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New England Arabidopsis Meeting - April 3rd 2002. A MAPK cascade in Arabidopsis innate immunity. Guillaume Tena. Signal. Input. The prototypical MAPK cascade. Sensor. MAPKKK. Filter Amplify. MAPKK. MAPK. Primary Response Genes. Output. Secondary Response Genes.

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A MAPK cascade in Arabidopsis innate immunity

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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)

MKK8

MKK7

MKK9

MKK5

MKK4

MKK10

MKK2

MKK3

MKK1

MKK6

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

At3g24720

At1g04700

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

Restricts 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. Boller’s lab)


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A Model for Defense Gene Induction

(From previous work in T. Boller’s lab)

Signal Transduction

Inactive TF

Active TF

TF

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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…MAPKsin 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…

… Promoters+LUC

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

MKP1

MPK3/6

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

and

constitutively active

(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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Kinase

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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N

C

Target Sequence : W box

(T)(T)TGAC(C/T)

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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WRKY29::GFP 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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Complexity of interactions

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 Sheen’s gang

Fred Ausubel’s gang

Everybody

Joulia Plotnikova

Tsuneaki Asai

Thomas Boller

Lourdes Gomez-Gomez

For the FLS2 seeds

Kazuya Ichimura

Kazuo Shinozaki

For the MAPKKK slide


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