New England Arabidopsis Meeting - April 3rd 2002
A MAPK cascade in
Arabidopsis innate immunity
Guillaume Tena
Signal
The prototypical
MAPK cascade
Input
Sensor
MAPKKK
MAPKK
Filter
Amplify
MAPK
Primary Response Genes
Secondary Response Genes
Output
How many genes involved?
20 MAPKs in Arabidopsis
(structurally homogenous)
B
MPK12
A
MPK5
MPK4
MPK6
MPK3
MPK11
MPK10
MPK13
MPK1
MPK15
MPK2
MPK8
MPK7
MPK9
MPK14
MPK20
MPK17
D
MPK16
MPK19
MPK18
C
10 MAPKKs in Arabidopsis
(structurally homogenous)
D
MKK8
MKK7
C
MKK9
MKK5
MKK4
MKK10
MKK2
MKK3
A
MKK1
MKK6
B
A
60 MAPKKK
in Arabidopsis
MAP3Ke1
MAP3Ke2
MEKK4
MEKK3
At1g63700
MEKK2
MAP3Kg
MAP3Ka
MEKK1
ANP2
ANP1
At5g01850
At5g50180
ATN1 At5g40540
At5g66710
At3g50720
ANP3
C1
At4g18950
ATN1-like
At4g38470
At3g58760
At3g59830
At2g17700
At4g35780
At2g43850
At2g24360
At4g31170
At2g31800
At3g46920
At1g14000
At3g06630
At5g57610
At3g06620
At3g06640
At2g35050
MAP3Ke3
At3g24720
At1g04700
At1g67890
At1g16270
MAP3Kd4
B
CTR1
MRK1
MAP3Kd5
At3g58640 MAP3Kt2
MAP3Kt1
At1g73660
At1g18160
MAP3Kd1
At1g79570
At3g01490
At5g50000
At3g22750
At4g14780
At1g62400
At3g46930
EDR1 At5g58950
C
Structural
heterogeneity
of plant
MAPKKKs
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
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
QuickTime™ and a
Intel Indeo® Video 4.4 decompressor
are needed to see this picture.
Functional Genomic Analyses
of
Plant Signal Transduction
in
Arabidopsis protoplasts
The early defense responses
1. Local defense
Pathogen
First line of protection = innate immunity
Very early detection of invaders in infected
cells through specific or generic interactions
Restricts pathogen growth and spread
(PCD, production of defensive molecules)
2. Systemic defense
Protects non-infected tissues from
secondary infection
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
The starting point model
(From previous work in T. Boller’s lab)
A Model for Defense Gene Induction
FLS2
Flag22
(From previous work in T. Boller’s lab)
(LRR)
(Kinase)
Signal Transduction (MAPK6)
Inactive TF
Active TF
TF
Early Defense Genes
Late Defense Genes
Are early defense responses
activated by flag22 in
our protoplast system?
Flg22
receptor
MAPKKK
MAPKK
MPK
Genes
…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
Which MAP kinases?
... MAPK 3 and 6
(Immunocomplex assay, MBP as substrate)
(35S Methionine labelling)
Flg22
receptor
MAPKKK
MAPKK
MPK3/6
Genes
Flg22 activates transfected…
… Promoters+LUC
Flg22
receptor
MAPKKK
MAPKK
MPK
Genes
Through which receptor?
... 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
What happens if we block the MAPK?
(there is no specific inhibitor of plant MAPKs : use a mouse MAPK phosphatase)
... totally the MAPK3/6 activation by flg22
Flg22
(protoplasts from wild-type)
(Immunocomplex assay,
MBP as substrate)
(35S Methionine labelling)
FLS2
MAPKKK
MAPKK
MKP1
MPK3/6
Genes
A MAPK phosphatase blocks…
… half of the gene activation by flg22
Flg22
FLS2
MAPKKK
MAPKK
MKP1
?
