Lessons from fungal effector proteins:
Paving the road to discover cereal rust
effectors
Rahim Mehrabi
Seed and Plant Impr...
PAMP
PTI
Resistance
ETS
Susceptibility
ETI
Resistance
ETS
Susceptibility
What we know in rust-wheat pathosystem
Rust Genes No.
Pst Yr1, Yr2, Yr3, Yr3a, Yr3b, Yr3c, Yr4, Yr4a, Yr4b, Yr5, Yr6, Yr7,...
TMLRRSP
Lr34/Yr18
LRRNBSCC
Lr1
Lr10
Lr21 (Genetics, 2003)
Sr33 (Science, 2013)
Sr35
STARTKinase
Yr36
Defense
? ? ? ?
TaRLP...
5
Susceptible host Resistant host
AvrAvr22 is a cysteine protease inhibitoris a cysteine protease inhibitor
Cf-2 RLP
Avr2
...
6
Susceptible host Resistant host
Protect pathogen
Avr4 is a chitin-bindingAvr4 is a chitin-binding proteinprotein
Cf-4 RL...
Combining three approaches to mine candidate effectors
Bioinformatics
Expressionprofile
 Bioinformatics
 Homologs
 Poss...
Genome features of cereal rusts
 Launched in 2006; draft genome Pst 2011
 Richness in repetitive and transposable elemen...
Genome mining of effectors
Effectorome criteria
Small and secreted
No TM domain
No GPI anchored signature
Cysteine ri...
Genome comparison
Race 1 Race 2
Mutated
Transposon insertion
Deleted
Secretome analysis of stripe rust races
Cantu et al. 2013
160 14 18 20 22 24 26 36
40 2 6 8
40 2 6 8 10 12 14 20
Avr4E
Avr4
Avr2
Avr9
40 2 6 8 10 12 13
Ecp1
80 4 26 30 34 38 42 48
...
Agricultural Applications:
effector-assisted breeding
Overexpression
BSMV
Yr1 Yr2 Yr3 Yr4 Yr5 Yr6 Yr7 Yr8 Yr9 Yr10 Yr11 Yr12 Yr13 Yr14 Yr15
TT3S Protein infiltration
Screening...
Quick screening of wheat germplasm (Effector
assay instead of infection assay)
pRM152
attL1 attL2ToxA
pRM152
attL1 attL2To...
Exploiting effectors provides a tool to accelerate R gene cloning
TMLRR
TMLRR
GST pull-down assay
HaloTag-based pull-down ...
Durable resistance
 Select R gene that recognizes effector with high
fitness penalty (those that are required for full
vi...
Durable resistance
Select R genes that recognize more, or
ideally all, allelic forms of Avr genes 
provide a broad spect...
Durable resistance
Select R genes that recognize conserved
effectors that presented in more races
Races
Effector Race1 Ra...
Breeding direction
Avoid redundant breeding or cloning efforts
R1
R2
Avr1
Identification of new R gene
Detection of new resistance alleles to allelic
diversity of effectors
X
X
X
WT
A2
A3
Effecto...
Circumvent crossing barriers
Functional allele-mining with Avr gene in
resistant germplasm
How many years?
TMLRRCC
Avr1
T...
Summary
The importance of the effectors in plant
susceptibility or resistance
The lessens from other fungal pathogens
G...
