A MAPK cascade in

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New England Arabidopsis Meeting - April 3rd 2002
A MAPK cascade in Arabidopsis innate immunity
Guillaume Tena
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Signal
Input
The prototypical MAPK cascade
Sensor
MAPKKK
Filter Amplify
MAPKK
MAPK
Primary Response Genes
Output
Secondary Response Genes
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How many genes involved?
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20 MAPKs in Arabidopsis (structurally homogenous)
B
MPK12
A
MPK5
MPK4
MPK6
MPK3
MPK11
MPK10
MPK13
MPK1
MPK15
C
MPK2
MPK8
MPK7
MPK9
MPK14
MPK20
MPK17
D
MPK16
MPK18
MPK19
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10 MAPKKs in Arabidopsis (structurally homogenous)
D
C
A
B
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A
60 MAPKKK in Arabidopsis
MAP3Ke1
MAP3Ke2
MEKK4
MEKK3
MEKK2
At1g63700
MAP3Kg
MAP3Ka
MEKK1
ANP2
ANP1
At5g01850
At5g50180
ATN1
At5g40540
ANP3
At5g66710
At3g50720
C1
At4g18950
ATN1-like
At4g38470
At3g58760
At3g59830
At2g17700
At4g35780
At2g43850
At2g24360
At4g31170
At2g31800
At3g46920
At1g14000
At3g06630
At5g57610
At3g06620
At3g06640
At2g35050
MAP3Ke3
At1g04700
At3g24720
At1g67890
At1g16270
MAP3Kd4
CTR1
At1g79570
MRK1
B
C
MAP3Kd5
At3g01490
At5g50000
At3g22750
At4g14780
At3g58640
MAP3Kt2
At1g62400
MAP3Kt1
At1g73660
At1g18160
At3g46930
MAP3Kd1
EDR1
At5g58950
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Structural heterogeneity of plant MAPKKKs
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Complexity of MAPK networks
20 x 10 x 60 a lot of virtual combinations
How to work with such a high number of
possibilities?
a simplified biological system possibility to do
hundreds of functional analyses quickly
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SIMPLIFIED BIOLOGICAL SYSTEM (to quickly select
interesting genes for later analyses in whole
plant)
From an entire plant to a tissue to a
single type of cell
Arabidopsis mesophyll cells protoplasts an
homogenous and easily manipulable material
Complexity
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Functional Genomic Analyses of Plant Signal
Transduction in Arabidopsis protoplasts
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The early defense responses
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1. Local defense
First line of protection innate immunity Very
early detection of invaders in infected cells
through specific or generic interactions Restrict
s pathogen growth and spread (PCD, production of
defensive molecules)
Pathogen
2. Systemic defense
Protects non-infected tissues from secondary
infection
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SIMPLIFIED PATHOGENIC SIGNAL
From bacterium to flagellum to
flagellin to flag22
Biologically active synthetic peptide acting as
a general elicitor just one pathway involved
QRLSTGSRINSAKDDAAGLQIA
Complexity
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The starting point model (From previous work in
T. Bollers lab)
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A Model for Defense Gene Induction (From
previous work in T. Bollers lab)
FLS2
Flag22
(LRR)
(Kinase)

(MAPK6)
Early Defense Genes
Late
Defense Genes
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Are early defense responses activated by flag22
in our protoplast system?
Flg22
receptor
MAPKKK
MAPKK
MPK
Genes
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MAPKs in 10 minutes (In-gel kinase assay, MBP
as substrate)
Flg22
receptor
MAPKKK
MAPKK
MPK
Flg22 activates endogenous
Genes
Genes in 30 minutes (RT-PCR on genes
identified from a substracted cDNA library)
Flg22
receptor
MAPKKK
MAPKK
MPK
Genes
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Which MAP kinases?
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... MAPK 3 and 6 (Immunocomplex assay, MBP as
substrate) (35S Methionine labelling)
Flg22
receptor
MAPKKK
MAPKK
MPK3/6
Genes
Flg22 activates transfected
PromotersLUC
Flg22
receptor
MAPKKK
MAPKK
MPK
Genes
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Through which receptor?
