Fig. 2 Active kinase His-Rpg1 can not transphosphorylate kinase deficient GST-Rpg1. 1, inactive GST-Rpg1 ( kDa); 2, active His- Rpg1 (92. 2 kDa) and the inactive GST-Rpg1( kDa) and 3, active His-Rpg1 (92. 2 kDa). Proteins were analysed by SDS-PAGE and autoradiography. The autoradiograph (bottom panel) shows the protein species phosphorylated while the Coomassie-stained gel (top panel) shows the protein species present in each reaction. Robert Brueggeman*, Jayaveeramuthu Nirmala*, Christina Maeir*, Christine Clay †, Nils Rostoks ‡, Gamini Kannangara*, Diter von Wettstein* †, Brian Steffenson § and Andris Kleinhofs* † * Department of Crop and Soil Sciences and †School of Molecular Biosciences, Washington State University, Pullman, WA, 99164; ‡Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5AD; and §Department of Plant Pathology, university of plant Pathology, university of Minnesota, St. Paul, MN IN VIVO AND IN VITRO CHARACTERIZATION OF THE BARLEY STEM RUST RESISTANCE GENE RPG1 Fig. 1. A, The K152N and K153Q mutations of the pK1 domain in pJN01K1(B. lane 2) or the K462Q mutation of the pK2 domain in pJN01K5 (C. lane 2) did not affect in vitro autophosphorylation. The K461N and K462Q mutations of the pK2 domain in pJN01K2 (B. lane 3) or the K461Q mutation of the pK2 domain in pJN01K4 (C. lane1) resulted in catalytically inactive enzyme. The double mutant was also inactive (B. lane 4). K:lysine, N: asparagine and Q: gluatamine. Top panel, Coomassie blue stained SDS-PAGE and bottom panel, autoradiograph. C, Lysine 461 is required for autophosphorylation while lysine 462 is not. 1 – pJN01K4 and 2 – pJN01K5. Top panel, Coomassie blue stained gel and bottom panel, autoradiograph. Fig. 4. Disease reaction of Rpg1 kinase mutants to the pathogen, P. graminis f.sp tritici pv. MCC. The pK1 and pK2 mutants were all susceptible with a IT=3, similar to the susceptible cv. Golden Promise (GP). The substitution of the critical lysine in the ATP anchor of either of the domains abolishes resistance to the stem rust fungus. The barley stem rust resistance gene Rpg1 conferring resistance to numerous strains of the rust fungus Puccinia graminis f. sp.tritici was recently cloned by a map based approach (Brueggeman et al, 2002). The predicted Rpg1 protein is a novel receptor-like non-RD kinase characterized by the presence of dual kinase domains, not found to date in any other described plant disease resistance genes. Though the pK1 and pK2 catalytic domains share 58.4% amino acid sequence homology, the pK2 domain has all the functional features of a typical S/T kinase while pK1 lacks several critical residues in the glycine motif, S/T kinase active signature motif and the ATP phosphotransfer domain. The nearly invariant aspartic acid involved in the phosphotransfer in the pK2 domain is substituted by glutamic acid in the pK1 domain. Here we present evidence that only the pK2 domain is essential for in vitro autophosphorylation, but both kinase domains are essential for Rpg1 mediated disease resistance. Fig. 3 A. Southern blot; B, Western blot and C, Autophosphorylation of the pK1 and pK2 mutant transformants. Rpg1 is transcribed (data not shown) and translated in all the pK1 and pK2 mutant transformants. The Rpg1 protein from the pK1 mutants (lanes 2-12) autophosphorylate while the pK2 mutants (lanes 13, 14) do not. CONCLUSIONS K461 of the Rpg1 pK2 domain is the catalytically active lysine and is essential for in vitro autophosphorylation and disease resistance demonstrating that phosphorylation and hence signal transduction play an important role in Rpg1 mediated resistance to stem rust. The K152 and K153 in the Rpg1 pK1 domain do not affect autophosphorylation in vitro, but are essential for Rpg1 mediated disease resistance. What is their function in disease resistance remains to be discovered. MATERIALS AND METHODS Rpg1 WT and mutant cDNA were expressed and purified in Pichia pastoris system as a His- tag protein. GST-Rpg1 was expressed in E.coli BL21 strain. The wild type and mutant forms were assayed using γ- 32 P ATP. The susceptible cultivar, Golden promise was transformed with in vitro generated mutant Rpg1 genes. Stable transformants of the mutated Rpg1 pK1 and pK2 mutants were tested for resistance /susceptibility to the stem rust fungus, Puccinia graminis f. sp.tritici pv. MCC and characterized for their ability to transcribe, translate and autophosphorylate. ACKNOWLEDGEMENTS The study was supported by the USDA-NRI grant # to A.K. and B.S. REFERENCES 1. Brueggeman, R., et al., The barley stem rust resistance gene Rpg1 is a novel disease resistance gene with homology to receptor kinases. Proc. Natl. Acad. Sci. USA 99: RESULTS 1. Rpg1 pK2 domain is essential for in vitro autophosphorylation, while the pK1 domain is not (Fig. 1). 2. Rpg1 autophosphorylates by an intramolecular mechanism and requires Mg ++ or Mn ++ ions, but not Ca ++ (data not shown). 3. Rpg1 can not transphosphorylate itself (Fig. 2). 4. Transgenic plants with mutant pK1 or pK2 domains express the Rpg1 gene at the mRNA and protein level. The mutant pK1 domain Rpg1 can autophosphorylate while the mutant pK2 domain Rpg1 can not autophosphorylate (Fig. 3). 5. Transgenic plants with mutant pK1 and pK2 domain are susuceptible to stem rust (Fig. 4).