1. Evolution and function of the P. sojae effectorome: What we’ve learned from next generation sequencing Brett Tyler Virginia Bioinformatics Institute Virginia Tech

2. Coevolutionary struggle between plants and pathogens pathogen plant SUSCEPTIBLE

3. Coevolutionary struggle between plants and pathogens pathogen plant RESISTANT

4. Coevolutionary struggle between plants and pathogens pathogen plant SUSCEPTIBLE

5. Coevolutionary struggle between plants and pathogens RESISTANT pathogen plant

6. Coevolutionary struggle between plants and pathogens pathogen plant SUSCEPTIBLE

7. Plant defenses and effectors Effectors suppress PAMP-triggered immunity (PTI) and Effector-triggered immunity (ETI) Extracellular effector intracellular effector R R immunity PRR Pathogen-associated molecular pattern (PAMP) Pattern-recognition receptor (PRR) PTI R R ETI Some effectors suppress ETI Some effectors suppress PTI

8. Effector – R gene Coevolutionary struggle Hein et al Molecular Plant Path (2009)

10. Loss of Avr gene function by mutation P. infestans Avr4 (van Poppel et al., 2008) Additional examples: C. fulvum Avr4

12. New functional variants Hyaloperonospora arabidopsidis ATR13 P. sojae Avr1b

14. Huge superfamily of Avr-like genes with accelerated divergence P. sojae ~ 400 P. ramorum ~ 370 P. infestans ~ 550 H. arabidopsidis ~ 130 Includes all 15 cloned oomycete avirulence genes PsAvr1a , PsAvr1b, PsAvr1k , PsAvr3a , PsAvr3c , PsAvr4/6 , PsAvr5 PiAvr1, PiAvr2 , PiAvr3a, PiAvr4, PiAvrPlb1/ ipiO1, PiAvrPlb2 , HpAtr1, HpAtr13 Jiang et al (2008) PNAS 105(12), 4874-4879 Signal Peptide RXLR dEER 27aa 10-67aa 14aa 7-55aa 126aa 28-835aa median range

16. Sequencing the variation in P. sojae Forster et al.(1994) MPMI 7 , 780-791

17. Distribution of variation in the effectorome

19. Effector Gene Expression Program Affymetrix GeneChip data quantile-normalized to an external reference E E

20. Expression Patterns of Elicitors and Suppressors log2 expression levels 0 3 6 12 hours post-inoculation (biotrophic phase) elicitors suppressors suppression Early expressed effectors suppress plant response to later effectors

21. 1.GFP+GFP+INF1 2.Avh172+GFP+INF1 3.GFP+Avh238+INF1 4.GFP+Avh238+INF1 5.Avh172+Avh238+INF1 6.GFP+Avh238 7.Avh172+Avh238 Avh172 1 6 4 5 2 3 7 Cooperation among effectors Avh238 INF1 Yuanchao Wang Nanjing Agricultural University immediate-early early PAMP

22. 1.GFP+GFP+INF1 2.Avh172+GFP+INF1 3.GFP+Avh238+INF1 4.GFP+Avh238+INF1 5.Avh172+Avh238+INF1 6.GFP+Avh238 7.Avh172+Avh238 Avh172 1 6 4 5 2 3 7 Cooperation among effectors Avh238 INF1 Yuanchao Wang Nanjing Agricultural University immediate-early early PAMP

23. 1.GFP+GFP+INF1 2.Avh172+GFP+INF1 3.GFP+Avh238+INF1 4.GFP+Avh238+INF1 5.Avh172+Avh238+INF1 6.GFP+Avh238 7.Avh172+Avh238 Avh172 1 6 4 5 2 3 7 Cooperation among effectors Avh238 INF1 Yuanchao Wang Nanjing Agricultural University immediate-early early PAMP

24. 1.GFP+GFP+INF1 2.Avh172+GFP+INF1 3.GFP+Avh238+INF1 4.GFP+Avh238+INF1 5.Avh172+Avh238+INF1 6.GFP+Avh238 7.Avh172+Avh238 Avh172 1 6 4 5 2 3 7 Cooperation among effectors Avh238 INF1 Yuanchao Wang Nanjing Agricultural University immediate-early early PAMP

25. 1.GFP+GFP+INF1 2.Avh172+GFP+INF1 3.GFP+Avh238+INF1 4.GFP+Avh238+INF1 5.Avh172+Avh238+INF1 6.GFP+Avh238 7.Avh172+Avh238 Avh172 1 6 4 5 2 3 7 Cooperation among effectors Avh238 INF1 Yuanchao Wang Nanjing Agricultural University immediate-early early PAMP

26. Avh172 and Avh238 are essential for infection Stable and transient silencing of effector genes in P. sojae

27. RNA seq reveals that disproportionately few genes contribute most transcripts P. sojae RXLR effector genes ranked by expression during infection

28. Expression and variation of the P. sojae effectorome Mutations Per gene > 10 5-10 1-4 0 Expression during infection (log 2 RPKM) Expression in mycelia (log 2 RPKM) Avh172 Avh238

30. Evolution of effector gene number Computer modeling Seasons 400 350 300 250 200 150 100 50 0 0 5000 10000 15000 20000 Effector Genes

31. Predictions: a few genes contribute most to virulence many gene duplications and pseudogenes Individual contributions cumulative contribtions Contributions of genes to virulence in model Effector genes ranked by virulence contribution DEATH Virulence Contribution 50% 90% BIRTH

32. Predictions: a few genes contribute most to virulence P. sojae RXLR effector genes ranked by expression during infection Avh172 Avh238

33. Many effector genes are drifting under neutral selection

34. Duplications and pseudogenes in the Avr3a/5 region Predictions: many gene duplications and pseudogenes