Signaling Network in Plant Cells

Plant Signal Transduction Pathways

Signal Receptor
?

? ? ? ? Responses

Transcription

Plant Signal Transduction An Integrated Approach

Genes Proteins

Cells

Mutants

Transgenics

Arabidopsis Mesophyll Protoplasts Are Viable and Responsive

High Transfection Efficiency of Mesophyll Protoplasts

Maize Arabidopsis PEG-Calcium Maize Electroporation

Functional Genomic Analyses of Plant Signal Transduction in Arabidopsis

Light & Glucose Signaling in Maize Mesophyll Protopalsts

Dark

Light

Light & Glucose

ABA Signaling in Maize Mesophyll Protoplasts

Control

ABA

CDPK

Auxin Signaling in Maize Mesophyll Protoplasts

Cytokinin Signaling in Arabidopsis Mesophyll Protoplasts

H2O2 Signaling in Arabidopsis Mesophyll Protoplasts

Flg22 Signaling in Arabidopsis Mesophyll Protoplasts

Functional Analysis of Plant MAPK Cascades in Stress and Hormonal Signaling

Jen Sheen, MGH Frederick M. Ausubel, MGH Kan Wang, ISU

Plant Systems Arabidopsis, maize, soybean

Sponsor NSF Plant Genome Research Project

Why MAPK cascades?

MAPK cascades are central signaling components that connect diverse receptors/sensors to cellular and nuclear activities in plants

Stress
wounding, H
2 O 2,

O 3 , cold, heat, salinity, drought/osmotic

Hormones
ethylene, ABA, auxin

Pathogens
virus, bacterial and fungal elicitors

Objectives
Develop protoplast assays Clone all Arabidopsis MAPK cascade genes Functional analysis in protoplasts Functional analysis in transgenic Arabidopsis Functional analysis in transgenic maize & soybean Web-accessible database

People & Assignments

Jen Sheen Binary constructs, Transgenic Arabidopsis & Affychips

Wan-Ling Chiu MAKPs & PTPs Guillaume Tena MAPKKKs, MAPKKs & MAPKs Tsuneaki Asai Early response genes & Transcription factors

Joulia Plotnikova Fungal infection & Transgenic Arabidopsis Sylvia Shou Kan Wang Senthil Ramu Cheri Chen Transgenic maize Transgenic soybean GTPase & Receptors Website & Database

Shuichi Yanagisawa Transcription factors Rahul Patharkar MAPK substrates, Scaffold proteins & Microarrays

Stimuli Sensors/Receptors
Arabidopsis Genes /Groups

MitogenA ctivated P rotein K inase C ascades

MAPKKKs MAPKKs MAPKs

> 25 9 23

7 4 5

Transcription Factors Primary Response Genes Secondary Response Genes

Functional Genomic Approach

NPK1(MAPKKK) Suppresses Auxin Signaling

Protein kinase Activity Assay

Protein Expression

Kinase Activity

MKP Blocks NPK1 Repression of Auxin Signaling

ANP1(MAPKKK) Suppresses Auxin Signaling

Embryogenesis Defect in NPK1 Transgenic Plants

H2O2 Activates Transcription & MAPKs

Redundant ANPs Mediate H2O2 Signaling & MAPK Activation

ANP1 Activates H2O2 Responsive Promoters

Cross Protection Phenomena in Plants Cold Heat Drought Wounding Ozone Pathogens Common Messengers H 2O 2 Calcium

Drought Resistance

Transgenic NPK1 Plants Display Salt, Freezing & Heat Tolerance

Crosstalk between Stress & Auxin Signaling AUXIN H 2 O 2 , Heat

COP9 Signalosome SCF (TIR1)

PKs IAA/AUX ARFs

MAPKKK (ANP1-3, NPK1 MAPKK ? MAPK (AtMPK3 & 6)

auxin genes
(GH3, IAAs)

stress genes
(GST6, HSP18.2)

