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

PNAS April 13, 2011

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INTRODUCTION

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Introduction

Plant defense against pathogens

MAMPS receptor

Transmembrane pattern

recognition receptors

Plant express

MAMPs

receptors to

detect

microbe

associated

molecularpatterns

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Introduction

Plant defense against pathogens

MAMPS receptors

MAMPS receptor

Recognition

lead to

intracellular

signaling and

transcriptional

output

responses

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Introduction

Nucleotide binding leucine rich repeat (NB-LRR) receptors

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Introduction

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

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Introduction

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

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Introduction

Subcellular localization

Preactivated

NB-LRR

proteins have

been localized

to multiple

subcellular

compartments.

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Introduction

Nucleotide binding leucine rich repeat (NB-LRR) receptors

RPM1 is a CC-NB-LRR protein

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Introduction

Subcellular localization

RPM1

recognize

effectormediated

modifications

of RIN4

AvrRpm1or

AvrB

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MATERIAL AND

METHODS

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Generation ofautoactive alleles

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MATERIAL AND METHODS

Generation of autoactive alleles

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MATERIAL AND METHODS

Generation of autoactive alleles

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MATERIAL AND METHODS

Generation of autoactive alleles

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Vector constructs

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MATERIAL AND METHODS

Vector constructs

pGPTV-HPT

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MATERIAL AND METHODS

Vector constructs

Myc

PCRMyc-tag

N-EQKLISEEDL-C

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MATERIAL AND METHODS

Vector constructs

Myc NES

PCR

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MATERIAL AND METHODS

Vector constructs

MYB nes

PCR

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MATERIAL AND METHODS

Vector constructs

CBL

PCR

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MATERIAL AND METHODS

Vector constructs

gRPM1-myc

T7-RIN4

RPM1-myc induced by estradiol

gRPM1-myc-NES

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Agroinfiltrationand generation oftransgenic plants

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MATERIAL AND METHODS

Agroinfiltration

Agrobacterium

were infiltrated

into leaves of 5 to6 weeks old

Nicotiana

benthamiana

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Conductivitymeasurements

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MATERIAL AND METHODS

Conductivity measurents

Conductivity

measurements

were performedto estimate the

state of the

plant

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RESULTS

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RESULTS

Autoactive alleles analysis

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RESULTS

Autoactive alleles analysis

Affected in the

coiled coil domain

Affected in spacer

region

Affected in the

P-loop domain

Lost of function mutants

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RESULTS

Autoactive alleles analysis

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RESULTS

Autoactive alleles analysis

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RESULTS

Autoactive alleles analysis

The disappearance of RPM1 is correlated with effector-

mediated activation

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RESULTS

RIN4 role as supressor

RPM1 wild type

is weakly

autoactive in a

rin4 null mutant

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RESULTS

RIN4 role as supressorRIN4 expression delayed and diminished cell death

RPM1(D505V) + rin4

5

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RESULTS

RIN4 role as supressor

The transgenic lines gRPM1(D505V) rmp1

rin4 were difficult to recover

g

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RESULTS

Localization of RPM1

RPM1 is associated to membranes

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MATERIAL AND METHODS

Cellular localization

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RESULTS

Localization of RPM1

RPM1 is predominantly localized on plasmatic membrane

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RESULTS

Localization of RPM1

It is highly unlike that activated RPM1

relocalizes to the nucleus

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RESULTS

Localization of RPM1

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MATERIAL AND METHODS

Generation of autoactive alleles RESULTS

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Localization of RPM1

RPM1 neither relocalizes to, nor

function in the nucleus

MATERIAL AND METHODS

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Generation of autoactive alleles RESULTS

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Localization of RPM1Plasma membrane-tethered RPM1 retains HR function

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CONCLUSIONS

CONCLUSIONS

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4) The results indicate that activated RPM1 resides

solely on the plasma membrane.

3) Preactivated RPM1 resides on the plasma

membrane.

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

localization.

2) The CC domain is critical for function.