learning from the pathogen towards tailored-sustainable resistance : the case study of xanthomonas...

Learning from the pathogen towards tailored-sustainable resistance : the case study of Xanthomonas TAL effectors

[email protected]

Boris SZUREKMontpellier

France

GroupGTIPP,forGenomics&TranscriptomicsofPlant-ProcaryotesInterac>ons

Learning from the pathogen towards tailored-sustainable resistance : the case study of Xanthomonas TAL effectors

OutlineØ SomebackgroundaboutTALeffectors

Ø StoryI:searchforSWEET14loss-of-S

Ø StoryII:ablastNLRconfersRagainstXo

Tomato

Arabidopsis

Cabbage

Rice Pepper

Cassava

Citrus

Plant pathogenic Xanthomonas

TAL (Transcription Activator-Like) type III effectors

TAL (Transcription Activator-Like) type III effectors

Ø  They localize to the plant nucleus

Ø  They induce expression of host genes

Ø  How do they bind to DNA ?

TALsarebonafideplantTFs:

•12+13:“hypervariable”

•4repeattypesinAvrBs3HD,NI,NG,NS

modifiedfrom:Schornacketal.(2006)J.PlantPhysiol.163:256.

TAL repeat types

picture:AdamBogdanove

Bochetal.(2009)BreakingthecodeofDNAbindingspecificityofTAL-typeIIIeffectors.Science326:1509-1512;publishedonline29October2009(10.1126/science.1178811).

MoscouandBogdanove(2009)AsimpleciphergovernsDNArecogni>onbyTALeffectors.Science326:1501;publishedonline29October2009(10.1126/science.1178817).

The TAL code revealed

Correlation between repeat types and UPA box

UPAboxconsensus

AvrBs3COOHNH2

17.5x34aa

LTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGNINGNS

1 12/13 34

T A T A T A A A C C T N N C C C T C TDNA

HD NG NS NG NI NI NI HD HD NG NS NS HD HD HD NG HD NGRepeats

1 AvrBs3-repeat corresponds to 1 bp

Different RVDs associate preferentially with different bases

Testthemodel!

Target specificity of Hax2, Hax3, and Hax4

Bochetal.,Science,2009

Artificial TAL effectors with novel specificities


GUSgeneArtX-box

GUSgeneArtX-box


Artificial TAL effectors with novel specificities

Crystal structure of a TAL bound to its DNA

Mak et al., Science, 2012

Onecanpredicttargets!

T

C

G

G

C

AT

C

A

T

C

TC

A

T

C

A

G

Xanthomonasoryzaepv.oryzae(Xoo)

The Rice-Xoo interaction

The Rice-Xoo interaction

Sgene

TAL EBE

DISEASE

Roles of TAL effectors in compatible interactions

5dpi1cm

5 kb

4 kb

2 kb

MAI1 BAI3

3 kb

talC

Yuetal.,MPMI,2011

TalC is a major virulence TAL effector

5dpi1cm

5 kb

4 kb

2 kb

MAI1 BAI3

3 kb

talC

Yuetal.,MPMI,2011

SWEET14talC

TalCBAI3

TalC is a major virulence TAL effector

SWEET/nodulin-3 proteins

. 22 members in rice

. Membrane Transporters

. Glucose & sucrose export

Talbot,2010,Nature

Clade III SWEET genes confer TAL-ETS to Xoo

Streubel,Pesce,etal.,2013,NewPhytol.

SWEET14talC

TalCBAI3

Burkina Faso

SWEET14 is targeted by multiple strains..

SWEET14talC

TalCBAI3

tal5

Tal5MAI1

Burkina Faso

Mali


pthXo3 SWEET14avrXa7 talC

TalCBAI3

PthXo3PXO61

tal5

Tal5MAI1

AvrXa7PXO86

Philippines Burkina Faso

…of different geographic origins and lineages

Mali

Susceptibility


SWEET14

TalCBAI3

PthXo3PXO61

Tal5MAI1

AvrXa7PXO86

Philippines Burkina Faso

Mali

? ? ?

