关键词: 葡萄霜霉菌, 糖基水解酶, 生物信息学, 功能分析

Abstract:

【Objective】The objective of this study is to clone glycosyl hydrolase genes from Plasmopara viticola (PvGHs), analyze their characteristics and expression patterns in infected grape leaves, and the abilities to inhibit or promote programmed cell death (PCD) and affect Phytophthora nicotianae infection in tobacco leaves, so as to provide a theoretical basis for further study on mechanism of regulating host plant immunity.【Method】Eight PvGHs with full-length were amplified by RT-PCR from P. viticola Pv5-27 strain. The sequences of these eight PvGHs and encoded proteins were analyzed by bioinformatics. Yeast signal peptide trap system (SST) was used to verify the secretory activity of the PvGH proteins. The expression pattern of PvGHs during infection grape leaves was detected by qRT-PCR. At the same time, eight PvGH effector proteins were transiently expressed in Nicotiana benthamiana by Agrobacterium-mediated PVX virus expression system. Moreover, their inhibitory abilities to inhibit INF1- and BAX-triggered PCD and to promote P. nicotianae infection were also analyzed.【Result】The sequences of eight PvGHs were completely consistent with the prediction in genome sequence, with the length of 1 092 to 1 392 bp, encoding 364-464 amino acids, respectively. The similarity with homologous proteins from other oomycetes was as high as 60.62%-86.36%. None of them had transmembrane domains. Their secondary structures were quite different from each other, and their tertiary structures were less similar to those of other proteins, which showed a very unique tertiary structure. SingalP 5.0 software was used to predict signal peptide of these proteins. It was found that all the PvGH proteins contained signal peptide sequences of 20-26 aa in length. However, the SST verification results showed that PvG09279 and PvG13517 do not have secretion activity. The retention, deletion or replacement of signal peptide sequences of the eight PvGH proteins could inhibit the BAX-triggered PCD and all these PvGH proteins could promote P. nicotianae infection in tobacco leaves. It suggests that the potential virulence of eight PvGH effectors does not dependent on the signal peptide. The up-regulated expression of PvGHs in the early stage of infection of P. viticola further indicated that PvGHs play an important role in the interaction between pathogen and host.【Conclusion】During downy mildew infection, PvGHs secreted by P. viticola are involved in pathogenic process by inhibiting the host PTI response.

Key words: Plasmopara viticola, glycosyl hydrolase, bioinformatics, function analysis