Identification of RXLR effectors from Phytophthora infestans that suppress PAMP-triggered immunity in Arabidopsis and

tomato

Liu XiaoyuZMBP, Universität Tübingen

Supervisors:Frédéric Brunner

Phytophthora infestans

Symptom of Phytophthora infestans on the potato leaf

Potato infected with Phytophthora infestans

Phytophthora infestans is an oomycete that causes the serious potato disease known as late blight or potato blight

In 2009 scientists completed the sequencing of the genome of P. infestans. The genome is much larger (240 Mb) compared to other Phytophthora species

Pathogen-Associated Molecular Patterns (PAMPs) as triggers of innate immune responses in Arabidopsis thaliana

Oomycete RXLR effectors as virulence factors that contribute to plant susceptibility

modified from Schornack et al., 2009

plant cell

oomycetehaustorium

extrahaustorialmatrix

for secretion into extrahaustorial space

for targeting into plant cell

for host-manipulatingeffector function

signalpeptide

RXLR motif

effectordomain

N

C

Screening for RXLR effectors repressing PAMP-Triggered innate immunity (PTI) in Arabidopsis

Arabidopsis leaf protoplast

p35S w/wo GFP effector pFRK1 luciferase

luminescence measurement

effector

luciferase

luciferase

MAPK

WRKYpFRK1

FRK1

FRK1

MAPK

WRKYpFRK1

MAPKKK

MAPK

MAPKK

FLS2

flg22

method adapted fromYoo et al., 2007

Phytophthora infestans RXLR effectors repressing PTI in Arabidopsis

• About 100 effectors are screened for repression of flg22-induced pFRK1-Luc activity

• ~ 20 repress pFRK1-Luc activity

• Best candidates: piRXLR15125, piRXLR16737, piRXLR04145, piRXLR14833, piRXLR21388, piRXLR20303, piRXLR00821, piRXLR05750, piRXLR04097

RXLR RNAi Silencing phenotype

Screened by Y2H Localisation

PITG_15125 no many spots-PM association?

PITG_16737.1 less path.? HR?

no nuclear/nucleolar and membranes

PITG_04145.1 less path.? no nuclear/nucleolar

PITG_14833.1 less path.? HR? no nuclear/nucleolar and membranes

PITG_21388 (IPIO-4,Avrblb1)

several interactors cytoplasmic

PITG_20303 (Avrblb2 recognised)

smooth cytoplasmic labelling, excluded from nucleolus

PITG_00821no interactors

grainy-PM association

PITG_04097 nuclear/nucleolar

PITG_05750.1 No penetr.

excluded from nucleus, PM associated?

priority list from Paul Birch’ s lab

RXLR effector candidates block flg22-induced pFRK1-Luc activity

4,89

1,11 1,11

Prom

oter

acti

vity

(pFR

K1::L

uc/G

US)

5,43

pFRK1::Luc reporter gene assay

Prom

oter

acti

vity

(pFR

K1::L

uc/G

US)

1.40.6

1.5

piRXLR04145, 20303, (14833) repress endogenous FRK1 activation upon flg22 treatment

FRK1

rela

tive

expr

essi

on

leve

l

piRXLR04097 repress endogenous FRK1 activation upon flg22 treatment

FRK1

rela

tive

expr

essi

on

leve

l

RXLR effector candidates do not block flg22-induced MAP kinase activation in Arabidopsis protoplasts

Post-translational MAP kinase activation

0 15 30 0 15 30 0 15 30 0 15 30ev AvrPto PiRXLR04145 PiRXLR20303

+Flg22

Ponceau S

@-phosphop42/p44

ev AvrPto PiRXL16737 PiRXLR148330 15 30 0 15 30 0 15 30 0 15 30

Ponceau S

@-phosphop42/p44

+Flg22

ev PiRXLR04097 PiRXLR05750 PiRXLR2138815 0 15 30 0 15 30 0 15 30

Ponceau S

@-phosphop42/p44

+Flg22

ev AvrPto PiRXLR00821 PiRXLR1515250 15 30 0 15 30 0 15 30 0 15 30

Ponceau S

@-phosphop42/p44

+Flg22

sub-cellular localisation of piRXLR20303, 21388 and 14833 in Arabidopsis protoplasts

GFP-piRXLR20303(cytoplasm and nucleus)

GFP

GFP-piRXLR21388(nucleus)

GFP-piRXLR14833(nucleus)

GFP-piRXLR04145(nucleus)

GFP-piRXLR15125(cytoplasm)

GFP-piRXLR16737(plasma membrane)

sub-cellular localisation of piRXLR15125, 04145 and 16737 in Arabidopsis protoplasts

sub-cellular localisation of piRXLR00821, 05750 and 04097 in Arabidopsis protoplasts

GFP-piRXLR00821(cytoplasm and PM?)

GFP-piRXLR05750(cytoplasm and PM?)

GFP-piRXLR04097(cytoplasm and PM?)

Conclusions

~100 P.infestans RXLR effectors have been screened for PTI repression in Arabidopsis and several interesting candidates were identified.

piRXLR15125, piRXLR16737, piRXLR04145, piRXLR14833, piRXLR21388, piRXLR20303 piRXLR00821, piRXLR04097, piRXLR05750 repress flg22-induced pFRK1-Luc activity but do not block MAP kinase activation.

piRXLR04145,20303,04097,(14833) abort the up-regulation of FRK1 expression after PAMP treatment.

GFP fusion of piRXLR15125, piRXLR16737, piRXLR04145, piRXLR14833, piRXLR21388, piRXLR20303 piRXLR00821, piRXLR04097, piRXLR05750 reveal different sub-cellular localisations.

Outlook

Test sub-cellular localisation, repression of MAP kinase activation and of reporter gene activation in tomato protoplasts.

Identify and characterise plant interactors for the RXLRs via approaches like immunoprecipitation, BiFC/FACS in protoplasts...

What is the effect of RXLRs in planta?- Generate Dex-inducible RXLR-transgenic Arabidopsis and tomato plants (w/wo tag)- Analyse immune response with PTI bioassays and test susceptibility to phytopathogens- Perform microarray study to detect gene expression changes