Zhiyong Gao, Eui-Hwan Chung, Timothy K. Eitas and Jeffery L. Dangl.

PNAS April 13, 2011

INTRODUCTION

Introduction

MAMPS receptor

Transmembrane pattern recognition receptors

Plant express MAMPs receptors to detect microbe associated molecular patterns

Plant defense against pathogens

Introduction

MAMPS receptor

MAMPS receptors

Recognition lead to intracellular signaling and transcriptional output responses

Plant defense against pathogens

Introduction

Nucleotide binding leucine rich repeat (NB-LRR) receptors

Introduction

Coiled coil nucleotide binding leucine rich repeat (CC NB-LRR)

Introduction

Hypersensitive-response (HR)
Nucleotide binding leucine rich repeat (NB-LRR) receptors

Introduction

Preactivated NB-LRR proteins have been localized to multiple subcellular compartments.

Subcellular localization

Introduction

RPM1 is a CC-NB-LRR protein

Nucleotide binding leucine rich repeat (NB-LRR) receptors

Introduction

AvrRpm1 or AvrB

RPM1 recognize effector mediated modifications of RIN4

Subcellular localization

MATERIAL AND METHODS

Generation of autoactive alleles

MATERIAL AND METHODS

Generation of autoactive alleles

MATERIAL AND METHODS

Generation of autoactive alleles

MATERIAL AND METHODS

Generation of autoactive alleles

Vector constructs

MATERIAL AND METHODS

pGPTV-HPT

Vector constructs

MATERIAL AND METHODS

PCR

Myc-tag N-EQKLISEEDL-C

Myc

Vector constructs

MATERIAL AND METHODS

PCR

Myc

NES

Vector constructs

MATERIAL AND METHODS

PCR

MYB

nes

Vector constructs

MATERIAL AND METHODS

PCR
CBL

Vector constructs

MATERIAL AND METHODS

gRPM1-myc

gRPM1-myc-NES

RPM1-myc induced by estradiol

T7-RIN4

Vector constructs

Agroinfiltration and generation of transgenic plants

MATERIAL AND METHODS

Agrobacterium were infiltrated into leaves of 5 to 6 weeks old Nicotiana benthamiana

Agroinfiltration

Conductivity measurements

MATERIAL AND METHODS

Conductivity measurements were performed to estimate the state of the plant

Conductivity measurents

RESULTS

RESULTS

Autoactive alleles analysis

RESULTS
Lost of function mutants

Affected in the coiled coil domain

Affected in spacer region Affected in the P-loop domain

Autoactive alleles analysis

RESULTS

Autoactive alleles analysis

RESULTS

Autoactive alleles analysis

RESULTS

The disappearance of RPM1 is correlated with effectormediated activation

Autoactive alleles analysis

RESULTS

RPM1 wild type is weakly autoactive in a rin4 null mutant

RIN4 role as supressor

RESULTS
5

RPM1(D505V) + rin4

RIN4 expression delayed and diminished cell death

RIN4 role as supressor

RESULTS

The transgenic lines gRPM1(D505V) rmp1 rin4 were difficult to recover

RIN4 role as supressor

RESULTS

RPM1 is associated to membranes

Localization of RPM1

MATERIAL AND METHODS

Cellular localization

RESULTS

RPM1 is predominantly localized on plasmatic membrane

Localization of RPM1

RESULTS

It is highly unlike that activated RPM1 relocalizes to the nucleus

Localization of RPM1

RESULTS

Localization of RPM1

MATERIAL AND METHODS

Generation of autoactive alleles

RESULTS

RPM1 neither relocalizes to, nor function in the nucleus

Localization of RPM1

MATERIAL AND METHODS

Generation of autoactive alleles

RESULTS

Plasma membrane-tethered RPM1 retains HR function

Localization of RPM1

CONCLUSIONS

CONCLUSIONS

1) The P-loop is important for RPM1 function and localization.

2) The CC domain is critical for function.

3) Preactivated RPM1 resides on the plasma membrane. 4) The results indicate that activated RPM1 resides solely on the plasma membrane.
Localization of RPM1