supervisors: frédéric brunner
Post on 01-Feb-2016
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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
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