Table_4_Genome-Wide Identification and Analysis of Chitinase GH18 Gene Family in Mycogone perniciosa.docx

Mycogone perniciosa causes wet bubble disease in Agaricus bisporus and various Agaricomycetes species. In a previous work, we identified 41 GH18 chitinase genes and other pathogenicity-related genes in the genome of M. perniciosa Hp10. Chitinases are enzymes that degrade chitin, and they have diverse functions in nutrition, morphogenesis, and pathogenesis. However, these important genes in M. perniciosa have not been fully characterized, and their functions remain unclear. Here, we performed a genome-wide analysis of M. perniciosa GH18 genes and analyzed the transcriptome profiles and GH18 expression patterns in M. perniciosa during the time course of infection in A. bisporus. Phylogenetic analysis of the 41 GH18 genes with those of 15 other species showed that the genes were clustered into three groups and eight subgroups based on their conserved domains. The GH18 genes clustered in the same group shared different gene structures but had the same protein motifs. All GH18 genes were localized in different organelles, were unevenly distributed on 11 contigs, and had orthologs in the other 13 species. Twelve duplication events were identified, and these had undergone both positive and purifying selection. The transcriptome analyses revealed that numerous genes, including transporters, cell wall degrading enzymes (CWDEs), cytochrome P450, pathogenicity-related genes, secondary metabolites, and transcription factors, were significantly upregulated at different stages of M. perniciosa Hp10 infection of A. bisporus. Twenty-three out of the 41 GH18 genes were differentially expressed. The expression patterns of the 23 GH18 genes were different and were significantly expressed from 3 days post-inoculation of M. perniciosa Hp10 in A. bisporus. Five differentially expressed GH18 genes were selected for RT-PCR and gene cloning to verify RNA-seq data accuracy. The results showed that those genes were successively expressed in different infection stages, consistent with the previous sequencing results. Our study provides a comprehensive analysis of pathogenicity-related and GH18 chitinase genes’ influence on M. perniciosa mycoparasitism of A. bisporus. Our findings may serve as a basis for further studies of M. perniciosa mycoparasitism, and the results have potential value for improving resistance in A. bisporus and developing efficient disease-management strategies to mitigate wet bubble disease.

有害疣孢霉（Mycogone perniciosa）可引发双孢蘑菇（Agaricus bisporus）及多种伞菌纲（Agaricomycetes）物种感染湿泡病。在前期研究中，我们已在有害疣孢霉Hp10的基因组中鉴定出41个糖苷水解酶家族18（GH18）几丁质酶基因及其他致病相关基因。几丁质酶是一类可降解几丁质的酶类，在营养获取、形态发生及致病过程中具备多样功能。然而，有害疣孢霉中的这类重要基因尚未得到充分表征，其具体功能仍未明确。本研究针对有害疣孢霉的GH18家族基因开展全基因组分析，并解析了其侵染双孢蘑菇过程中不同时间节点的转录组图谱与GH18基因的表达模式。将41个GH18基因与另外15个物种的同源基因进行系统发育分析后发现，依据保守结构域，这些基因可划分为3个大类群与8个亚类群。同一大类群的GH18基因虽基因结构存在差异，但具有相同的蛋白质基序。所有GH18基因均定位于不同细胞器，在11条重叠群上呈不均匀分布，且在另外13个物种中存在直系同源基因。本研究共鉴定出12次基因复制事件，这些复制事件同时经历了正向选择与纯化选择。转录组分析结果显示，在有害疣孢霉Hp10侵染双孢蘑菇的不同阶段，包括转运蛋白、细胞壁降解酶（CWDEs）、细胞色素P450、致病相关基因、次生代谢产物及转录因子在内的大量基因均出现显著上调表达。41个GH18基因中有23个呈现差异表达，这些基因的表达模式各不相同，且在有害疣孢霉Hp10接种双孢蘑菇后的第3天开始显著表达。研究选取5个差异表达的GH18基因进行逆转录聚合酶链式反应（RT-PCR）与基因克隆实验，以验证RNA测序（RNA-seq）数据的准确性。结果表明，这些基因在不同侵染阶段依次表达，与此前的测序结果一致。本研究全面解析了致病相关基因与GH18家族几丁质酶基因对有害疣孢霉重寄生双孢蘑菇过程的影响。本研究成果可为后续有害疣孢霉重寄生机制的研究提供理论基础，同时为提升双孢蘑菇抗病性、开发高效病害管理策略以防控湿泡病提供潜在应用价值。