To identify chitinase genes from the genome of the mycoparasitic Cladosporium sp. strain SYC23, bioinformatical analyses and real-time quantitative PCR (RT-qPCR) were employed to screen mycoparasitism-associated genes at 12, 24, 48, and 72 h post-induction with Aecidium pourthiaea rust spores. A total of eight chitinase genes were identified from SYC23 via bioinformatics analysis and designated CaChi34, CaChi40, CaChi45, CaChi67, CaChi82, CaChi92, CaChi93, and CaChi286 based on sequence and phylogenetic analyses. Analysis of the chitinase protein sequence characteristics revealed molecular weights ranging from 33.86 to 286.03 kDa and theoretical isoelectric points from 4.48 to 7.7. All CaChi genes contained the conserved GH18 domain, and promoter analysis showed that each harbored MYB-binding sites and pathogen-responsive elements. Mycoparasitism-related sequence clustering analysis indicated that the chitinase sequences of SYC23 shared the closest phylogenetic relationship with those from Trichoderma sp. RT-qPCR results following rust spore induction showed that five CaChi genes reached their highest expression levels at 24 h post-induction, CaChi45 was most highly expressed at 72 h post-induction, CaChi93 was continuously upregulated, and CaChi82 was continuously downregulated throughout the induction period. His-tagged recombinant CaChi93 protein was purified from E. coli and characterized. The results demonstrate that the enzymatic activity of CaChi93 was 0.929 U/mg, with optimal reaction conditions at 65 °C and pH 7. Treatment of A. pourthiaea rust spores with the recombinant CaChi93 chitinase confirmed that CaChi93 could effectively dissolve rust spore walls. In conclusion, this study confirms that the mycoparasitic Cladosporium sp. strain SYC23 can secrete chitinase to degrade the rust spore wall and induce spore death, thereby providing novel gene resources and a theoretical basis for the biological control of A. pourthiaea.
Chen et al. (Thu,) studied this question.