Acknowledgments
Seed and Plant Improvement Institute
Cereal Research Department
Symposium organizers
Dr. Kumarse Nazari
Izmir rust final final
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Izmir rust final final

  1. 1. Lessons from fungal effector proteins: Paving the road to discover cereal rust effectors Rahim Mehrabi Seed and Plant Improvement Institute Iran
  2. 2. PAMP PTI Resistance ETS Susceptibility ETI Resistance ETS Susceptibility
  3. 3. What we know in rust-wheat pathosystem Rust Genes No. Pst Yr1, Yr2, Yr3, Yr3a, Yr3b, Yr3c, Yr4, Yr4a, Yr4b, Yr5, Yr6, Yr7, Yr8, Yr9, Yr10, Yr11, Yr12, Yr13, Yr14, Yr15, Yr16, Yr17, Yr18, Yr19, Yr20, Yr21, Yr22, Yr23, Yr24, Yr25, Yr26, Yr27, Yr28, Yr32, Yr29, Yr30, Yr31, Yr33, Yr34, Yr35, Yr36, Yr37, Yr38, Yr39, Yr40, Yr41, Yr42, Yr43, Yr44, Yr45, Yr46, Yr47, Yr48, Yr49, Yr50, Yr51, Yr52, Yr53, YrCle, YrD, YrH46, YrHVII, YrMin, YrMor, YrND, YrS, YrTye, YrTr1, YrTr2, YrYam, YrV23, Yrns-B1, YrH52, YrSP, YrDa1, YrDa2, YrDru, YrDru2, YrSte, YrSte2, YrCK, YrAlp, YrSp, YrZH84, YrCN17, YrC591, YrExp1, YrExp2, YrR212, YrS2199, YrC142, YrP81, YrAS2388, YrR61, Yrxy1, Yrxy2, 97 Pgt Sr2, Sr3 & Sr4, Sr5, Sr6, Sr7, Sr7a, Sr7b, Sr8, Sr8a, Sr8b, Sr9, Sr9a, Sr9b, Sr9c, Sr9d, Sr9e, Sr9f, Sr9g, Sr10, Sr11, Sr12, Sr13, Sr14, Sr15, Sr16, Sr17, Sr18, Sr19, Sr20, Sr21, Sr22, Sr23, Sr24, Sr25, Sr26, Sr27, Sr28, Sr29, Sr30, Sr31, Sr32, Sr33, Sr34, Sr35, Sr36, Sr37, Sr38, Sr39, Sr40, Sr41, Sr42, Sr43, Sr44, Sr45, SrA, SrTmp, SrWld, SrZdar, Sr1, SrCad, Sr46, Sr47, Sr48, Sr49, Sr50, Sr51, Sr52, Sr53, Sr54, Sr55, Sr56, Sr57, SrAes7t, SrWeb, Sr1RSAmigo 75 Pt Lr1, Lr2, Lr2a, Lr2b, Lr2c, Lr3a, Lr3b, Lr3c, Lr4, Lr5, Lr6, Lr7, Lr8, Lr9, Lr10, Lr11, Lr12, Lr13, Lr14, Lr14a, Lr14b, Lr14ab, Lr15, Lr16, Lr17, Lr17a, Lr17b, Lr18, Lr19, Lr20, Lr21, Lr22, Lr22a, Lr22b, Lr23, Lr24, Lr25, Lr26, Lr27, Lr28, Lr29, Lr30, Lr31, Lr32, Lr33, Lr34, Lr35, Lr36, Lr37, Lr38, Lr41, Lr42, Lr43, Lr44, Lr45, Lr46, LrTb, LrVPM, Lr52, LrW2, Lr48, Lr49, Lr47, Lr39, Lr50, LrTm, LrTr, Lr51, Lr3, LrTt1, Lr53, Lr54, Lr55, Lr56, LrKr1, LrKr2, LrMq1, Lr57, Lr58, Lr59, Lr60, Lr40, Lr61, Lr62, Lr63, Lr64, Lr65, Lr66, LrZH84, Lr67, LrWo, Lr68, Lr69, Lr70, Lr71, Lr72 95
  4. 4. TMLRRSP Lr34/Yr18 LRRNBSCC Lr1 Lr10 Lr21 (Genetics, 2003) Sr33 (Science, 2013) Sr35 STARTKinase Yr36 Defense ? ? ? ? TaRLP1.1 2013 PGTAUSPE-10-1 Upadhyaya 2014
  5. 5. 5 Susceptible host Resistant host AvrAvr22 is a cysteine protease inhibitoris a cysteine protease inhibitor Cf-2 RLP Avr2 Rcr3 HR
  6. 6. 6 Susceptible host Resistant host Protect pathogen Avr4 is a chitin-bindingAvr4 is a chitin-binding proteinprotein Cf-4 RLP HR
  7. 7. Combining three approaches to mine candidate effectors Bioinformatics Expressionprofile  Bioinformatics  Homologs  Possible Host cell translocation motifs  Domain analyses Proteomics Effector candidate  Expression profile  In vitro  In planta over time  Proteomics  Shotgun protein sequencing  apoplatic fluid  in vitro secreted proteins
  8. 8. Genome features of cereal rusts  Launched in 2006; draft genome Pst 2011  Richness in repetitive and transposable elements  Expansions of gene families  Oligopeptide and amino acid transporters (important role to play in rust fungi biology)  Large multigene families (small secreted proteins)  Contractions of gene families  Secondary metabolites  Toxins synthesis  Glycosyl hydrolases, lipases and peptidases  High number of predicted genes  Pst=65Mb  (>20,000)  Blumeria graminis 120Mb 5,854  Ustilago maydis 18.7Mb 6,902  69 % of the Pst predicted genes show significant homology to Pgt genes (Large number of lineage-specific genes)
