|SHERWOOD, ROBERT - Cornell University - New York|
Submitted to: Molecular Plant Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/10/2014
Publication Date: 4/15/2014
Publication URL: https://handle.nal.usda.gov/10113/59509
Citation: Lu, S., Faris, J.D., Sherwood, R., Friesen, T.L., Edwards, M.C. 2014. A dimeric PR-1-type pathogenesis-related protein interacts with ToxA and potentially mediates ToxA-induced necrosis in sensitive wheat. Molecular Plant Pathology. 15(7):650-663.
Interpretive Summary: Stagonospora nodorum blotch and tan spot are two major diseases of wheat. Isolates of the fungal pathogens causing these diseases often produce a small secreted protein (ToxA) "toxic" to wheat cultivars carrying a sensitivity gene which, ironically, is known to encode a disease-resistance protein not directly targeted by ToxA. In this study, we demonstrate that ToxA interacts with a wheat protein belonging to the type-1 pathogenesis-related (PR-1) group of proteins known to play roles in plant defense responses; this ToxA-interacting protein (PR-1-5) exists as a "dimer" (consisting of two identical polypeptides of the same protein), and binds to a unique site on the surface of the ToxA protein that is essential for the toxin to induce necrosis in sensitive wheat. We also show that the native PR-1-5 protein is induced by ToxA; treating the plants with recombinant PR-1-5 protein along with ToxA "promotes" cell death only in sensitive wheat. This work provides direct evidence that disease resistance gene-controlled defense pathways can be exploited by certain pathogens to induce diseases in the host plants that may be otherwise resistant to other pathogens. It also suggests that the protein dimerization may be an important mechanism in regulating biological activities of the PR-1 type defense proteins.
Technical 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.