A MAPK cascade in Arabidopsis innate immunity England Arabidopsis Meeting - April 3rd 2002 Guillaume Tena
Dec 19, 2015
A MAPK cascade in Arabidopsis innate immunity
New England Arabidopsis Meeting - April 3rd 2002
Guillaume Tena
Signal
Sensor
MAPKKK
MAPKK
MAPK
Primary Response Genes
Secondary Response Genes
The prototypical MAPK cascade
Input
Output
FilterAmplify
How many genes involved?
MPK1
MPK2
MPK7
MPK14
MPK20
MPK18MPK19MPK16
MPK17
MPK9
MPK8
MPK15
MPK10
MPK3MPK6
MPK5
MPK12
MPK4
MPK11
MPK13
20 MAPKs in Arabidopsis(structurally homogenous)
AB
C
D
MKK10
MKK3
MKK6
MKK1
MKK2
MKK4
MKK5
MKK8
MKK7
MKK9
10 MAPKKs in Arabidopsis(structurally homogenous)
A
C
D
B
60 MAPKKK in Arabidopsis
At3g46920
At5g57610
At2g35050
At1g04700At3g24720
At1g16270
At1g79570MRK1
At3g01490At5g50000
At3g22750At4g14780At1g62400
At3g46930
At5g58950EDR1MAP3Kd1
At1g18160At1g73660
MAP3Kt1
MAP3Kt2At3g58640
MAP3Kd5
CTR1MAP3Kd4
At1g67890
MAP3Ke3
At3g06640
At3g06620
At3g06630At1g14000
At2g31800
At2g43850
At3g59830
At3g58760
At4g18950
ANP3
ANP1ANP2
MAP3Ka
At1g63700MAP3Kg
MAP3Ke2
MAP3Ke1
MEKK4
MEKK3
MEKK2
MEKK1At5g01850
At5g50180ATN1 At5g40540
At5g66710At3g50720
ATN1-like
At4g38470
At2g17700
At4g35780
At2g24360At4g31170
B
A
C
C1
Structural heterogeneity
of plant MAPKKKs
How to work with such a high number of possibilities?
20 x 10 x 60 = a lot of virtual combinations
a simplified biological system
possibility to do hundreds of functional analyses quickly
Complexity of MAPK networks
From an entire plant… to a tissue… to a single type of cell
SIMPLIFIED BIOLOGICAL SYSTEM(to quickly select interesting genes for later analyses in whole plant)
Arabidopsis mesophyll cells
protoplasts: an homogenous and easily manipulable
material
Complexity
Functional Genomic Analysesof
Plant Signal Transductionin
Arabidopsis protoplasts
QuickTime™ and aIntel Indeo® Video 4.4 decompressor
are needed to see this picture.
The early defense responses
Pathogen
1. Local defense
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
From bacterium… to flagellum… to flagellin… to flag22
SIMPLIFIED PATHOGENIC SIGNAL
QRLSTGSRINSAKDDAAGLQIA
Biologically active synthetic peptide
acting as a general elicitor:
just one pathway involved
Complexity
The starting point model(From previous work in T. Boller’s lab)
A Model for Defense Gene Induction
(From previous work in T. Boller’s lab)
Late Defense Genes
TF
Inactive TF Active TF
Signal Transduction
Early Defense Genes
Flag22FLS2
(LRR)
(Kinase)
(MAPK6)
Are early defense responses activated by flag22 in our protoplast system?
Flg22
receptor
MAPKKK
MAPKK
MPK
Genes
Flg22
receptor
MAPKKK
MAPKK
MPK
Genes
…MAPKs in 10 minutes(In-gel kinase assay, MBP as substrate)
… Genes in 30 minutes(RT-PCR on genes identified from a substracted cDNA library)
Flg22
receptor
MAPKKK
MAPKK
MPK
Genes
Flg22 activates endogenous…
Which MAP kinases?
... MAPK 3 and 6 (Immunocomplex assay, MBP as substrate)(35S Methionine labelling)
Flg22
receptor
MAPKKK
MAPKK
MPK3/6
Genes
… Promoters+LUC
Flg22
receptor
MAPKKK
MAPKK
MPK
Genes
Flg22 activates transfected…
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
... Gene activation by flg22 Flg22
FLS2
MAPKKK
MAPKK
MPK
Genes
FLS2 receptor is needed for…
What happens if we block the MAPK?
(there is no specific inhibitor of plant MAPKs : use a mouse MAPK phosphatase)
A MAPK phosphatase blocks…
… half of the gene activation by flg22 Flg22
FLS2
MAPKKK
MAPKK
MPK3/6
Genes
MKP1
... 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
Genes
MKP1
?
