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Identification of Pgt effectorsthat are recognized in riceFumiaki Katagiri1, Yayoi Tsuda1, Thomas Stoddard1,Kenichi Tsuda1...
Why rice? No rust pathogen is known to infect rice  – durable and broad-spectrum resistance It is a cereal – the resista...
Is R gene-mediated resistanceinvolved in non-host resistance? Non-host resistance could be associated  with hypersensitiv...
Long-term goals Isolate Pgt effectors that are recognized  by rice R genes. Isolate the corresponding rice R genes. Tra...
Strategy1.   Identify Pgt effector gene candidates     using bioinformatics.2.   Clone the effector gene candidates into  ...
Importance of using isolated effector genes             Effector1 Effector2                R1        R2
Importance of using isolated effector genes             Effector1 Effector2                R1        R2
Importance of using isolated effector genes             Effector1                R1        R2
Importance of using isolated effector genes             Effector1                R1        R2
Bioinformatic identification ofPgt effector gene candidates
Criteria to select 1,386 predictedsecreted proteins   17,773 predicted protein-coding genes in Pgt    CRL 75-36-700-3 (“7...
Candidate gene selection 1386 predicted secreted protein genes in  Pgt 7a. Expression criteria (NimbleGen array  data): ...
Cloning of Pgt effector genecandidates
Candidate gene cloning Template: cDNA from wheat infected  with 7a 5’-end: add CACCATG to the predicted  coding sequence...
Breakdown of cloned genes   121 primer sets (one added manually)    ◦ 79 primer sets with products with ORFs of      reas...
An example of mis-prediction of intronsGenome sequencePredicted transcript sequenceActual cDNA sequence
An example of highly homologous genesPredicted transcript sequencecDNA clone group 1cDNA clone group 2                    ...
Testing the Pgt effector gene candidatesin transient assays
Transient assay forR gene- effector gene interactionsEffector geneR geneGUS reporter    GUS enzyme                        ...
The origin of the assay.       Bombarded with RPS2 + GUS     Psp / vector            Psp / avrRpt2          Mindrinos, Kat...
It works for fungal effectors in rice                      Rice blast AVR-Pita                                      and   ...
We reproduced the AVR-Pita:Pi-taresults in rice.   AVR-Pita + GUS              GFP + GUS               YT14 (Pi-ta+)
Pgt effectors potentially recognized by ricewere identified in the biolistic assay. A representative of most of the    PGT...
The same principle works withtransient expression in protoplasts                 Leister and Katagiri (2000) Plant J. 22, ...
The protoplast assay works withfungal effectors and rice                Yoshida, et al. (2009) Plant Cell 21, 1573.
Pgt effectors potentially recognized by ricewere identified in the protoplast assay.
The Effector-to-Host-Analyzer(EtHAn) system Thomas et al. 2009, Plant J. 60, 919                                      Pseu...
EtHAn can deliver CyaA protein    into the rice cellcAMP (pmol/cm2)                  CyaA 0h   CyaA 8h   GFP 0h   GFP 8h
Limitations R gene-dependent discovery of effector  genes  → These are the ones we want anyway. HR-dependent discovery  ...
Pgt effectors that suppressplant cell death?
Some Pgt effector gene candidatesresulted in higher reporter activitiesthan the negative control.                 rLUC/LUC...
Some Pgt effector gene candidatesresulted in higher reporter activitiesthan negative controls.
Some Pgt effector candidates maysuppress RPS2-induced cell death inNicotiana benthamiana                                  ...
Would cell death suppressor genescomplicate the situation?   Promote pathogenicity by suppressing the    HR?   Suppress ...
Summary 120 Pgt effector gene candidates were  bioinformatically identified from Pgt 7a. 65 of them were tested in trans...
Acknowledgements Fumi Katagiri and Jane Glazebrook Labs (U of  Minnesota)  ◦ Kenichi Tsuda, Yayoi Tsuda, Thomas Stoddard...
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Katagiri i 110614

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Fumiaki Katagiri, U of Minn.
Identification of Pgt effectors that are recognized in rice

