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

Author:
Lu, Shunwen, Faris, Justin D., Sherwood, Robert, Friesen, Timothy L., Edwards, Michael C.
Source:
ARS USDA Submissions 2014 v.15 no.7 pp. 650
ISSN:
1464-6722
Subject:
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
Abstract:
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.
Agid:
59509
Handle:
10113/59509