Pathogenic Function Of Three Pectinase Genes In Valsa Mali

Apple valsa canker, caused by the Ascomycete Valsa mali, is a severe branches canker disease. It occurs in China commonly, impacting the yield and quality of apple seriously. The pathogenesis of V. mali is not clear up to now, bringing some difficulty in prevention and treatment of apple valsa canker. To explore the pathogenesis of V. mali can provide theoretical basis for disease prevention and treatment. Transcriptome analysis of V. mali showed that there were three highly up-regulated pectinase genes during the pathogen infection progress, two polygalacturonase(PG) genes Vmpg7 and Vmpg8, and one pectate lyase(PL) gene Vmpl4. To clear the role of the three pectinase genes in pathogenesis and growth of V. mali can further explore the pathogenesis of apple valsa canker.qRT-PCR was used to detected the expression levels of the three pectinase genes during the pathogen infection progress, the expression levels of other genes in PG family when Vmpg7/Vmpg8 were deleted and the expression levels of other genes in PL family when Vmpl4 was deleted. Double-joint PCR, gap-repair technique and PEG-mediated protoplast transformation technique were used to construct the deletion and complement cassettes. The mutants were verified by PCR and Southern blot detection, and then, inoculated on PDA medium to observe the vegetable growth. The activities of extracellular pectinase of the mutants were detected through 3, 5-dinitrosalicylic acid(DNS) colorimetric method. In vitro inoculation to apple leaves and twigs of Malus domestica borkh. cv. ‘Fuji’ was used to detect the pathogenicity of the mutants. The utilization of the pectin was observed by inoculation the mutants on pectin medium. The research results are as follows:1. Compared to mycelium of wild-type strain 03-8, the PG genes Vmpg7, Vmpg8 and the PL gene Vmpl4 were up-regulated to 27.73, 8.19 and 10.20 folds in pathogen infection process,respectively.2. Three deletion mutants of Vmpg7, one deletion mutant of Vmpg8, one deletion mutant of Vmpl4 and one double deletion mutant of Vmpg7/Vmpg8, together with complemented mutants of Vmpg7 and Vmpg8 were obtained through protoplast transformation technique and detection, respectively.3. The colony morphologies and sizes of the mutants on PDA medium suggested that Vmpg7, Vmpg8 and Vmpl4 did not affect the vegetable growth of the pathogen. Compared to 03-8, the activities of extracellular pectinase of Vmpg8 deletion mutant and Vmpg7/Vmpg8 double deletion mutant were reduced 44.40% and 33.55%, respectively. But the activities of extracellular pectinase of Vmpg7 deletion mutant and Vmpl4 deletion mutant have no significantly changes. Pathogenicity analysis showed that, the pathogenicities of Vmpg7 deletion mutant, Vmpl4 deletion mutant and Vmpg7/Vmpg8 double deletion mutant on leaves and twigs were significantly reduced compared to 03-8. The pathogenicities of Vmpg7 mutant on leaves and twigs were reduced 16.97% and 8.70%, respectively. The pathogenicities of Vmpl4 mutant on leaves and twigs were reduced 16.82% and 18.59%, respectively. The pathogenicities of Vmpg7/Vmpg8 mutant on leaves and twigs were reduced 13.12% and 14.27%, respectively. The pathogenicity of Vmpg8 mutant on leaves was reduced 9.26%, but had no change on twigs. The colony sizes of Vmpg7, Vmpg8, Vmpl4 and Vmpg7/Vmpg8 deletion mutants on pectin medium for 3 days were reduced 9.58%, 14.01%, 8.79% and 10.42%, respectively. The activities of extracellular pectinase, pathogenicities and the colony sizes on pectin medium of the complemented mutants of Vmpg7 and Vmpg8 were all back to the levels of wild-type 03-8.4. Three genes in PG family were significantly up-regulated when Vmpg7 and Vmpg8 both were deleted, and four genes in PL family were significantly up-regulated when Vmpl4 was deleted.In conclusion, polygalacturonase genes Vmpg7、Vmpg8 and pectate lyase gene Vmpl4 participant in the pathogenic process of V. mali by degradation of pectin. The knockout of the target genes caused some changes in expression levels of other genes in the same family, which may synergized with the target genes during the pathogen infection process.