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