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Identification of RXLR effectors from Phytophthora infestans that suppress PAMP-triggered immunity in Arabidopsis and tomato Liu Xiaoyu ZMBP, Universität Tübingen. Supervisors: Frédéric Brunner. Phytophthora infestans.

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Supervisors fr d ric brunner

Identification of RXLR effectors from Phytophthora infestans that suppress PAMP-triggered immunity in Arabidopsis and tomatoLiu XiaoyuZMBP, Universität Tübingen

Supervisors:

Frédéric Brunner


Supervisors fr d ric brunner

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

Symptom of Phytophthora infestans on the potato leaf

Potato infected with Phytophthora infestans


Supervisors fr d ric brunner

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
Oomycete RXLR effectors as virulence factors that contribute to plant susceptibility

N

signal

peptide

for secretion into

extrahaustorial space

oomycete

haustorium

RXLR

motif

for targeting

into plant cell

extrahaustorial

matrix

effector

domain

for host-manipulating

effector function

plant cell

C

modified from Schornack et al., 2009


Screening for rxlr effectors repressing pamp triggered innate immunity pti in arabidopsis

w/wo GFP to plant susceptibility

effector

p35S

flg22

luciferase

pFRK1

FLS2

MAPKKK

WRKY

WRKY

effector

MAPKK

luciferase

FRK1

MAPK

MAPK

MAPK

FRK1

pFRK1

luciferase

luminescence

measurement

pFRK1

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

Arabidopsis

leaf protoplast

method adapted from

Yoo et al., 2007


Phytophthora infestans rxlr effectors repressing pti in arabidopsis
Phytophthora infestans to plant susceptibility 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

priority list from Paul Birch’ s lab


Rxlr effector candidates block flg22 induced pfrk1 luc activity
RXLR effector candidates block to plant susceptibility flg22-induced pFRK1-Luc activity

pFRK1::Luc reporter gene assay

4,89

5,43

Promoter activity

(pFRK1::Luc/GUS)

1,11

1,11

Promoter activity

(pFRK1::Luc/GUS)

1.5

1.4

0.6


Pirxlr04145 20303 14833 repress endogenous frk1 activation upon flg22 treatment
piRXLR04145, 20303, (14833) to plant susceptibility repress endogenous FRK1 activation upon flg22 treatment

FRK1 relative expression level


Pirxlr04097 repress endogenous frk1 activation upon flg22 treatment
piRXLR04097 to plant susceptibility repress endogenous FRK1 activation upon flg22 treatment

FRK1 relative expression level


Supervisors fr d ric brunner
RXLR effector candidates to plant susceptibility do not block flg22-induced MAP kinase activation in Arabidopsis protoplasts

ev AvrPto PiRXL16737 PiRXLR14833

Post-translational MAP kinase activation

+Flg22

0 15 30 0 15 30 0 15 30 0 15 30

@-phosphop42/p44

Ponceau S

@-phosphop42/p44

ev AvrPto PiRXLR04145 PiRXLR20303

+Flg22

0 15 30 0 15 30 0 15 30 0 15 30

ev AvrPto PiRXLR00821 PiRXLR151525

+Flg22

0 15 30 0 15 30 0 15 30 0 15 30

Ponceau S

@-phosphop42/p44

Ponceau S

ev PiRXLR04097 PiRXLR05750 PiRXLR21388

+Flg22

15 0 15 30 0 15 30 0 15 30

@-phosphop42/p44

Ponceau S


Supervisors fr d ric brunner

GFP to plant susceptibility

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

GFP-piRXLR20303

(cytoplasm and nucleus)

GFP-piRXLR21388

(nucleus)

GFP-piRXLR14833

(nucleus)


Supervisors fr d ric brunner

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

GFP-piRXLR15125

(cytoplasm)

GFP-piRXLR04145

(nucleus)

GFP-piRXLR16737

(plasma membrane)


Sub cellular localisation of pirxlr00821 05750 and 04097 in arabidopsis protoplasts
sub-cellular localisation of to plant susceptibility piRXLR00821, 05750 and 04097 in Arabidopsis protoplasts

GFP-piRXLR00821

(cytoplasm and PM?)

GFP-piRXLR05750

(cytoplasm and PM?)

GFP-piRXLR04097

(cytoplasm and PM?)


Conclusions
Conclusions to plant susceptibility

  • ~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 blockMAP 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
Outlook to plant susceptibility

  • 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


Supervisors fr d ric brunner

Thank you! to plant susceptibility

Questions?