rpg1-mediated Durable Stem Rust Resistance: Mechanisms of action

Rpg1-mediated durable stem rust resistance: mechanisms of action

A. Kleinhofs, J. Nirmala, R. Brueggeman and B. Steffenson

AcknowledgmentsMultiyear support by USDA/NRI/AFRI to

A. Kleinhofs and B. J. Steffenson and collaboration with D. von Wettstein and S. Hulbert

Dave KudrnaAndrzej KilianFeng HanArnis DrukaNils RostoksHenriette HorvathStephanie Dahl

Jayaveeramuthu NirmalaRobert BrueggemanTom Drader

past current

Research was supported by the United States Department of AgricultureCooperative State Research, Education, and Extension Service Grants

#2007-35301-18205 and #2010-65108-20568. to A.K. and B.J.S.

GP vs. Chevron Morex vs. Chevron

Morex/Chevron GP/transgenic

Novel gene structure

Exon 1 2 3 4 5 6 7 8 9 10 11 12 13 14

TATA ATG TGA

5’UTR 3’UTR

Genomic DNA

cDNA

(Brueggeman et al. (2002) PNAS)

Pkinase2Pkinase1 Pkinase2Pkinase1 Pkinase2Pkinase1

1 110 837 a.a.Protein

TMD

Rpg1 is constitutively transcribed at low levels in all tissues tested

Quantification of the Rpg1 mRNA

6

9 8

3

11

00.002.004.006.008.00

10.0012.0014.0016.00

Seed

ling l

eaf

Seed

ling r

oot

Etiolat

ed se

edlin

g

Matu

re lea

f

Inflor

escen

ce

Golden

Promise

Tissue type

Per

cent

of a

ctin

mR

NA

Except the epidermis

43.41

72.08

3.346.54

0.310.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Morex epidermis G02-448Depidermis

Morexmesophyll

G02-448Dmesophyll

Morex wholeleaf

Tissue type

RPG1 protein in leaf epidermis (LE) compared to leaf after removal of epidermis (LARE)

A: positive control, U : uninoculated control

PS: Phosphoserine, PT: Phosphothreonine

In vivo RPG1 phosphorylates within 5 min. post inoculation with avirulent Pgt spores.

Viable but not inviable MCCF spores elicit RPG1 phosphorylation in cv. Morex

Lanes 1 and 3 inviable spores; 2 and 4 viable spores

PS PT

30 min post inoculation

RPG1 phosphorylation is race specific

1 – positive control2 – uninoculated3 – 15 min. samplePS – phosphoserine ABPT – phosphothreonine AB

In vivo phosphorylation of RPG1 is required, but not sufficient for resistance

Lane 1: Positive control

Lane 2: uninoculated control

Lane 3: 15 min. post inoculation

PS: Phosphoserine

PT: Phosphthreonine

Effect of protein kinase inhibitors on Rpg1- mediated disease reaction.

PKI: Protein kinase inhibitor, PS: Phosphoserine, PT: Phosphothreonine

Isolation and characterization of RGD-binding and VPS9-like proteins

RGD-binding and VPS9 proteins together induce in vivo phosphorylation and degradation of RPG1.

RGD-binding protein alone induced phosphorylation of RPG1

VPS9 protein alone did not induce phosphorylation of RPG1

VPS9 failed to induce RPG1 degradation up to 48 h after application

Application of both proteins together induced RPG1 phosphorylation within 0.25 h and degradation prior to 24 h

Morex Steptoe

A B C

RG

D+V

PS9

RG

D+V

PS9

GP-

Rpg

1/T1

GP-

Rpg

1/pK

1

GP-

Rpg

1/pK

2

Gol

den

Prom

ise

ASM

170

Step

toe

RG

D

RG

D

VPS9

VPS9

Mor

ex

RGD-binding and the VPS9 proteins together induce HR in barley harboring a functional Rpg1 gene

RGD-binding and VPS9 proteins interact with RPG1 and with each other in yeast

VPS9 interacts with the RGD-binding protein

RPG1 interacts with RGD-binding and VPS9 proteins. The RPG1 pK1 and pK2 mutants failed to interact with the RGD-binding and the VPS9 proteins.

Summary*RPG1 phosphorylates in vivo within 5 min post-spore landing on the leaf surface.

*RPG1 phosphorylation is a highly race specific event and takes place only with viable spores.

*RPG1 phosphorylation is required for resistance and probably acts as a very early signal in pathogen perception.

Summary continued

• Two effector proteins isolated from spores• Both effectors required to mimic spore

function• Phenotypes tested: RPG1 phosphorylation &

degradation &host hypersensitive response• RGD-binding and VPS9 proteins interact with

RPG1 and with each other in yeast• RGD-binding protein may interact with

unknown RGD motif protein(s)

Speculation• RGD-binding protein interacts with RPG1 and

possibly other proteins at the cell membrane• RPG1 does not have an RGD motif – another

protein may be the receptor• RPG1 is phosphorylated and signals to initiate

the defense response• RPG1 degradation may release defense

complex from negative regulation or it may suppress runaway HR

Conserved domains of Rpg1 and JAK

Protein degradation is not sufficient for disease resistance

0 12 16 20 24 480 12 200 12 20 0 12 16 20 24 480 12 200 12 20

0 12 16 20 24 480 12 200 12 20 0 12 16 20 24 480 12 200 12 20

0 12 16 20 24 480 12 200 12 20 0 12 16 20 24 480 12 200 12 20

OSU6Beacon

Morex rpr1

G03-448F-3R Chevron

0

1

2

3

4

5

6

7

LE LARE

RPG1 protein (micromoles ofRPG1/g total protein)

RPG1 protein elevated in Leaf Epidermis (LE) compared to rest of the tissue (LARE)

PS: Phosphoserine

PT: Phosphothreonine

Lane 1: Positive control

Lane 2: uninoculated control

Lane 3: 15 min. post inoculation

RPG1 phosphorylation is race specific