Plant Defense Mechanisms Recognize Enemies

Gene-for-Gene Interactions
Host Receptor: R genes (Pto, N, Rps2, Cf9, Xa21) Pathogen Elicitor: Avr genes (avrPto, Avr9)

General Interactions
Host Receptor: FLS2 Pathogen Elicitor: Flagellin (Flg22), OS, LP

Plant Defense Mechanisms Signal Transduction

Specific Elicitors
LRR R LRR

General Elicitors
R LRR

Convergent Signaling
Ca++, PKs H2O2, ion flux

Early Genes:
TFs, SA, ET, JA

Late Genes:
PRs, PL

Flg22 Signaling Cascade in Plant Pathogen Defense

Convergent Signaling ? Cascade

? ?

Defense

Transcription

Arabidopsis Leaf Protoplast System

Flg22 Signaling in Arabidopsis Mesophyll Protoplasts

Flg22 Signaling Is Mediated by LRR Receptor Kinase FLS2

Flg22 Activation of MAPKs Is FLS2 Dependent

Flg22 MAPK Signaling Cascade in Plant Pathogen Defense

Flg22 peptide FLS2 (LRR-RLK)

MAPKKK MAPKK MAPK Defense MAPK WRKY Transcription

Arabidopsis
LRR Receptor

Mammals
LRR Receptor

Drosophila
LRR Receptor

Elicitors

FLS2

TLR2, 4, 5, 9

Toll

Other LRR Receptors ? FLS2 Kinase IRAK Kinase Pelle Kinase

? AtMEKK1 AtMKK4/AtMKK5 AtMPK3/AtMPK6 R ? WRKY 22/29 Jun Fos MEKK MEK JNK ERK,p38 TAK NIK IKK IkB NF-kB ? JNK p38 ? Cactus Dif/Rel

Immune Response Gene Transcription W W W Disease Resistance

Positive Feedback Control by WRKY 22/29

Plant MAPK Signaling Cascades 2001

Plant
Arabidopsis Tobacco Alfalfa Arabidopsis Tobacco

Signal Sensor

Bacterial Elicitor FLS2 ?

Oxidative Stress

Fungal Elicitor

Salt Stress

Abiotic Sresses AtHK1?

Mitosis Cytokinesis

MAPKKK

AtMEKK1

AtANP1
(NtNPK1)

AtMEKK1

NtNPK1
(AtANP1)

MAPKK

AtMKK4/5
(NtMEK2) (MsSIMKK)

NtMEK2
(AtMKK4/5) (MsSIMKK)

MsSIMKK
(AtMKK4/5) (NtMEK2)

AtMKK1/2
(NtSIPKK)

NtMEK1 **

MAPK

AtMPK3/6

AtMPK3/6

NtSIPK/WIPK
(AtMPK3/6) (MsSAMK/SIMK *)

MsSIMK
(AtMPK6) (NtSIPK)

AtMPK4
(MsMMK2)

NtNtf6 **
(MsMMK3)

(NtWIPK/SIPK) (MsSAMK/SIMK *)

Target Genes Negative Regulator

PAL(+)

GST6, HSP18.2 (+) GH3, ER7 (-) AtMKP1? AtPTP1?

HMGR(+) PAL (+)

MsMP2C?

AtMKP1? AtPTP1?

Antagonistic Interactions between MAPK Cascades

Auxin H2O2 Stress Ethylene Unknown Signal
ctr1 mutant

SA

JA

PD98059 U0126

mpk4 mutant

Unknown MAPK cascade

AtANP1 MAPK cascade

etr1 mutant

Unknown MAPK cascade

AtCTR1 MAPK cascade

NtSIPK AtMPK6 MAPK cascade

AtMPK4 MAPK cascade

Signal integration

Signal integration

Signal integration

OUTPUT

OUTPUT

OUTPUT

Summary
MAPK cascades play a central role in intracellular signaling MAPK cascades are involved in stress and hormonal signaling MAPK cascades mediate both positive and negative controls Manipulation of MAPK cascades confers multiple stress protection

MAPK Signaling Group

NSF/DIP