Abstract: 【Objective】Transcriptome analysis showed that Vmpg7 and Vmpg8 are two significantly up-regulated polygalacturonase genes of Valsa mali during the pathogen infection progress. The objective of the study is to explore the role of these two genes in pathogenicity and lay the foundation for further pathogenesis of V. mali by analyzing the vegetative growth, pathogenicity, pectinase activity, the utilization of pectin, and the effect of gene knock-out of Vmpg7 and Vmpg8 to the expression levels of the other PG genes in PG family.【Method】qRT-PCR was used to detected the expression levels of Vmpg7 and Vmpg8 during the pathogen infection progress and the expression levels of other PG genes in PG family when Vmpg7 and Vmpg8 were deleted. The single and double deletion mutants were constructed and confirmed using double-joint PCR, PEG-mediated protoplast transformation technique, PCR with four pairs of primers and Southern blot, and the gene complemented mutants were constructed by gap repair technology. The PDA routine culture was selected to analyze the vegetative growth of mutants. In vitro inoculation to apple leaves and twigs was used to detect the pathogenicity. The 3,5-dinitrosalicylic acid (DNS) colorimetric method was used to detect the activity of extracellular pectinase, and the Czapek culture medium was selected to observe the utilization of pectin.【Result】The expression levels of Vmpg7 and Vmpg8 were assayed by qRT-PCR, and results showed that the two genes were up-regulated 27.73 and 8.19 folds at 3 days after infection, respectively. With the help of the gene knockout technique, three deletion mutants of Vmpg7, one deletion mutant of Vmpg8, three deletion mutants of Vmpg7/Vmpg8, together with the complemented mutants of Vmpg7 and Vmpg8 were constructed, respectively. When the mutants were cultured on PDA medium, the colony morphology and growth rate showed no significant change. When they were inoculated to apple leaves and twigs, the pathogenicity of the Vmpg7 deletion mutant and the Vmpg7/Vmpg8 double deletion mutant showed significant reduction, and the pathogenicity of the Vmpg8 deletion mutant was only reduced on apple leaves. Further analysis of pectinase activity and the utilization of pectin showed that the pectinase activities of the Vmpg8 deletion mutant and the Vmpg7/Vmpg8 double deletion mutant were significantly reduced, and all the mutants had a slow growth rate on pectin medium. Interestingly, the pathogenicity, pectinase activity, and the growth rate on pectin medium were back to the level of wild-type 03-8 when the genes were complemented back to the distinct mutants, respectively. More importantly, the expression levels of the other PG genes in PG family were obviously affected when Vmpg7and Vmpg8 were knockout, especially three genes that were significantly up-regulated.【Conclusion】Vmpg7 and Vmpg8 participate in pathogenic process of V. mali by adjusting the pectinase activity, which may synergized with the other PG genes in the same family.

Key words: Valsa mali, polygalacturonase, pectinase, gene knockout, pathogenicity