title: A dimeric PR-1-type pathogenesis-related protein interacts with ToxA and potentially mediates ToxA-induced necrosis in sensitive 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, 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.
authors.affiliation: Cereal Crops Research Unit, USDA-ARS, Fargo, ND, 58102, USA.
authors.forename: Shunwen
authors.initials: S
authors.lastname: Lu
authors.forename: Justin D
authors.initials: JD
authors.lastname: Faris
authors.forename: Robert
authors.initials: R
authors.lastname: Sherwood
authors.forename: Timothy L
authors.initials: TL
authors.lastname: Friesen
authors.forename: Michael C
authors.initials: MC
authors.lastname: Edwards
mesh_terms: D001216:Asparagine / Q000378:metabolism; D018076:DNA, Complementary; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011485:Protein Binding; D014908:Triticum / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
chemical_list: D018076:DNA, Complementary; D010940:Plant Proteins; D001216:Asparagine
keywords: host-selective toxin; inducible defence-related protein; protein dimerization; protein-protein interaction