Loss of Susceptibility

ü  Unique collection of 132 wild and cultivated rice accessions

ü Sequencingofa400-bpPCRproduct

3

65

pthXo3 SWEET14avrXa7 talC tal5

30 34

Search for polymorphism in the SWEET14 promoter

PATTERN1

PATTERN2

PATTERN3

AvrXa7 Tal5

Search for polymorphism in the SWEET14 promoter

SWEET14avrXa7 talC tal5

Δ

Does it work ?

Nodele>on

Δ18bp

5DPI


Δ

Does it work ?

Nodele>on

Δ18bp5DPI


Δ

Does it work ?

Nodele>on

Δ18bp2DPI


Δ

Does it work ?

•  Reduced disease symptoms

•  Impaired Xoo multiplication

•  No induction of SWEET14

Ø  The xa41(t) allele confers resistance by loss of susceptibility to Xoo strains relying on Tal5 and AvrXa7

Summary

Asia

Africa

50% of Xoo strains are defeated by xa40(t)

PATTERN1

PATTERN2

PATTERN3

AvrXa7 Tal5

What about pattern 3 ?

A portion of SWEET14 is duplicated in O. glaberrima..

A duplication of pSWEET14 occurred in O. barthii ancestor

A duplication of pSWEET14 occurred in O. barthii ancestor

Hu>netal.,2015,PlantJ

Conclusions – Part I

Ø  Based on the TAL code, an allele with an 18bp deletion was predicted to resist to Xoo strains relying on AvrXa7 and Tal5

Ø  New HTP methodology for the isolation of loss-of-S resistance alleles

Ø  Avoid laborious germplasm phenotyping

Hu>netal.,Fron>ersinPlantScience,2015

Ralf Koebnik Lionel Gagnevin Valérie Verdier

Sébastien Cunnac Servane Baufume Jonathan Jacobs Florence Auguy Mathilde Hutin Céline Pesce Alvaro Perez

Tu Tran Tuan Hinda Doucoure

Carolina Flores Cheick Tekete

Sandrine Fabre

Alain Ghesquière François Sabot

NewFron>erProject«Menergep»(IRD,Cirad,JIRCAS&AfricaRice)

Funding

A Rice Blast Resistance Gene Confers Resistance Against BLB and BLS

ThomasKrojStellaCesariVéroniqueChalvon

MathildeHu>nTuanTuTranRalfKoebnik

Xoo - X.oryzaepv.oryzae

Xoc - X.oryzaepv.oryzicola

BLB

BLS

HydathodeWounds

StomateWounds

Vascular bundle

Mesophyll intercellular space

Xanthomonas oryzae pathovars profiles



BLB

BLS

HydathodeWounds

StomateWounds

Vascular bundle



ClonedRgenes:Xa1Xa21Xa27Xa10Xa23



BLB

BLS

HydathodeWounds

StomateWounds

Vascular bundle



ClonedRgenes:Xa1Xa21Xa27Xa10Xa23

NoRgeneavailable(inrice)

Bogdanoveetal.,COPB,2010

TAL effectors during resistance and disease

Bochetal.,NewPhytol,2014

Engineered resistance against TALEs

Bs3,Xa27,Xa10

Ø Causal agent : Magnaporthe

oryzae

Ø Host : Oryza spp.

Ø Hemibiotrophic pathogen

Rice Blast

Rice

M.oryzae

Pi-CO39 pi-CO39

Avr1-CO39

avr1-CO39

The Avr1-C039 / Pi-CO39 interaction

Cesarietal.,PlantCell,2013Cesarietal.,EMBO,2014

Pi-CO39 resistance is mediated by a pair of NB-LRR proteins

KitaakePi-CO39

Kanto51pi-CO39

AVR1-CO39UAS

AVR1-CO39UAS

uidAUAS

uidAUAS

DEX-inducible AVR1-CO39 transgenic lines

Kitaake(Pi-CO39)

