1. Lessons from fungal effector proteins:
Paving the road to discover cereal rust
effectors
Rahim Mehrabi
Seed and Plant Improvement Institute
Iran

2. PAMP
PTI
Resistance
ETS
Susceptibility
ETI
Resistance
ETS
Susceptibility

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. 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
Susceptible host Resistant host
AvrAvr22 is a cysteine protease inhibitoris a cysteine protease inhibitor
Cf-2 RLP
Avr2
Rcr3
HR

6. 6
Susceptible host Resistant host
Protect pathogen
Avr4 is a chitin-bindingAvr4 is a chitin-binding proteinprotein
Cf-4 RLP
HR

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. 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. 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. Genome comparison
Race 1 Race 2
Mutated
Transposon insertion
Deleted

12. Secretome analysis of stripe rust races
Cantu et al. 2013

13. 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

14. Agricultural Applications:
effector-assisted breeding

15. 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

16. Quick screening of wheat germplasm (Effector
assay instead of infection assay)
pRM152
attL1 attL2ToxA
pRM152
attL1 attL2ToxA
Pichia pastoris

17. 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

18. 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

19. 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

20. 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

21. Breeding direction
Avoid redundant breeding or cloning efforts
R1
R2
Avr1

22. 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

23. 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

24. 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

25. Acknowledgments
Seed and Plant Improvement Institute
Cereal Research Department
Symposium organizers
Dr. Kumarse Nazari