rpg1-mediated Durable Stem Rust Resistance: Mechanisms of action
Rpg1-mediated durable stem rustresistance: mechanisms of action A. Kleinhofs, J. Nirmala, R. Brueggeman and B. Steffenson
Acknowledgments Multiyear support by USDA/NRI/AFRI toA. Kleinhofs and B. J. Steffenson and collaboration with D. von Wettstein and S. Hulbert past currentDave Kudrna Jayaveeramuthu NirmalaAndrzej Kilian Robert BrueggemanFeng Han Tom DraderArnis DrukaNils RostoksHenriette HorvathStephanie Dahl Research was supported by the United States Department of Agriculture Cooperative State Research, Education, and Extension Service Grants #2007-35301-18205 and #2010-65108-20568. to A.K. and B.J.S.
Morex/Chevron GP/transgenic GP vs. Chevron Morex vs. Chevron
Rpg1 is constitutively transcribed at low levels in all tissues tested Quantification of the Rpg1 mRNA Percent of actin mRNA 16.00 14.00 12.00 11 9 10.00 8 8.00 6 6.00 4.00 3 2.00 0 0.00 e f t ise g f o lea nc ea lin ro m el e ed g sc g ro in r in se atu re nP dl dl lo ed e M e e f Se Se ld lat In Go io Et Tissue type
RPG1 protein in leaf epidermis (LE) compared toleaf after removal of epidermis (LARE)
In vivo RPG1 phosphorylates within 5 min. post inoculation with avirulent Pgt spores.A: positive control, U : uninoculated controlPS: Phosphoserine, PT: Phosphothreonine
Viable but not inviable MCCF spores elicit RPG1 phosphorylation in cv. Morex 30 min post inoculation PS PT Lanes 1 and 3 inviable spores; 2 and 4 viable spores
RPG1 phosphorylation is race specific 1 – positive control 2 – uninoculated 3 – 15 min. sample PS – phosphoserine AB PT – 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 degradationof 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
RGD-binding and the VPS9 proteins together induce HR in barley harboring a functional Rpg1 gene A B C Golden Promise GP-Rpg1/pK1 GP-Rpg1/pK2 GP-Rpg1/T1 RGD+VPS9RGD+VPS9 ASM170 Steptoe Morex VPS9 VPS9 RGD RGD Morex Steptoe
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 minpost-spore landing on the leaf surface.*RPG1 phosphorylation is a highly race specificevent 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