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
 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
Potato infected with
Phytophthora infestans
Symptom of Phytophthora
infestans on the potato leaf
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
N
signal
peptide
RXLR
motif
for secretion into
extrahaustorial space
for targeting
into plant cell
oomycete
haustorium
effector
domain
for host-manipulating
effector function
extrahaustorial
matrix
plant cell
C
modified from Schornack et al., 2009
Screening for RXLR effectors repressing PAMP-Triggered innate
immunity (PTI) in Arabidopsis
flg22
p35S w/wo GFP
pFRK1
effector
luciferase
FLS2
MAPKKK
effector
MAPKK
MAPK
Arabidopsis
leaf protoplast
FRK1
MAPK
WRKY
FRK1
pFRK1
method adapted from
Yoo et al., 2007
luciferase
MAPK
WRKY
pFRK1
luciferase
luminescence
measurement
Phytophthora infestans RXLR effectors repressing PTI in Arabidopsis
•
About 100 effectors are screened for
repression of flg22-induced pFRK1Luc activity
RXLR
PITG_15125
PITG_16737.1
•
•
~ 20 repress pFRK1-Luc activity
Best candidates: piRXLR15125,
piRXLR16737, piRXLR04145,
piRXLR14833, piRXLR21388,
piRXLR20303, piRXLR00821,
piRXLR05750, piRXLR04097
RNAi Silencing
phenotype
PITG_04145.1
PITG_14833.1
PITG_21388
(IPIO-4,Avrblb1)
less path.? HR?
less path.?
less path.? HR?
no
no
nuclear/nucleolar
nuclear/nucleolar
and membranes
several
interactors cytoplasmic
PITG_20303
(Avrblb2
recognised)
smooth cytoplasmic
labelling, excluded
from nucleolus
no
grainy-PM
interactors association
PITG_00821
PITG_04097
PITG_05750.1
Screened
by Y2H
Localisation
many spots-PM
no
association?
nuclear/nucleolar
no
and membranes
No penetr.
priority list from Paul Birch’ s lab
nuclear/nucleolar
excluded from
nucleus, PM
associated?
RXLR effector candidates block flg22-induced pFRK1-Luc activity
pFRK1::Luc reporter gene assay
6
6
Promoter activity
(pFRK1::Luc/GUS)
Promoter activity
(pFRK1::Luc/GUS)
6
4,89
5,43
4
4
4
3
1,84
2
1,92
2
1,18
1,3
2
1,34
1,11
0,83
0
0
0
6
6
4,8
4
2
0
4
1,82
1,14
2
0
3.0
1.0
1.5
1.4
0.6
1,11
piRXLR04145, 20303, (14833) repress endogenous FRK1
activation upon flg22 treatment
9
0h
FRK1 relative expression level
1h
3h
6
3
0
piRXLR04097 repress endogenous FRK1 activation upon flg22
treatment
3
0h
1h
FRK1 relative expression level
3h
2
1
0
RXLR effector candidates do not block flg22-induced MAP kinase
activation in Arabidopsis protoplasts
Post-translational MAP kinase activation
+Flg22
0
ev
15
30
0
AvrPto
15
30
PiRXL16737
0
15
30
0
PiRXLR14833
15
30
@-phosphop42/p44
Ponceau S
ev
+Flg22
0
AvrPto
15
30
0
15
PiRXLR04145
30
0
15
PiRXLR20303
30
0
15
30
@-phosphop42/p44
Ponceau S
ev
+Flg22
@-phosphop42/p44
0
15
AvrPto
30
0
15
PiRXLR00821
30
0
15
PiRXLR151525
30
0
15
Ponceau S
+Flg22
@-phosphop42/p44
Ponceau S
ev
15
0
PiRXLR04097
15
30
0
PiRXLR05750
15
30
PiRXLR21388
0
15
30
30
sub-cellular localisation of piRXLR20303, 21388 and 14833 in
Arabidopsis protoplasts
GFP
GFP-piRXLR20303
(cytoplasm and nucleus)
GFP-piRXLR21388
(nucleus)
GFP-piRXLR14833
(nucleus)
sub-cellular localisation of 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
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