rpg1-mediated Durable Stem Rust Resistance: Mechanisms of action

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Andris Kleinhofs

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rpg1-mediated Durable Stem Rust Resistance: Mechanisms of action

  1. 1. Rpg1-mediated durable stem rustresistance: mechanisms of action A. Kleinhofs, J. Nirmala, R. Brueggeman and B. Steffenson
  2. 2. 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.
  3. 3. Morex/Chevron GP/transgenic GP vs. Chevron Morex vs. Chevron
  4. 4. Novel gene structureGenomic DNAExon 1 2 3 4 5 6 7 8 9 10 11 12 13 14 5’UTR 3’UTRTATA ATG TGAcDNAProtein 1 110 837 a.a. Pkinase1 Pkinase1 Pkinase2 Pkinase2 TMD (Brueggeman et al. (2002) PNAS)
  5. 5. 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
  6. 6. Except the epidermis90.0080.00 72.0870.0060.0050.00 43.4140.0030.0020.0010.00 6.54 3.34 0.31 0.00 Morex epidermis G02-448D Morex G02-448D Morex whole epidermis mesophyll mesophyll leaf Tissue type
  7. 7. RPG1 protein in leaf epidermis (LE) compared toleaf after removal of epidermis (LARE)
  8. 8. In vivo RPG1 phosphorylates within 5 min. post inoculation with avirulent Pgt spores.A: positive control, U : uninoculated controlPS: Phosphoserine, PT: Phosphothreonine
  9. 9. 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
  10. 10. RPG1 phosphorylation is race specific 1 – positive control 2 – uninoculated 3 – 15 min. sample PS – phosphoserine AB PT – phosphothreonine AB
  11. 11. 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
  12. 12. Effect of protein kinase inhibitors on Rpg1- mediated disease reaction. PKI: Protein kinase inhibitor, PS: Phosphoserine, PT: Phosphothreonine
  13. 13. Isolation and characterization of RGD-binding and VPS9-like proteins
  14. 14. 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
  15. 15. 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
  16. 16. 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.
  17. 17. 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.
  18. 18. 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)
  19. 19. 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
  20. 20. Conserved domains of Rpg1 and JAK
  21. 21. Protein degradation is not sufficient for disease resistance0 12 16 20 24 48 0 12 16 20 24 48G03-448F-3R Chevron0 12 16 20 24 48 0 12 16 20 24 48 Morex rpr10 12 16 20 24 48 0 12 16 20 24 48 Beacon OSU6
  22. 22. RPG1 protein elevated in Leaf Epidermis (LE) compared to rest of the tissue (LARE)7 RPG1 protein (micromoles of6 RPG1/g total protein)543210 LE LARE
  23. 23. RPG1 phosphorylation is race specific PS: Phosphoserine PT: Phosphothreonine Lane 1: Positive control Lane 2: uninoculated control Lane 3: 15 min. post inoculation

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