Kanto51(pi-co39)

pUAS

gal4::u

idA

pUAS

gal4::AV

R1-CO39

DEX

+

-

+

-

Line1 Line2


pINDE

X::AVR

1-CO

39:HA

pINDE

X::uidA

Kanto51(pi-co39)

DEX + - + - + -

Line1 Line2

Kitaake(Pi-CO39)

DEX-induction of AVR1-CO39 triggers cell death and ROS

Kitaake(Pi-C

O39)

Kanto5

1(pi-co3

9)

+ - + -

pINDEX::AVR1-CO39 pINDEX::uidA

DEX

DEX-induction of AVR1-CO39 triggers cell death and ROS

KitaakePi-CO39

Kanto51pi-CO39

AVR1-CO39

dTALEUAS

UAS

AVR1-CO39

dTALEUAS

UAS

uidA

dTALEUAS

UASuidA

dTALEUAS

UAS

Using dTALEs to induce AVR1-CO39

0,00

0,05

0,10

0,15

0,20 mockEVdTALE

RelaNv

eAV

R1CO

39expression

∅ ∅∅

Kitaake(Pi-CO39)

Kanto51(pi-co39)

uidAAVR1-CO39Line1

AVR1-CO39Line2

AVR1-CO39


0,00

0,05

0,10

0,15

0,20 mockEVdTALE

RelaNv

eAV

R1CO

39expression

∅ ∅∅

Kitaake(Pi-CO39)

Kanto51(pi-co39)

uidAAVR1-CO39Line1

AVR1-CO39Line2

AVR1-CO39

TheAVR1-CO39anduidAgenesareinducedbyXanthomonasoryzaecarryingthedTALEUAS


The induction of AVR1-CO39 confers resistance to Xoo

.. and Xoc


0

5

10

15

20

25

30

35

Line1 Line2 Line1 Line2 Line1 Line1

pUASgal4::AVR1-CO39 pUASgal4::uidA pUASgal4::AVR1-CO39

pUASgal4::uidA

Kitaake Kanto51

lesion

length(cm)

PXO99EV

PXO99ArTAL

PXO99

PXO99(EV)

PXO99(dTALE)

PXO99 PXO99Kitaake(Pi-CO39) Kanto51(pi-CO39)


0

1

2

3

4

5

6

7

8

9

Line1 Line2 Line1 Line2 Line1 Line1

pUASgal4::AVR1-CO39 pUASgal4::uidA pUASgal4::AVR1-CO39

pUASgal4::uidA

Kitaake Kanto51

Bacterialpop

ualNon

slog10(C

FU)

PXO99EV

PXO99ArTAL

PXO99PXO99(EV)

PXO99(dTALE)

PXO99 PXO99Kitaake(Pi-CO39) Kanto51(pi-CO39)2

1

Cesarietal.,PlantCell,2013Cesarietal.,EMBO,2014

Pi-CO39 resistance is mediated by a pair of NB-LRR proteins

Induction of RGA4 in Pi-CO39 lines confers resistance to Xoo

RGA4

dTALERGA4

0

2

4

6

8

10

12

lesion

length(cm)

Co39

02468

101214161820

Kitaake

PXO99

Lesio

nlength(cm.)

Lesio

nlength(cm.)

PXO99

PXO99PXO99

PXO99

PXO99PXO99PXO99

PXO99Co39 Kitaake

0

5

10

15

20

25

Kanto51PXO99Kanto51

PXO99PXO99

PXO99

PXO99

Lesio

nlength(cm.)

Hu>netal.,PlantJ,2016

Ø  TheRGA4/RGA5-mediatedresistanceisabletocontrolBLBandBLS

Ø  Autoac>veformsofNB-LRRsandAVRscanbeusedfor

synthe>cpromoter-trapsresistancestrategies

tal2 RGA4tal1

Tal1

Tal2

tal3

Tal3

Conclusions

ThomasKrojStellaCesariVéroniqueChalvon

MathildeHu>nTuanTuTranRalfKoebnik

Thanks !