant vs susceptible Medicago root transcriptomes post Fusariuminfection 4466 differentially regulated genes >1500 up >2-fold in resistant Resistant Susceptible GO Term Description p-value GO: defense response 1.80E-43 GO: response to stress 3.30E-38 GO: response to stimulus 3.80E-37 GO: signal transduction 3.00E-12 GO: signaling process 3.00E Australasian Plant Pathology Conference, September 2015 Louise Thatcher

10 Genome sequencing and assembly Medicago Isolate Forma specialis Total assembly length (M bp) average length(bp) maximum length (bp) L50 (bp) N50 total scaffolds coverage Proteins Fom-5190a medicaginis ,674 4,225,797 1,632, ,034 ~170 16,858 F. oxysporum f.sp. lycopersici (FOL 4287, race2) scaffolds F. oxysporum f.sp. medicaginis (Fom5190a) scaffolds Australasian Plant Pathology Conference, September 2015 Louise Thatcher

11 F. oxysporum genome shows unique chromosomes Lineage specific (LS) sequences found in F. oxysporum strains Rich in transposable elements Enriched for pathogenicity factors, TFs, signal transduction Can be transferred to other strains or f.sp. Chr 14 virulence chromosome Basal genome conserved in all Fusaria Slide from Ma et al and D. Gardiner Australasian Plant Pathology Conference, September 2015 Louise Thatcher

12 Prediction of Fomdispensable chromosomes Outer ring- Fol chrs highlighting CDCs(red) a - gene density (100kb windows) b - repeat density (100 kb windows) c - GC content (50kb windows) Region of Fol chrs overlapped by d Fom5190a Dispensable sequences >50% hypothetical Effector properties Small, secreted proteins Thatcher, Williams, Hane and Singh Australasian Plant Pathology Conference, September 2015 Louise Thatcher

13 High priority candidate effector set 4 Secreted In Xylem genes SIX proteins first identified in xylem sap of Fol infected tomato Fom5190a homologs SIX1, SIX8, SIX9, SIX13 Fom5190a SIX1 and SIX8 best matches to other legume-infecting f. sp. qrtpcrverified in planta expression 6 novel effectors Hypothetical / uncharacterized proteins Best matches to proteins from other F. oxysporum f. sp. F. fujikuroi F. solani Colletotrichum species Host specific Thatcher, Williams and Singh Two candidates most conserved with other legume-infecting f. sp Australasian Plant Pathology Conference, September 2015 Louise Thatcher

14 Summary High coverage in planta RNA sequencing High confidence candidate effector set Host resistance mechanisms Includes Fusarium, F. oxysporumf.sp., Fom5190a, and legume-conserved genes Transcription factors for manipulation and RFO1 candidates Current and future work functional characterization, focusing on effectors highly conserved amongst legume-infecting f. sp. characterize host resistance / susceptibility cloning major Resistance allele in Medicago A Australasian Plant Pathology Conference, September 2015 Louise Thatcher

15 Thank you CSIRO Angela Williams (UWA) Gagan Garg Elaine Smith Lars Kamphuis (UWA) Karam Singh (UWA) Curtin James Hane Judith Lichtenzveig ICRISAT Mamta Sharma Sarwar Azam Rajeev Varshney CSIRO AGRICULTURE FLAGSHIP