Functional Characterization Of Cytochrome P450 Genes In Valsa Mali

Apple tree valsa canker is a serious disease in apple production of China which leads to decay of apple tree stems cortex caused by the fungus Valsa mali Mayabe et Yamada.It makes the apple trees weak,the yields and quality decreased which cause severe economic loss.Phytopathogen produces some micromolecular metabolite with pathogenic activity named fungaltoxin or phytotoxin in the process of infection and pathopoiesia.The research of comparative genomics indicates there are abundant genes with related toxin metabolism in the genome of necrotrophic fungus.The objective of the study is to reveal that cytochrome P450(CYP)plays a significant role in mytotoxin metabolism of necrotrophic fungus and may be related to pathogenicity.To analyse the function of CYP genes during infection process,we explore the functions of 28 CYP genes(VM1G＿00042、VM1G＿00043、VM1G＿02313、VM1G＿02575、VM1G＿03094、VM1G＿03153、VM1G＿03188、VM1G＿03596、VM1G＿04956、VM1G＿04960、VM1G＿04962、VM1G＿04973、VM1G＿04974、VM1G＿06772、VM1G＿06783、VM1G＿07082、VM1G＿07088、VM1G＿07096、VM1G＿07333、VM1G＿07348、VM1G＿08239、VM1G＿08245、VM1G＿08826、VM1G＿09622、VM1G＿09625、VM1G＿09631、VM1G＿09634、VM1G＿09643)in Valsa mali toxin gene cluster to reveal the effection of CYP genes to pathogenicity and lay the foundation for further pathogenesis of Cytochrome P450 gene families.The knockout cassette was constructed using Double-joint PCR and mutants were obtained and confirmed by PEG-mediated transformation of protoplasts,PCR and Southern Blotting analysis.The original strain 03-8 and mutants were performed experiements of phenotype and pathogenicity separately,and the gene complemented mutants were constructed by gap repair technology.Finally,salient differences and the expression levels detections of 6 genes(VM1G＿00287,VM1G＿00288,VM1G＿00289,VM1G＿00290,VM1G＿00291,VM1G＿00292)from melanin gene cluster were analysed by SPSS and q RT-PCR.The results are as follows:1.Knockout cassetes of 28 CYP genes were successfully constructed.30 positive transformations of 21 CYP genes were obtained by PEG-mediated transformation of Valsa mali protoplasts,and 18 single copy mutants of 15 CYP genes were got respectively after PCR and Southern Blotting.2.The study of mutants phenotype observation showed thatΔVM1G＿02575-59 grew slowly and was sensitive to Na~+and K~+stress.The amount ofΔVM1G＿04956-58 andΔVM1G＿04974-222 propagulum reduced significantly by 57.6%and 73.5%respectively.Besides,the colony color of deletion mutant changed from light yellow to white,and the amount of propagulum reduced by 51.3%significantly.And complementation mutant was almost back to the level of original strain 03-8 in colony color and propagulum.3.The study of mutants pathogenicity detection showed that the pathogenicity of deletion mutantΔVM1G＿03094-5 was decreased for 24.5%obviously compared with original strain03-8.And complementation mutant was almost back to the pathogenicity level of original strain 03-8.4.The q RT-PCR analysis showed that the relative expression levels of 6 genes fromΔVM1G＿03094-5 melanin gene cluster were down-regulated to 4.0,8.9,1.8,2.2,4.8 and 2.9folds respectively compared with original strain 03-8.In conclusion,the research demonstrated that VM1G＿03094 is related to melanin biosynthesis and propagulum formation,and participates in pathogen process of V.mali.VM1G＿02575,VM1G＿04956,VM1G＿04974 are related with pathogenic growth and development,and VM1G＿02575 participates in response to stress of V.mali.