Evolution and function of the P. sojae effectorome: What we’ve learned from next generation sequencing Brett Tyler Virgini...
Coevolutionary struggle between plants and pathogens pathogen plant SUSCEPTIBLE
Coevolutionary struggle between plants and pathogens pathogen plant RESISTANT
Coevolutionary struggle between plants and pathogens pathogen plant SUSCEPTIBLE
Coevolutionary struggle between plants and pathogens RESISTANT pathogen plant
Coevolutionary struggle between plants and pathogens pathogen plant SUSCEPTIBLE
Plant defenses and effectors Effectors suppress PAMP-triggered immunity (PTI) and Effector-triggered immunity (ETI) Extrac...
Effector – R gene Coevolutionary struggle  Hein et al Molecular Plant Path (2009)
Loss of Avr gene transcription <ul><li>Loss of transcription allows later reactivation of the gene </li></ul><ul><li>May i...
Loss of Avr gene function by mutation P. infestans Avr4 (van Poppel et al., 2008) Additional examples: C. fulvum Avr4
New functional variants <ul><li>P. infestans  Avr3a </li></ul><ul><li>Avr3a is essential for infection  </li></ul><ul><ul>...
New functional variants Hyaloperonospora arabidopsidis ATR13 P. sojae Avr1b
<ul><li>Positive selection occurs when a mutation that causes an amino acid change is favored over one that does not </li>...
Huge superfamily of Avr-like genes with accelerated divergence P. sojae  ~ 400 P. ramorum  ~ 370 P. infestans  ~ 550 H. ar...
Sequencing of  P. sojae  genomes <ul><li>P6497 reference strain. Genotype I </li></ul><ul><ul><li>2002-2004 DOE JGI </li><...
Sequencing the variation in  P. sojae Forster et al.(1994) MPMI  7 , 780-791
Distribution of variation in the effectorome
Characterization of  P. sojae  transcriptomes <ul><li>3000 Sanger ESTs 1998 </li></ul><ul><ul><li>Mycelia, zoospores, infe...
Effector Gene Expression Program Affymetrix GeneChip data quantile-normalized to an external reference E E
Expression Patterns of Elicitors and Suppressors log2 expression levels 0 3 6 12 hours post-inoculation (biotrophic phase)...
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 Avh...
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 Avh...
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 Avh...
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 Avh...
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 Avh...
Avh172 and Avh238 are essential for infection Stable and transient silencing of effector genes in  P. sojae
RNA seq reveals that disproportionately few genes contribute most transcripts P. sojae RXLR effector genes ranked by expre...
Expression and variation of the  P. sojae  effectorome Mutations Per gene > 10 5-10 1-4 0 Expression during infection (log...
Hypothesis : balancing selection results in expansion and divergence of effector gene family <ul><li>Gene duplication incr...
Evolution of effector gene number Computer modeling Seasons 400 350 300 250 200 150 100 50 0 0 5000 10000 15000 20000 Effe...
Predictions: a few genes contribute most to virulence   many gene duplications and pseudogenes Individual contributions cu...
Predictions: a few genes contribute most to virulence P. sojae RXLR effector genes ranked by expression during infection A...
Many effector genes are drifting under neutral selection
Duplications and pseudogenes in the Avr3a/5 region Predictions: many gene duplications and pseudogenes
Genomics of oomycete effectors <ul><li>Co-evolutionary conflict between pathogens and their hosts drives rapid change in e...
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Tyler impact next gen fri 0900

  1. 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. 2. Coevolutionary struggle between plants and pathogens pathogen plant SUSCEPTIBLE
  3. 3. Coevolutionary struggle between plants and pathogens pathogen plant RESISTANT
  4. 4. Coevolutionary struggle between plants and pathogens pathogen plant SUSCEPTIBLE
  5. 5. Coevolutionary struggle between plants and pathogens RESISTANT pathogen plant
  6. 6. Coevolutionary struggle between plants and pathogens pathogen plant SUSCEPTIBLE
  7. 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. 8. Effector – R gene Coevolutionary struggle Hein et al Molecular Plant Path (2009)
  9. 9. Loss of Avr gene transcription <ul><li>Loss of transcription allows later reactivation of the gene </li></ul><ul><li>May involve rapid epigenetic changes </li></ul>P. sojae Avr1b P. sojae Avr3a
  10. 10. Loss of Avr gene function by mutation P. infestans Avr4 (van Poppel et al., 2008) Additional examples: C. fulvum Avr4
  11. 11. New functional variants <ul><li>P. infestans Avr3a </li></ul><ul><li>Avr3a is essential for infection </li></ul><ul><ul><li>silencing reduces infection (Bos et al., 2010) </li></ul></ul><ul><ul><li>R genes are being sought against EM allele </li></ul></ul>Avr vir Avr vir Avr vir
  12. 12. New functional variants Hyaloperonospora arabidopsidis ATR13 P. sojae Avr1b
