Cloning And Functional Analysis Of Phytophthora Boehmeriae Gene Coding Elicitor That Induces Hypersensitive Response In Nonhost Plant

We have devised a novel, high-throughput functional cloning method to isolate cDNAs from Phytophthora boehmeriae of which the productions elicit a hypersensitive response (HR) in tobacco.The cDNAs were cloned into a binary potato virus X(PVX)-based expression vector and transfermed to Agrobacterium tumefeciens(Mog101). 4100 colonies were individually toothpick-inoculated onto leaflets of Nicotiana benthamiana. 12 cDNAs were identified whose expression induced formation of a necrotic lesion around the inoculation site. 7 of these clones have different sequence. one of these clones PBC43 encodes specific elicitin. Clone PBC163 encodes a protein highly homologous to Rab; PBC241 encodes a prohibitin protein; PBN62 encodes a heat shock protein 60(HSP60). The other five cDNAs reveals no homology to known genes and are that considered novel. These observations confirm that this functional screening method is a versatile strategy to identify cDNAs of pathogens that encode elicitor and other HR-inducing protein.Functioning as an elicitin inducing hypersensitive cell death in tobacco, named Pbelicitin(Phytophthora boehmeriae elicitin) was obtained from the library of Phytophthora boehmeriae. Southern blot analysis indicated that Pbelicitin genes occur at least five copies in P. boehmeriae. Bacterial expression of the cloned elicitin gene as translational funsion protein yielded a biologically active protein capable of inducing a hypersensitive response in wile-type tobacco, suggesting that Phytophthora -specific posttranslational modifications of elicitin are not required for its activity. The elicitor induced HR of a consistent shape and size on tobacco plants expressing the bacterial gene nahG. Salicylate hydroxylase is encodec by nahG and inactivates SA by converting it into catechol. The mutant NahG does not accumulate SA. HR appears to be mediated by a SA-independent signaling pathway. And elicitin treatment resulted in enhanced resistance of wild-type and Tetre tobacco plants to infection by black shank fungus, P. nicotianae, and TMV, suggest that ethylene do not mediate HR and SAR in tobacco treated with Pbelicitin. The Pbelicitin could induce expression of an SAR marker gene encoding PR-1a was suppressed in NahG plants. These results indicate that SA mediates SAR but not HR in tobacco treated with Pbelicitin. And...