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