Lessons from fungal effector proteins:
Paving the road to discover cereal rust effectors
Rahim Mehrabi
Seed and Plant Improvement Institute
Iran
PAMP
PTI
Resistance
ETS
Susceptibility
ETI
Resistance
ETS
Susceptibility
What we know in rust-wheat pathosystemRust 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
TM
LRR
SP
Lr34/Yr18
LRRNBSCCLr1Lr10Lr21 (Genetics, 2003)Sr33 (Science, 2013)Sr35
STARTKinaseYr36
Defense
? ? ? ?
TaRLP1.12013
PGTAUSPE-10-1Upadhyaya 2014
5
Susceptible host Resistant host
AvrAvr22 is a cysteine protease inhibitor is a cysteine protease inhibitor
Cf-2 RLP
Avr2Rcr3
HR
6
Susceptible host Resistant host
Protect pathogen
Avr4 is a chitin-binding Avr4 is a chitin-binding proteinprotein
Cf-4 RLP
HR
Combining three approaches to mine candidate effectors
Bioinform
atics
Exp
ress
ion
prof
ile
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
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)
Genome mining of effectors
Effectorome criteriaSmall and secreted
No TM domain
No GPI anchored signature
Cysteine rich
Pst Pgt Pt
1344 1213 845
Genome comparisonRace 1 Race 2
MutatedTransposon insertionDeleted
Secretome analysis of stripe rust racesCantu 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 13Ecp1
80 4 26 30 34 38 42 48Ecp2-1
40 2 40 42 44 46 48 165Ecp4
40 2 6 8 10 12 14
Ecp5175
80 4 36 40 44 48 76 80Ecp6
1700 168 172 174 176 178 180 213
Ecp7120 8 32 70 74 78 82
Retro
Copia
Unknown
Gypsy
16
40 2 18 20 22 24 116 118Ecp2-2
Line
40 2 74 94 128 130 138Ecp2-3
136
Effector genome structure
Agricultural Applications:
effector-assisted breeding
OverexpressionBSMV
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
Quick screening of wheat germplasm (Effector assay instead of infection assay)
pRM152attL1 attL2ToxA
pRM152attL1 attL2ToxA
Pichia pastoris
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
Durable resistance Select R gene that recognizes effector with high
fitness penalty (those that are required for full virulence)
TM
LRRC
C
Effector
Resistance
Effector TM
LRRC
C
X
Susceptible
Low fitness penalty
Some level ofresistance
Effector TM
LRRC
C
X
high fitness penalty
Reduced virulence
Durable resistanceSelect R genes that recognize more, or
ideally all, allelic forms of Avr genes provide a broad spectrum of resistance
TM
LRRC
C
Effector
Resistance
WT form
Effector
Effector
Effector
Isoform 1
Isoform 2
Isoform 3
TM
LRRC
CResistance
TM
LRRC
C
Susceptible
Effector
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
1913 17
0
50
100
150
200
250
Total Unique With IPS/GO Conserved
M. graminicola
M. fijiensis
C. fulvum
Breeding direction
Avoid redundant breeding or cloning efforts
R1
R2
Avr1
Identification of new R geneDetection of new resistance alleles to allelic
diversity of effectors
X
XX
WT
A2
A3
Effector alleles
A1
AVR3aKI
AVR3aEMR3a
X
X
WT
A2
A3
Effector alleles
A1
Circumvent crossing barriers
Functional allele-mining with Avr gene in resistant germplasm
How many years?
TM
LRRC
C
Avr1
TM
LRRCC
Functional homolog
AVRblb1
RB/Rpi-blb1
Rpi-sto1
SummaryThe importance of the effectors in plant
susceptibility or resistanceThe lessens from other fungal pathogens
Genome comparisonNew techniquesStructural genomics Integrated –omics approaches
Lots of agricultural applications
Acknowledgments
Seed and Plant Improvement InstituteCereal Research Department
Symposium organizersDr. Kumarse Nazari