  13. 13. <ul><li>Positive selection occurs when a mutation that causes an amino acid change is favored over one that does not </li></ul><ul><li>Synonymous mutations are frequently lost by genetic drift </li></ul><ul><li>Non-synonymous mutations are preserved by selection </li></ul><ul><li>Ability to grow on a plant containing a resistance gene is an example of positive selection </li></ul><ul><li>Measured by a statistical test comparing the frequencies of synonymous mutations (dS) to that of non-synonymous ones (dN), adjusted for the frequency of codons available for each type of substitution. </li></ul><ul><li>ATR1 and Avr1b display dN/dS >> 1 </li></ul><ul><li>dN/dS ratio is a valuable tool for finding pathogen genes involved in co-evolutionary conflict with host </li></ul>Positive (divergent) selection
  14. 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
  15. 15. Sequencing of P. sojae genomes <ul><li>P6497 reference strain. Genotype I </li></ul><ul><ul><li>2002-2004 DOE JGI </li></ul></ul><ul><ul><li>Sanger sequencing with Megabase </li></ul></ul><ul><ul><li>9X draft assembly </li></ul></ul><ul><li>P6497 finishing </li></ul><ul><ul><li>2007-2010 Hudson Alpha Institute </li></ul></ul><ul><ul><li>Gap filling; BAC finishing; genetic mapping </li></ul></ul><ul><li>Resequencing. 2009 VBI CLF </li></ul><ul><ul><li>P7076 (genotype II) </li></ul></ul><ul><ul><li>P7074 (genotype III) </li></ul></ul><ul><ul><li>P7064 (genotype IV) </li></ul></ul><ul><ul><li>454 Titanium sequencing. $25,000 each </li></ul></ul><ul><ul><li>9x coverage. Newbler assembly </li></ul></ul>
  16. 16. Sequencing the variation in P. sojae Forster et al.(1994) MPMI 7 , 780-791
  17. 17. Distribution of variation in the effectorome
  18. 18. Characterization of P. sojae transcriptomes <ul><li>3000 Sanger ESTs 1998 </li></ul><ul><ul><li>Mycelia, zoospores, infected hypocotyls </li></ul></ul><ul><ul><li>Qutob, D.et al (2000) Plant Phys. 123, 243-253. </li></ul></ul><ul><li>27,000 Sanger ESTs 2002 </li></ul><ul><ul><li>Mycelia (various treatments), zoospores, infected hypocotyls </li></ul></ul><ul><ul><li>Torto-Alalibo et al (2007). Mol. Plant-Microbe Interact. 20, 781-793. </li></ul></ul><ul><li>Affymetrix microarrays 2006 </li></ul><ul><ul><li>15,800 probe sets (along with soybean) </li></ul></ul><ul><ul><li>About half the effector genes are missing </li></ul></ul><ul><ul><li>Various treatments including infection </li></ul></ul><ul><li>ABI Solid™ sequence tags 2009 </li></ul><ul><ul><li>50 million from mycelia </li></ul></ul><ul><ul><li>200 million from infected soybean (~40m from P. sojae ) </li></ul></ul>
  19. 19. Effector Gene Expression Program Affymetrix GeneChip data quantile-normalized to an external reference E E
  20. 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. 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. 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. 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. 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. 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. 26. Avh172 and Avh238 are essential for infection Stable and transient silencing of effector genes in P. sojae
  27. 27. RNA seq reveals that disproportionately few genes contribute most transcripts P. sojae RXLR effector genes ranked by expression during infection
  28. 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
  29. 29. Hypothesis : balancing selection results in expansion and divergence of effector gene family <ul><li>Gene duplication increases virulence and is selected for </li></ul><ul><li>Gene duplication increases selection pressure from R genes </li></ul><ul><ul><li>Gene divergence or loss is selected for </li></ul></ul><ul><li>Genes damaged by mutation are replaced as functional genes are duplicated </li></ul><ul><ul><li>Gene birth and death model </li></ul></ul>Jiang et al (2008) PNAS 105(12), 4874-4879 Evolution of RXLR effector gene family
  30. 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. 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. 32. Predictions: a few genes contribute most to virulence P. sojae RXLR effector genes ranked by expression during infection Avh172 Avh238
  33. 33. Many effector genes are drifting under neutral selection
  34. 34. Duplications and pseudogenes in the Avr3a/5 region Predictions: many gene duplications and pseudogenes
  35. 35. Genomics of oomycete effectors <ul><li>Co-evolutionary conflict between pathogens and their hosts drives rapid change in effector genes </li></ul><ul><li>Effector gene repertoires can change through gene deletion, transcriptional inactivation and rapid mutation </li></ul><ul><li>A small number of effector genes contributes disproportionately large number of transcripts </li></ul><ul><li>Birth-and-death evolution may shape the oomycete RXLR effector repertoire </li></ul>Summary
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