GST1
one MAPK dependent (blocked by MKP1, MKK inhibitors, dominant-negative)
one MAPK independent (unknown)
Probably two pathways involved in the response:
After genes, signal, receptor, MAPK
Next step:
MAPKK
Kinase-dead mutant
MKK1 ...LVKHKLTQQFFALKVIQLNTEEST...ITDFGVSKILTSTSSLANSFVGTYPYMSPERISG...MKK2 ...LVQHKWTGQFFALKVIQLNIDEAI...ITDFGVSTVMTNTAGLANTFVGTYNYMSPERIVG...MKK6 ...LVRHKWVGKFFAMKVIQMNIQEEI...ISDFGVSASLASSMGQRDTFVGTYNYMSPERISG...MKK4 ...KVIHRPSSRLYALKVIYGNHEETV...IADFGVSRILAQTMDPCNSSVGTIAYMSPERINT...MKK5 ...KVIHTPTSRPFALKVIYGNHEDTV...IADFGVSRILAQTMDPCNSSVGTIAYMSPERINT...MKK7 ...KVHHKTTGEIYALKSVNGDMSPAF...IADFGVSKIITRSLDYCNSYVGTCAYMSPERFDS...MKK9 ...KVRHKTTSEIYALKTVNGDMDPIF...IADFGVSKILVRSLDSCNSYVGTCAYMSPERFDS...MKK8 ...KVKDKTTSEIYALKKVKENWDSTS...IADFGVSKIVVRSLNKCNSFVGTFAYMSPERLDS...MKK3 ...RAIHIPNHRILALKKINIFEREKR...ITDFGISAGLENSMAMCATFVGTVTYMSPERIRN...MKK10 ...KTRHRRTKTLYALKVLRPNLNTTV...IADFGASRIVAGGDYGSN---GTCAYMSPERVDL...
constitutively activeand
(dominant-negative)
M D or E
Manipulation of MAPKK activity
Constitutively active MKK5 activates MAPK 3 and 6Without the need for Flg22 signal
(Immunocomplex assay, MBP as substrate)(35S Methionine labelling)
Flg22
FLS2
MAPKKK
MKK5
MPK3/6
Genes
MKK4 and 5 are redundant for...
... The MAPK3/6 activation (protoplasts from wild-type) (Immunocomplex assay, MBP as substrate) (35S Methionine labelling)
… Gene activation … Downstream of the receptor
Flg22
FLS2
MAPKKK
MKK4/5
MPK3/6
Genes
Flg22
FLS2
MAPKKK
MKK4/5
MPK3/6
Genes
Next step:
MAPKKK
Manipulation of MAPKKK activity
Kinase
Kinase
Removal of regulatory domains (auto-inhibitory, interaction with upstream factors, …)
constitutively active MAPKKK
Substrate
Substrate
MEKK1 activates...
... MKK5 (Immunocomplex assay, GST-MPK6km as substrate)(35S Methionine labelling)
... MPK3/6 (Immunocomplex assay, MBP as substrate)
MAPKMEKK1
Flg22
FLS2
MEKK1
MKK4/5
MPK3/6
Genes
Flg22
FLS2
MEKK1
MKK4/5
MPK3/6
Genes
… Downstream gene activation No need for the receptor
Going deeper in the pathway:
The effect of WRKY29 itself
(T)(T)TGAC(C/T)Target Sequence : W box
N C
WRKYdomain
NLS
----WRKY------------C-----C-----------------H-H--Zinc-finger motif(Trp-Arg-Lys-Tyr)
~ 60 a.a.
General Features of WRKY Transcription Factors
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 happensdownstream of FLS2
Flg22
FLS2
MEKK1
MKK4/5
MPK3/6
WRKY29/22
Final signaling pathway analyzed in protoplasts
MAPK cascade
Receptor
Signal
Early responses
early genes (FRK1)
late genes (PR)
What about in plants?
Flag22QRLSTGSRINSAKDDAAGLQIA
Going back to the real world
Complexity of interactions
100 times reduction of bacterial growth after 4 days
Agrobacterium-mediated transient transformation of Arabidopsis leaves to activate the pathway protects from...
… bacterial infection with Pseudomonas syringae
… fungal infection with Botrytis cinereaMKK1a (control) MKK4a
Fungal resistance of transgenic Arabidopsis
MEKK1 MKK4a WRKY29 vector
Conclusion:
the innate immune system isvery conserved between
Arabidopsis, mammals and Drosophila
Conservation of innate immunity signaling pathways
ArabidopsisMammalsFLS2FlagellinIRAKImmune Response GenesTLR4LPSTLR5FlagellinTLR2PGNWRKY22/29Inhibitor?MKK4/5MAPK3/6AtMEKK1?Fungus-derivedelicitor
Immune Response GenesIκB-NF κBNIKIKK 1TAKTollPelle( )kinaseSpaetzle( )LRR( )TIRDrosophilaCactus/Dif Rel Immune Response GenesJunFosMEK, , 38JNK ERK pMEKK???
Acknowledgments
Jen Sheen’s gang Fred Ausubel’s gang
Thomas BollerLourdes Gomez-Gomez
Everybody Joulia PlotnikovaTsuneaki Asai
Kazuya IchimuraKazuo Shinozaki
For the FLS2 seeds
For the MAPKKK slide