Pathogenicity and Gene Expression Pattern of the Exocrine Protein LtGH61A of Grape Canker Fungus

【Objective】Grape canker disease, caused by Botryosphaeria genus fungi, occurs in a wide range of grape-producing areas in China and seriously threatens the yield and quality of grape. The objective of this study is to analyze the function of a hypothetical exocrine protein, LtGH61A, in grape canker fungus Lasiodiplodia theobromae, and to lay a foundation for in-depth analysis of the pathogenic mechanism and disease control of grape canker fungus.【Method】The signal peptide of LtGH61A protein was predicted by SignalP 4.0. The function of LtGH61A protein was predicted by the homologous comparison and functional annotation. The exocrine characteristic of LtGH61A protein was analyzed by yeast complementary experiment. The quantitative real-time PCR (qRT-PCR) was used to analyze the expression of LtGH61A in vegetative hyphae and different infection processes. The expression of LtGH61A was inhibited through RNA interference (RNAi). The effect of LtGH61A protein on the pathogenicity of L. theobromae was analyzed by in vitro inoculation test of grape shoots. The effect of LtGH61A protein on the hyphal growth rate of L. theobromae was analyzed by comparing the colony diameter.【Result】Amino acid sequence analysis predicts that the N-terminal of the LtGH61A protein contains a signal peptide with a length of 18 amino acids. The gene function annotation suggests that LtGH61A belongs to glycoside hydrolase family 61 (GH61) and can degrade cellulose as a substrate. Yeast complementary experiments showed that the signal peptide of LtGH61A protein could guide the secretion of invertase of yeast YTK12. Compared with the vegetative hyphae, the expression of LtGH61A was increased significantly at the infectious stages, and the mRNA accumulation of LtGH61A at 48 h post inoculation was 19 times of that in the vegetative hyphae. Moreover, RNAi lines were constructed for LtGH61A and two lines RNAi-LtGH61A1 and RNAi-LtGH61A2 were confirmed by qRT-PCR. The results of in vitro inoculation test of wild-type and RNAi transformants on wounded grape shoots showed that the lesion length caused by both RNAi-LtGH61A1 and RNAi-LtGH61A2 was significantly shorter than that of wild type (WT) CSS-01s, which was about 55% of WT, indicating that LtGH61A affected the pathogenicity of L. theobromae. The colony diameter comparison showed that compared with WT, the colony diameter of RNAi-LtGH61A1 and RNAi-LtGH61A2 transformants became smaller, about 85% of WT, indicating that LtGH61A affected the hyphal growth rate of L. theobromae.【Conclusion】LtGH61A affects the pathogenicity and hyphal growth of grape canker pathogen. LtGH61A protein can be secreted outside the cell. The expression level of LtGH61A during infectious stages is significantly increased, suggesting that LtGH61A can destroy the host plant tissue by exerting its own enzyme activity function, thus promoting pathogen infection.