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Mapping the Elicitor and Necrotic Sites of Phytophthora Elicitins with Synthetic Peptides and Reporter Genes Controlled by Tobacco Defense Gene Promoters

Elicitins are 10-kDa proteins secreted by Phytophthora and Pythium fungi that elicit a hypersensitive-like necrotic reaction, leading to resistance against fungal and bacterial plant pathogens. Induction of necrosis and resistance were previously shown to be borne by different sites of the molecule. Furthermore, sequence comparison indicated several potential residues necessary for necrosis. The role of one of these residues was previously evidenced with site-directed mutagenesis. In order to locate other necrosis-determining sites and reveal the defense-eliciting sites, we synthesized a series of synthetic peptides. Tests were performed on two types of transgenic tobacco plants, both transformed with a construction containing the β-glucuronidase reporter gene, in one case controlled by the promoter of the multiple stimulus response gene str 246C and in the other by the promoter of the pathogenesis-related gene PR1a. We report that only certain peptides were found to be active. Whereas PR1a induction was consistently correlated with induction of necrosis, four peptides were observed to induce only str 246C expression without necrosis, which led to differentiate the defense-eliciting sites from the necrotic sites. From the structure-function relationships thus obtained, two different defense pathways were inferred to be independently induced by elic-itins.