Subcellular localization and functions of the barley stem rust resistance receptor-like serine/threonine-specific protein kinase Rpg1

Abstract

The Rpg1 gene confers resistance to many pathotypes of the stem rust fungus Puccinia graminis f. sp. tritici and has protected barley from serious disease losses for over 60 years. Rpg1 encodes a constitutively expressed protein with two tandem kinase domains. Fractionation by differential centrifugation and aqueous twophase separation of the microsome proteins located Rpg1 mainly in the cytosol but also in the plasma membrane and intracellular membranes. Recombinant Rpg1 autophosphorylates in vitro intramolecularly only serine and threonine amino acids with a preference for Mn 2؉ cations and a Km of 0.15 and a Vmax of 0.47 nmol⅐min ؊1 ⅐mg ؊1 protein. The inability of wild-type Rpg1 to transphosphorylate a recombinant Rpg1 inactivated by sitedirected mutation confirmed that Rpg1 autophosphorylation proceeds exclusively via an intramolecular mechanism. Site-directed mutagenesis of the two adjacent lysine residues in the ATP anchor of the two-kinase domains established that the first of the two tandem kinase domains is nonfunctional and that lysine 461 of the second domain is the catalytically active residue. Transgenic barley, expressing Rpg1 mutated in either the kinase 1 or 2 domains, were fully susceptible to P. graminis f. sp. tritici revealing requirement of both kinase domains for resistance. In planta-expressed Rpg1 mutant protein confirmed that mutation in domain 2, but not 1, rendered the protein incapable of autophosphorylation.cultivar ͉ protein kinase 1 domain ͉ protein kinase 2 domain

Plant Innate Immunity: Perception of Conserved Microbial Signatures

“…The R gene corresponding to RPG1 was identified by map-based cloning and found to encode a dual protein kinase (22,92). Both kinases of RPG1 carry the non-RD motif and are required for disease resistance, yet only protein kinase 2 has catalytic activity in vitro (152). RPG1 localizes to the plasma membrane and cytosol, where it is phosphorylated within minutes after urediniospore attachment to the leaf surface (154).…”

Proteolysis of the barley receptor-like protein kinase RPG1 by a proteasome pathway is correlated with Rpg1 -mediated stem rust resistance

“…The K2 mutant has lost RPG1 kinase catalytic activity, whereas the K1 mutant retains autophosporylation activity (24). Both mutants were susceptible to infection by pathotype MCC as demonstrated (24), and the RPG1 protein was not degraded (Fig.…”

“…This difference in the timing of the RPG1 protein disappearance may simply be a reflection of the quantity of protein present in these lines compared with the somewhat less resistant lines. The RPG1 protein in a susceptible accession containing the suppressor mutation rpr1 also disappeared [20][21][22][23][24] hai. An experiment designed to determine more precisely the time of RPG1 protein disappearance in cv.…”

“…Polyclonal antibody was raised in rabbits by Alpha Diagnostics (Austin, TX) against an RPG1 synthetic peptide NKLTATPLEEKSRSC, representing residues 834-848 (24). Antibody against the full-length recombinant RPG1 with a C-terminal His-tag (24) was raised at Washington State University in New Zealand White rabbits with RIBI Adjuvant (Corixa, Hamilton, MT).…”

“…Whether Rpg1 fits into the receptor-ligand model or the guard hypothesis is not known, because the AvrRpg1 gene has not been identified. However, it is evident that the kinase activity of RPG1 is required, but not sufficient, for resistance (24). So could RPG1 phosphorylation and/or proteolysis act as an initial signal in recognizing the avirulent fungus?…”