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Published

Peter Dodds, CSIRO …

Peter Dodds, CSIRO
Effectors from flax rust and Puccinia graminis

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  • 1. University of MinnesotaEffectors from flax rust and Puccinia graminis
  • 2. Gene-for-gene resistance in flax: Basic Research Questions 1. What are rust resistance genes and how do they work? 2. How do rusts overcome resistance? 3. Can we apply knowledge from the model system to cereal rust diseases?
  • 3. Gene-for-gene resistance in flaxR R genes encode recognition components of plant immune systemS ~30 Rust resistance genes in flax Cloned 20 R genesR Locus Cloned L L, L1-L11 TIR NB LRR M M, M1, M3 N N, N1, N2 P P, P2 Signalling Activation Recognition
  • 4. Flax rust Avirulence Genes: Haustorially expressed secreted proteinsIdentified Avr genes from four loci - encode small secreted proteins - expressed in haustoria HR R AVR - recognised inside plant cells cell death L5 Bison Avr Locus Matching R genes AvrL567 L5, L6, L7 AvrM M AvrP123 P, P1, P2, P3 35S promoter AvrL567 Nos AvrP4 P4 No signal peptide (cytoplasmic)
  • 5. R-Avr recognition occurs by direct interaction Receptor ligand model Avr proteins escape recognition R by altering surface residues Avr AvrL567-A Yeast-2-hybrid assays AvrL567 A B DL5 AvrL567-D AvrM avrMM
  • 6. Flax rust AvrM protein secreted and translocated to host cell Anti-haustoria Anti-AvrM Plant Cytoplasm Haustorium 0.5 μm Rafiqi et al Plant Cell 2010 22:2017
  • 7. AvrM protein is taken up in host vesicles Haustorium Plant cytoplasm Plant Haustorium cytoplasm Pamela Gan and Adrienne Hardham et al unpublished
  • 8. AvrM in the plant endomembrane system Plant cytoplasm Haustorium Pamela Gan and Adrienne Hardham et al unpublished
  • 9. Effector delivery is pathogen-independent and requires N-terminal uptake signals Plasmolysed cells SP-GFPSP-AvrM-GFP SP GFP Uptake signal MKFLKPDQVQKLSTDDLITYMAEKDKNVRDL FL V Ls LI Yqaekd Rafiqi et al Plant Cell 2010 22:2017
  • 10. Effector delivery from transgenic rust also requires the N-terminal uptake signalM CH5F2-96 AvrM-YFP AvrM-∆106-153-YFP
  • 11. Secreted effectors in rust infection and hostimmunity R Immune Host Recognition Manipulation
  • 12. Stem rust effectoromics• What is the set of stem rust effector proteins? > Secreted (have signal peptide) > Expressed in haustoria• Which of these are recognised by wheat R genes? > Genetic association > Functional assays
  • 13. Wheat stem rust genomic resourcesReference genome – strain CRL75-36-700 (7a) Broad Institute (Cuomo, Szabo) - 81.5 Mbp; ~92% coverage - 20,567 genes annotated - 1342 have signal peptides for secretionFour Australian isolates – represent founder strains - 21-0 - 126 - 194 - 326
  • 14. Identifying stem rust effector genes Isolated haustoria• Haustorial isolation (21-0) haustoria > 13K ESTs > Illumina RNAseq• Genome sequence - 21-0 454/Illumina - 126 Illumina - 194 Illumina - 326 Illumina• Illumina RNAseq – infected tissue - 21-0 - 194 - 126 - 326
