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, whereas two surface-exposed asparagine residues are essential for the interaction: the N102 residue of the turning loop between β2 and β3 in ToxA and the N141 residue of the turning loop between βC and βD in PR-1-5. Recombinant PR-1-5 and ToxA mutant proteins carrying alanine substitutions at the interacting sites were expressed in Pichia pastoris, together with the wild-type proteins. Native polyacrylamide gel electrophoresis (PAGE) 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. Reverse transcriptase-polymerase chain reaction and Western blot analyses revealed that the native PR-1-5 protein is differentially expressed between ToxA-sensitive and ToxA-insensitive wheat lines in response to ToxA treatment. These results suggest that PR-1-5 is a potential target of ToxA and the site-specific interaction between PR-1-5 and ToxA may mediate ToxA-induced necrosis in sensitive wheat.