Characterization of cytochrome P450 gene Vmcyp5 in Valsa mali

Abstract:

[Objective] Cytochrome P450 (CYP) plays a significant role in mycotoxin metabolism of necrotrophic fungus and may be related to pathogenicity. The objective of the study was to reveal the function of CYP genes in infection process.[Methods] 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. 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 twigs was used to detect the pathogenicity. Salient differences were analyzed by SPSS and qRT-PCR was used to detect the expression of melanin gene cluster.[Results] Compared with the original strain 03-8, the colony color of deletion mutant changed from light yellow to white, and the amount of propagulum reduced by 51.3%. The relative expression level of Vmcyp5-knockout mutant was assayed by qRT-PCR, and mutant's melanin gene cluster was down-regulated. More importantly, the pathogenicity of deletion mutant was decreased by 24.5%. Complementation mutant was almost back to the level of the original strain 03-8 in colony color, propagulum and pathogenicity.[Conclusion] Vmcyp5 may be related to melanin biosynthesis and propagulum formation, and participates in pathogen process of Valsa mali.