  • 15. Identifying stem rust effector genes21-0 Haustorial ESTs 7a reference genome 212 with SP - 84 predicted SP 56 no SP (incorrect annotation) 77 absent 105 no SP - predicted SP (incomplete clones) - Manually curate using genome and RNAseq data 369 candidates
  • 16. p7a genome-assisted building of 21-0 reference genome Assembled on 7a ref (79 Mbp) De novo assembly of non-aligned reads (18 Mbp) MbpGene calling based on Illumina RNAseq data Gene models 13,921 5370 Candidate effectors 339 30
  • 17. Sequence Variations Among Different Pgt Isolates 21-0anchored 100 pg7a % Similarity Plot 80 126 100 80 326 100 80 100 194 80 SuperContig1 Pgt-p7a genome assisted assembly of Pgt-21-0 genome and subsequent assembly of genomes of other Australian strains assisted by derived Pgt21-0 genome (Illumina GAII/HiSeq 100 bp paired-end sequencing)
  • 18. Strain variation in effector genesOnly 13 effector candidates show no differences between five strains ~40 have < 5 SNPs across five strains SNPs in secreted effector genes reference Protein strain length gene 7a 126 194 21-0 326 (aa) PGTT_02220 15 18 20 10 5 189 PGTT_19738 7 18 17 7 4 209 PGTT_01618 23 30 27 12 8 354 PGTT_07595 17 19 29 15 10 339 PGTT_09048 17 14 4 3 177 PGTT_05791 9 6 5 2 103 PGTT_13278 21 26 31 20 12 425 PGTT_10668 9 9 6 1 123 PGTT_12848 7 7 17 2 2 138 PGTT_00796 9 11 2 1 115 PGTT_02680 14 10 2 134 PGTT_06969 5 9 23 5 4 217 PGTT_08533 10 11 9 1 1 117
  • 19. Expression variation of candidate effectors Relative 18.00 absentExpression ~100fold absent (log2) 16.00 14.00 12.00 10.00 8.00 6.00 4.00 HS_210 2.00 210 0.00 126 194.00 326.00
  • 20. Functional Assay: bacterial type 3 delivery Pseudomonas fluorescens ‘Ethan’ (Pfo): Express as fusion to AvrRPM1 bacterial effector Adenylate cyclase AvrM delivery delivery to wheat to tobacco (M) cAMP mg/protein700060005000 Agro:4000 mock avrM30002000 Agro:1000 Pfo AvrM 0 Pfo: Pfo: avrM AvrRPM1 Pfo: AvrM
  • 21. Wheat accessions with different R genes Pfo inoculation Differentials R genesBW56 Sr31Acme Sr9g, SrXAgent Sr24Comb X Sr5b, Sr7b, Sr9bFestiguay Sr30Gatcher L06-139 Sr2, Sr5, Sr6, Sr8a, Sr12Kite Sr26Banks L09-94 Sr5, Sr8a, Sr9b, Sr12Line S L09_10 Sr13, Sr17 DAB stainMendos L09_89 Sr11,Sr17, Sr36Norka L09_95 Sr15Trident L09_91 Sr38W3534 Sr22
  • 22. Screening Effector Candidates 13 Wheat accessions1 2 34 5 67 8 910 11 12 Treatments: 5. Lcontig10 al4 1. Mock 6. Lcontig625_al1 2. Pfo Ethan/AvrRPM1:Cya 7. Lcontig75_al1 13 3. Lcontig10 _al1 8. Lcontig10 al6 4. Lcontig10 _al3 9. Lcontig10 al7
  • 23. Two potential effector-R gene recognition responsesKite Sr26 Norka Sr15 Treatments Mock G1LContig1_al1_G1#1-1 G1LContig1_al5_G1#1-9 G1LContig7_al1-al2_G1#2-4 G1LContig7_al3_G1#2-7 G1LContig7_al4_G1#2-3
  • 24. AcknowledgementsCSIRO-Plant Industry U. Sydney ANUNarayana Upadhyaya Robert Park Adrienne HardhamRobyn East Colin Wellings David JonesRohit Mago Pamela Gan U. Minnesota Maryam RafiqiJeff Ellis Les SzaboXiaodi Xia Jerry JohnsonKim Newell Jane GlazebrookJen Taylor Fumi KatagiriAndrew SpriggsMaud Bernoux Broad InstituteGreg Lawrence Christina Cuomo Jane Wilkinson Two Blades Foundation