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 Filter Amplify

How many genes involved?

MPK1 MPK2 MPK7 MPK14 MPK20 MPK18 MPK19 MPK16 MPK17 MPK9 MPK8 MPK15 MPK10 MPK3 MPK6 MPK5 MPK12 MPK4 MPK11 MPK13 20 MAPKs in Arabidopsis (structurally homogenous) A B 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 At1g04700 At3g24720 At1g16270 At1g79570 MRK1 At3g01490 At5g50000 At3g22750 At4g14780 At1g62400 At3g46930 At5g58950 EDR1 MAP3Kd1 At1g18160 At1g73660 MAP3Kt1 MAP3Kt2 At3g58640 MAP3Kd5 CTR1 MAP3Kd4 At1g67890 MAP3Ke3 At3g06640 At3g06620 At3g06630 At1g14000 At2g31800 At2g43850 At3g59830 At3g58760 At4g18950 ANP3 ANP1 ANP2 MAP3Ka At1g63700 MAP3Kg MAP3Ke2 MAP3Ke1 MEKK4 MEKK3 MEKK2 MEKK1 At5g01850 At5g50180 ATN1At5g40540 At5g66710 At3g50720 ATN1-like At4g38470 At2g17700 At4g35780 At2g24360 At4g31170 B A C C1C1

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 Analyses of Plant Signal Transduction in Arabidopsis protoplasts

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 TFInactive TFActive TF Signal Transduction Early Defense Genes Flag22 FLS2 (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 mutantconstitutively activeand (dominant-negative) MD or E Manipulation of MAPKK activity

Constitutively active MKK5 activates MAPK 3 and 6 Without 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 Removal of regulatory domains (auto-inhibitory, interaction with upstream factors, …) constitutively active MAPKKK Substrate

MEKK1 activates MKK5 (Immunocomplex assay, GST-MPK6km as substrate) (35S Methionine labelling)... MPK3/6 (Immunocomplex assay, MBP as substrate) MAPK MEKK1 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 NC WRKY domain 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 happens downstream 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?

Flag22 QRLSTGSRINSAKDDAAGLQIA 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 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 Thomas Boller Lourdes Gomez-Gomez EverybodyJoulia Plotnikova Tsuneaki Asai Kazuya Ichimura Kazuo Shinozaki For the FLS2 seeds For the MAPKKK slide