MPK3/6
Genes
GST1
Probably two pathways involved in the response:
one MAPK dependent
(blocked by MKP1, MKK inhibitors, dominant-negative)
one MAPK independent
(unknown)
After genes, signal, receptor, MAPK
Next step:
MAPKK
Manipulation of MAPKK activity
Kinase-dead mutant
(dominant-negative)
MKK1
MKK2
MKK6
MKK4
MKK5
MKK7
MKK9
MKK8
MKK3
MKK10
and
constitutively active
...LVKHKLTQQFFALKVIQLNTEEST...ITDFGVSKILTSTSSLANSFVGTYPYMSPERISG...
...LVQHKWTGQFFALKVIQLNIDEAI...ITDFGVSTVMTNTAGLANTFVGTYNYMSPERIVG...
...LVRHKWVGKFFAMKVIQMNIQEEI...ISDFGVSASLASSMGQRDTFVGTYNYMSPERISG...
...KVIHRPSSRLYALKVIYGNHEETV...IADFGVSRILAQTMDPCNSSVGTIAYMSPERINT...
...KVIHTPTSRPFALKVIYGNHEDTV...IADFGVSRILAQTMDPCNSSVGTIAYMSPERINT...
...KVHHKTTGEIYALKSVNGDMSPAF...IADFGVSKIITRSLDYCNSYVGTCAYMSPERFDS...
...KVRHKTTSEIYALKTVNGDMDPIF...IADFGVSKILVRSLDSCNSYVGTCAYMSPERFDS...
...KVKDKTTSEIYALKKVKENWDSTS...IADFGVSKIVVRSLNKCNSFVGTFAYMSPERLDS...
...RAIHIPNHRILALKKINIFEREKR...ITDFGISAGLENSMAMCATFVGTVTYMSPERIRN...
...KTRHRRTKTLYALKVLRPNLNTTV...IADFGASRIVAGGDYGSN---GTCAYMSPERVDL...
M
D or E
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)
... The MAPK3/6 activation
Flg22
(protoplasts from wild-type)
(Immunocomplex assay,
MBP as substrate)
(35S Methionine labelling)
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
Next step:
MAPKKK
Manipulation of MAPKKK activity
Kinase
Substrate
Removal of regulatory domains (auto-inhibitory, interaction with upstream factors, …)
constitutively active MAPKKK
Kinase
Substrate
... MPK3/6
(Immunocomplex assay,
MBP as substrate)
... MKK5
(Immunocomplex assay, GST-MPK6km as substrate)
(35S Methionine labelling)
Flg22
FLS2
MEKK1
MAPK
MEKK1
MKK4/5
MPK3/6
Genes
MEKK1 activates...
… Downstream gene activation
No need for the receptor
Flg22
FLS2
MEKK1
MKK4/5
MPK3/6
Genes
Going deeper in the pathway:
The effect of WRKY29 itself
General Features of WRKY Transcription Factors
NLS
WRKY
domain
N
C
~ 60 a.a.
----WRKY------------C-----C-----------------H-H-(Trp-Arg-Lys-Tyr)
Zinc-finger motif
Target Sequence : W box
(T)(T)TGAC(C/T)
WRKY29::GFP has a nuclear localization
WRKY29 activates its own
promoter and is a member
of a sub-family containing
WRKY22 as well
… and this happens
downstream of FLS2
Final signaling pathway analyzed in protoplasts
Signal
Receptor
Flg22
FLS2
MEKK1
MAPK cascade
MKK4/5
MPK3/6
Early responses
WRKY29/22
early genes (FRK1)
late genes (PR)
What about in plants?
Going back to the real world
Complexity of interactions
Flag22
QRLSTGSRINSAKDDAAGLQIA
… 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
Fungal resistance
of transgenic Arabidopsis
MEKK1
MKK4a
WRKY29
vector
Conclusion:
the innate immune system is
very conserved between
Arabidopsis, mammals and Drosophila
Conservation of innate immunity signaling pathways
Acknowledgments
Jen Sheen’s gang
Fred Ausubel’s gang
Everybody
Joulia Plotnikova
Tsuneaki Asai
Thomas Boller
Lourdes Gomez-Gomez
Kazuya Ichimura
Kazuo Shinozaki
For the FLS2 seeds
For the MAPKKK slide