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A dimeric PR-1-type pathogenesis-related protein interacts with ToxA and potentially mediates ToxA-induced necrosis in sensitive wheat

Lu, Shunwen, Faris, Justin D., Sherwood, Robert, Friesen, Timothy L., Edwards, Michael C.
ARS USDA Submissions 2014 v.15 no.7 pp. 650
Leptosphaeria nodorum, Pichia pastoris, Triticum aestivum, Western blotting, alanine, amino acid substitution, asparagine, cell membranes, genes, mutants, mycotoxins, necrosis, pathogenesis-related proteins, polyacrylamide gel electrophoresis, protein-protein interactions, site-directed mutagenesis, two hybrid system techniques, wheat
A dimeric PR-1-type pathogenesis-related protein (PR-1-5) recently identified in wheat was found to interact with Stagonospora nodorum ToxA in both yeast two-hybrid and co-immunoprecipitation assays. Site-specific mutational analyses revealed that the RGD motif of ToxA is not targeted by PR-1-5, while two surface-exposed asparagine residues are essential for the interaction: the N102 residue of the turning loop between ß2 and ß3 in ToxA and the N141 residue of the turning loop between ßC and ßD in PR-1-5. Recombinant PR-1-5 and ToxA mutant proteins carrying alanine substitutions at the interacting sites were expressed in Pichia pastoris along with the wild type proteins. Native PAGE analysis confirmed that the PR-1-5-N141A mutant retains the ability to form dimers. Plant assays indicated that the ToxA-N102A mutant fails to induce necrosis whereas the PR-1-5-N141A mutant is impaired in the "necrosis-promoting" activity shown by the wild type PR-1-5 when co-infiltrated with ToxA in sensitive wheat. Western blot analyses revealed that the native PR-1-5 protein accumulates in ToxA-treated sensitive wheat and is likely associated with membranes. These results suggest that the PR-1-5-ToxA interaction is potentially involved in ToxA internalization or activation of cell death pathway(s) governed by the cognate sensitivity gene in wheat.