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Katagiri i 110614

  1. 1. Identification of Pgt effectorsthat are recognized in riceFumiaki Katagiri1, Yayoi Tsuda1, Thomas Stoddard1,Kenichi Tsuda1, Christina Cuomo2,Jane Glazebrook1, and Les Szabo31Department of Plant Biology, Microbial and PlantGenomics Institute, University of Minnesota2Broad Institute of MIT and Harvard3USDA-ARS, Cereal Disease Laboratory June 14, 2011 BGRI Technical Workshop St Paul, MN
  2. 2. Why rice? No rust pathogen is known to infect rice – durable and broad-spectrum resistance It is a cereal – the resistance may be transferable to wheat. A well established model plant with a well annotated genome.
  3. 3. Is R gene-mediated resistanceinvolved in non-host resistance? Non-host resistance could be associated with hypersensitive-like response (type II) – HR is a hallmark of R gene-mediated resistance. Even in type I, R gene-mediated resistance might be a backup mechanism. Some NB-LRR genes (a typical R gene class) confer broad-spectrum resistance.
  4. 4. Long-term goals Isolate Pgt effectors that are recognized by rice R genes. Isolate the corresponding rice R genes. Transgenically transfer the rice R genes into wheat. Investigate the genetic architecture of the Pgt effectors and the rice R genes to develop an effective R gene deployment strategy.
  5. 5. Strategy1. Identify Pgt effector gene candidates using bioinformatics.2. Clone the effector gene candidates into a plant transient expression vector.3. Test the effector gene candidates in transient expression assays in rice.
  6. 6. Importance of using isolated effector genes Effector1 Effector2 R1 R2
  7. 7. Importance of using isolated effector genes Effector1 Effector2 R1 R2
  8. 8. Importance of using isolated effector genes Effector1 R1 R2
  9. 9. Importance of using isolated effector genes Effector1 R1 R2
  10. 10. Bioinformatic identification ofPgt effector gene candidates
  11. 11. Criteria to select 1,386 predictedsecreted proteins 17,773 predicted protein-coding genes in Pgt CRL 75-36-700-3 (“7a”, sequenced isolate). SignalP to select proteins with potential signal peptides TargetP to filter out those potentially mitochodorion-targeted. TMHMM to filter out those potentially membrane integrated. big-PI to filter out those with potential GPI- anchor sites. 300 amino acids or smaller.
  12. 12. Candidate gene selection 1386 predicted secreted protein genes in Pgt 7a. Expression criteria (NimbleGen array data): highly expressed during infection of both wheat and barley, and not expressed in germinated or ungerminated urediniospores. 120 genes selected
  13. 13. Cloning of Pgt effector genecandidates
  14. 14. Candidate gene cloning Template: cDNA from wheat infected with 7a 5’-end: add CACCATG to the predicted coding sequence without the signal peptide. 3’-end: the predicted STOP codon.
  15. 15. Breakdown of cloned genes 121 primer sets (one added manually) ◦ 79 primer sets with products with ORFs of reasonable sizes 53 cloned genes that are expected ◦ 37 exact matches ◦ 16 intron mispredicted 22 clearly homologous variants that are not found in the genome sequence.
  16. 16. An example of mis-prediction of intronsGenome sequencePredicted transcript sequenceActual cDNA sequence
  17. 17. An example of highly homologous genesPredicted transcript sequencecDNA clone group 1cDNA clone group 2 Alleles (highly heterologus genome) Close gene family members
  18. 18. Testing the Pgt effector gene candidatesin transient assays
  19. 19. Transient assay forR gene- effector gene interactionsEffector geneR geneGUS reporter GUS enzyme HR DEAD R protein Low GUS activity Effector protein Leister et al. (1996) PNAS 93, 15497
  20. 20. The origin of the assay. Bombarded with RPS2 + GUS Psp / vector Psp / avrRpt2 Mindrinos, Katagiri,Yu, & Ausubel (1994) Cell 76, 1089.
  21. 21. It works for fungal effectors in rice Rice blast AVR-Pita and Rice Pi-ta R geneR+ r- Jia et al. (2000) EMBO J. 19, 4004
  22. 22. We reproduced the AVR-Pita:Pi-taresults in rice. AVR-Pita + GUS GFP + GUS YT14 (Pi-ta+)
  23. 23. Pgt effectors potentially recognized by ricewere identified in the biolistic assay. A representative of most of the PGT_62: a representative of Pgt effector gene candidates potentially positive Pgt effector genes
  24. 24. The same principle works withtransient expression in protoplasts Leister and Katagiri (2000) Plant J. 22, 345
  25. 25. The protoplast assay works withfungal effectors and rice Yoshida, et al. (2009) Plant Cell 21, 1573.
  26. 26. Pgt effectors potentially recognized by ricewere identified in the protoplast assay.
  27. 27. The Effector-to-Host-Analyzer(EtHAn) system Thomas et al. 2009, Plant J. 60, 919 Pseudomonas fluorescens TTSS Effector Need the type III secretion signal ← AvrRpm1 Thanks to Narayana Upadhyaya
  28. 28. EtHAn can deliver CyaA protein into the rice cellcAMP (pmol/cm2) CyaA 0h CyaA 8h GFP 0h GFP 8h
  29. 29. Limitations R gene-dependent discovery of effector genes → These are the ones we want anyway. HR-dependent discovery → Limits the types of resistance response to be detected. Candidate-based screen of Pgt genes → Only predicted secreted genes Cell-types, tissues, and developmental stages tested are limited.
  30. 30. Pgt effectors that suppressplant cell death?
  31. 31. Some Pgt effector gene candidatesresulted in higher reporter activitiesthan the negative control. rLUC/LUC PGT_25 PGT_41(A quantitative biolistic assay using two reporters: Leister et al. (1996) PNAS 93, 15497)
  32. 32. Some Pgt effector gene candidatesresulted in higher reporter activitiesthan negative controls.
  33. 33. Some Pgt effector candidates maysuppress RPS2-induced cell death inNicotiana benthamiana GFP PGT_411st: GFP 2nd: RPS2 1st: effector candidate GFP PGT_46
  34. 34. Would cell death suppressor genescomplicate the situation? Promote pathogenicity by suppressing the HR? Suppress the HR but not resistance efficiently, resulting in R gene-mediated resistance not associated with the HR?
  35. 35. Summary 120 Pgt effector gene candidates were bioinformatically identified from Pgt 7a. 65 of them were tested in transient assays for recognition in rice, resulting in 6 potential positives. 2 Pgt effector gene candidates are being characterized as potential cell death suppressors.
  36. 36. Acknowledgements Fumi Katagiri and Jane Glazebrook Labs (U of Minnesota) ◦ Kenichi Tsuda, Yayoi Tsuda, Thomas Stoddard Les Szabo (USDA-ARS, Cereal Disease Lab) Christina Cuomo (Broad Inst) Narayana Upadhyaya, Peter Dodds, Jeff Ellis, Mick Ayliffe (CSIRO) Ryohei Terauchi (Iwate Biotech Res Cntr) Yulin Jia (USDA-ARS, Dale Bumpers Natl Rice Res Cntr) Funding: Two Blades Foundation National Science Foundation

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