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... MAPK3/6 activation by flg22 (protoplasts from
fls2 mutant and wild-type) (Immunocomplex assay,
MBP as substrate) (35S Methionine labelling)
Flg22
FLS2
MAPKKK
MAPKK
MPK3/6
Genes
FLS2 receptor is needed for
... Gene activation by flg22
Flg22
FLS2
MAPKKK
MAPKK
MPK
Genes
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What happens if we block the MAPK?
(there is no specific inhibitor of plant MAPKs
use a mouse MAPK phosphatase)
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... totally the MAPK3/6 activation by
flg22 (protoplasts from wild-type) (Immunocomplex
assay, MBP as substrate) (35S Methionine
labelling)
Flg22
FLS2
MAPKKK
MAPKK
MPK3/6
MKP1
Genes
A MAPK phosphatase blocks
half of the gene activation by flg22
Flg22
FLS2
MAPKKK
?
MAPKK
MKP1
MPK3/6
Genes
GST1
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Probably two pathways involved in the response
one MAPK dependent (blocked by MKP1, MKK
inhibitors, dominant-negative) one MAPK
independent (unknown)
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After genes, signal, receptor, MAPK Next
step MAPKK
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Manipulation of MAPKK activity
Kinase-dead mutant
constitutively active
and
(dominant-negative)
M
D or E
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Constitutively active MKK5 activates MAPK 3 and
6 Without the need for Flg22 signal
Flg22
FLS2
MAPKKK
MKK5
MPK3/6
Genes
(Immunocomplex assay, MBP as substrate) (35S
Methionine labelling)
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... The MAPK3/6 activation (protoplasts from
wild-type) (Immunocomplex assay, MBP as
substrate) (35S Methionine labelling)
Flg22
FLS2
MAPKKK
MKK4/5
MPK3/6
Genes
MKK4 and 5 are redundant for...
Gene activation Downstream of
the receptor
Flg22
FLS2
MAPKKK
MKK4/5
MPK3/6
Genes
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Next step MAPKKK
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Manipulation of MAPKKK activity
Substrate
Removal of regulatory domains (auto-inhibitory,
interaction with upstream factors, )
constitutively active MAPKKK
Kinase
Substrate
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... MKK5 (Immunocomplex assay, GST-MPK6km as
substrate) (35S Methionine labelling)
... MPK3/6 (Immunocomplex assay, MBP as
substrate)
Flg22
FLS2
MEKK1
MAPK
MKK4/5
MEKK1
MPK3/6
Genes
MEKK1 activates...
Downstream gene activation No need for
the receptor
Flg22
FLS2
MEKK1
MKK4/5
MPK3/6
Genes
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Going deeper in the pathway The effect of
WRKY29 itself
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General Features of WRKY Transcription Factors
WRKY
NLS
domain
60 a.a.
----
WRKY
------------
C
-----
C
-----------------
H
-
H
--
Zinc-finger motif
(Trp-Arg-Lys-Tyr)
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WRKY29GFP has a nuclear localization
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WRKY29 activates its own promoter and is a
member of a sub-family containing WRKY22 as well
and this happens downstream of FLS2
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Final signaling pathway analyzed in protoplasts
Signal
Flg22
Receptor
FLS2
MEKK1
MAPK cascade
MKK4/5
MPK3/6
WRKY29/22
Early responses
early genes (FRK1)
late genes (PR)
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What about in plants?
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Going back to the real world
Flag22 QRLSTGSRINSAKDDAAGLQIA
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bacterial infection with Pseudomonas syringae
100 times reduction of bacterial growth after 4
days
Agrobacterium-mediated transient transformation
of Arabidopsis leaves to activate the pathway
protects from...
fungal infection with Botrytis cinerea
MKK1a (control) MKK4a
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Fungal resistance of transgenic Arabidopsis
MEKK1 MKK4a WRKY29 vector
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Conclusion the innate immune system is very
conserved between Arabidopsis, mammals and
Drosophila
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Conservation of innate immunity signaling pathways
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Acknowledgments
Jen Sheens gang
Fred Ausubels gang
Everybody
Joulia Plotnikova Tsuneaki Asai
Thomas Boller Lourdes Gomez-Gomez
For the FLS2 seeds
Kazuya Ichimura Kazuo Shinozaki
For the MAPKKK slide