The objective of this study is to clone the chalcone synthase gene CHS1 in different resistant grape varieties, clarify the expression pattern under the infection of Botrytis cinerea and Plasmopara viticola, verify the subcellular localization and function of CHS1, and to provide a basis for exploring the broad-spectrum resistance mechanism of CHS1.

CHS1 genes were cloned from the leaf cDNAs of Vitis quinquangularis ‘Yeniang-2’, Vitis vinifera ‘Thompson Seedless’ and Vitis amurensis ‘Shuanghong’. The differences in the nucleic acid sequence and physicochemical properties of CHS1 protein were analyzed by bioinformatics. Expression patterns of CHS1 in different varieties during B. cinerea and P. viticola infection were also detected by qRT-PCR. The expression vector was constructed, and the transient expression technology mediated by Agrobacterium was used to verify the subcellular localization and disease resistance function in the leaves of tobacco and ‘Thompson Seedless’ tissue culture seedlings.

The CHS1 is highly conserved among different grape varieties. The full length of ORF is 1 182 bp, which encodes 393 amino acids. The predicted molecular weight of the encoded protein is 42.92 kD, which is a stable hydrophilic protein. After being infected by B. cinerea, the expression patterns of VqCHS1 and VvCHS1 were similar, but the expression level of VqCHS1 in the resistant variety ‘Yejiang-2’ was significantly higher. After being infected by P. viticola, VaCHS1 and VvCHS1 showed different expression pattern changes. The expression level of VaCHS1 in the resistant variety ‘Shuanghong’ continued to increase, while the expression level of VvCHS1 gradually decreased. The results of subcellular localization showed that CHS1 protein is distributed in the cytoplasm and nucleus, but it is mainly localized in the nucleus. Compared with the negative control, the ‘Thompson Seedless’ leaves overexpressing VaCHS1 showed stronger resistance to gray mold and downy mildew after artificial inoculation.

The expression level of CHS1 in resistant varieties is higher than that in susceptible varieties, and overexpression of CHS1 can increase the resistance of susceptible grape varieties to gray mold and downy mildew.

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.

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.

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.

During downy mildew infection, PvGHs secreted by P. viticola are involved in pathogenic process by inhibiting the host PTI response.