  9. 9. Genome mining of effectors Effectorome criteria Small and secreted No TM domain No GPI anchored signature Cysteine rich Pst Pgt Pt 1344 1213 845
  10. 10. Genome comparison Race 1 Race 2 Mutated Transposon insertion Deleted
  11. 11. Secretome analysis of stripe rust races Cantu et al. 2013
  12. 12. 160 14 18 20 22 24 26 36 40 2 6 8 40 2 6 8 10 12 14 20 Avr4E Avr4 Avr2 Avr9 40 2 6 8 10 12 13 Ecp1 80 4 26 30 34 38 42 48 Ecp2-1 40 2 40 42 44 46 48 165 Ecp4 40 2 6 8 10 12 14 Ecp5 175 80 4 36 40 44 48 76 80 Ecp6 1700 168 172 174 176 178 180 213 Ecp7 120 8 32 70 74 78 82 Retro Copia Unknown Gypsy 16 40 2 18 20 22 24 116 118 Ecp2-2 Line 40 2 74 94 128 130 138 Ecp2-3 136 Effector genome structure
  13. 13. Agricultural Applications: effector-assisted breeding
  14. 14. Overexpression BSMV Yr1 Yr2 Yr3 Yr4 Yr5 Yr6 Yr7 Yr8 Yr9 Yr10 Yr11 Yr12 Yr13 Yr14 Yr15 TT3S Protein infiltration Screening Gene postulation Functional markers during breeding
  15. 15. Quick screening of wheat germplasm (Effector assay instead of infection assay) pRM152 attL1 attL2ToxA pRM152 attL1 attL2ToxA Pichia pastoris
  16. 16. Exploiting effectors provides a tool to accelerate R gene cloning TMLRR TMLRR GST pull-down assay HaloTag-based pull-down assayHaloTag-based pull-down assay Co-Immunoprecipitation assayCo-Immunoprecipitation assay
  17. 17. Durable resistance  Select R gene that recognizes effector with high fitness penalty (those that are required for full virulence) TMLRRCC Effector Resistance Effector TMLRRCC X Susceptible Low fitness penalty Some level of resistance Effector TMLRRCC X high fitness penalty Reduced virulence
  18. 18. Durable resistance Select R genes that recognize more, or ideally all, allelic forms of Avr genes  provide a broad spectrum of resistance TMLRRCC Effector Resistance WT form Effector Effector Effector Isoform 1 Isoform 2 Isoform 3 TMLRRCC Resistance TMLRRCC Susceptible Effector
  19. 19. Durable resistance Select R genes that recognize conserved effectors that presented in more races Races Effector Race1 Race2 Race3 Race4 Race5 Race6 Race7 Race8 Avr1 √ no √ no no no no no Avr2 no no no no √ no no no Avr3 √ √ √ √ √ √ √ no 230 137 234 152 68 120 59 56 97 19 13 17 0 50 100 150 200 250 Total Unique With IPS/GO Conserved M. graminicola M. fijiensis C. fulvum
  20. 20. Breeding direction Avoid redundant breeding or cloning efforts R1 R2 Avr1
  21. 21. Identification of new R gene Detection of new resistance alleles to allelic diversity of effectors X X X WT A2 A3 Effector alleles A1 AVR3aKI AVR3aEM R3a X X WT A2 A3 Effector alleles A1
  22. 22. Circumvent crossing barriers Functional allele-mining with Avr gene in resistant germplasm How many years? TMLRRCC Avr1 TMLRRCC Functional homolog AVRblb1 RB/Rpi-blb1 Rpi-sto1
  23. 23. Summary The importance of the effectors in plant susceptibility or resistance The lessens from other fungal pathogens Genome comparison New techniques Structural genomics  Integrated –omics approaches Lots of agricultural applications
  24. 24. Acknowledgments Seed and Plant Improvement Institute Cereal Research Department Symposium organizers Dr. Kumarse Nazari
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