Data from: Phylogenetic analyses reveal molecular signatures associated with functional divergence among Subtilisin like Serine Proteases are linked to lifestyle transitions in Hypocreales

Background: Subtilisin-like serine proteases or Subtilases in fungi are important for penetration and colonization of host. In Hypocreales, these proteins share several properties with other fungal, bacterial, plant and mammalian homologs. However, adoption of specific roles in entomopathogenesis may be governed by attainment of unique biochemical and structural features during the evolutionary course. Due to such functional shifts Subtilases coded by different family members of Hypocreales acquire distinct features according to respective hosts and lifestyle. We conducted phylogenetic and DIVERGE analyses and identified important protein residues that putatively assign functional specificity to Subtilases in fungal families/species under the order Hypocreales. Results: A total of 161 Subtilases coded by 10 species from five different families under the fungal order Hypocreales was included in the analysis. Based on the presence of conserved domains, the Subtilase genes were divided into three subfamilies, Subtilisin (S08.005), Proteinase K (S08.054) and Serine-carboxyl peptidases (S53.001). These subfamilies were investigated for phylogenetic associations, protein residues under positive selection and functional divergence among paralogous clades. The observations were co-related with the life-styles of the fungal families/species. Phylogenetic and Divergence analyses of Subtilisin (S08.005) and Proteinase K (S08.054) families of proteins revealed that the paralogous clades were clear-cut representation of familial origin of the protein sequences. We observed divergence between the paralogous clades of plant-pathogenic fungi (Nectriaceae), insect-pathogenic fungi (Cordycipitaceae/Clavicipitaceae) and nematophagous fungi (Ophiocordycipitaceae). In addition, Subtilase genes from the nematode-parasitic fungus Purpureocillium lilacinum made a unique cluster which putatively indicated that the fungus might have developed distinctive mechanisms for nematode-pathogenesis. Our evolutionary genetics analysis revealed evidence of positive selection on the Subtilisin (S08.005) and Proteinase K (S08.054) protein sequences of the entomopathogenic and nematophagous species belonging to Cordycipitaceae, Clavicipitaceae and Ophiocordycipitaceae families of Hypocreales. Conclusions: Our study provided new insights into the evolution of Subtilisin like serine proteases in Hypocreales, a fungal order largely consisting of biological control species. Subtilisin (S08.005) and Proteinase K (S08.054) proteins seemed to play important roles during life style modifications among different families and species of Hypocreales. Protein residues found significant in functional divergence analysis in the present study may provide support for protein engineering in future.# 背景 真菌类枯草杆菌蛋白酶样丝氨酸蛋白酶（Subtilisin-like serine proteases，又称Subtilases）在宿主侵染与定殖过程中发挥关键作用。在肉座菌目（Hypocreales）中，这类蛋白与其他真菌、细菌、植物及哺乳动物的同源蛋白共享多项共性特征。然而，其在昆虫致病过程中承担的特定功能，可能由进化历程中获得的独特生化与结构特征所决定。受此类功能转变的影响，肉座菌目不同科属成员所编码的Subtilases会根据各自宿主与生活方式呈现出差异化的分子特征。本研究开展了系统发育分析与DIVERGE功能趋异分析，成功鉴定出可赋予肉座菌目真菌科/物种Subtilases功能特异性的关键蛋白质残基。 # 结果 本分析共纳入来自肉座菌目5个不同科的10个物种所编码的161个Subtilases。依据保守结构域的存在情况，该类基因被划分为3个亚家族：枯草杆菌蛋白酶（Subtilisin，S08.005）、蛋白酶K（Proteinase K，S08.054）以及丝氨酸羧基肽酶（Serine-carboxyl peptidases，S53.001）。我们对这些亚家族的系统发育关联、受正向选择的蛋白质残基以及旁系同源进化枝间的功能趋异情况进行了系统研究，并将观测结果与对应真菌科/物种的生活方式进行了关联分析。针对枯草杆菌蛋白酶（S08.005）与蛋白酶K（S08.054）家族蛋白的系统发育及DIVERGE分析显示，旁系同源进化枝清晰对应了蛋白序列的科系起源。我们观测到植物病原真菌（赤霉科，Nectriaceae）、昆虫病原真菌（虫草科/麦角菌科，Cordycipitaceae/Clavicipitaceae）以及食线虫真菌（线虫草科，Ophiocordycipitaceae）的旁系同源进化枝间存在显著功能趋异现象。此外，来自线虫寄生真菌淡紫紫孢菌（Purpureocillium lilacinum）的Subtilase基因形成了独特的进化簇，这提示该真菌可能演化出了针对线虫致病过程的特有机制。本研究的进化遗传学分析结果显示，肉座菌目虫草科、麦角菌科及线虫草科的昆虫病原与食线虫物种的枯草杆菌蛋白酶（S08.005）与蛋白酶K（S08.054）蛋白序列存在正向选择信号。 # 结论 本研究为肉座菌目（该真菌目多数物种为生物防治剂）的枯草杆菌蛋白酶样丝氨酸蛋白酶的演化过程提供了全新的研究视角。在肉座菌目不同科与物种的生活方式转变过程中，枯草杆菌蛋白酶（S08.005）与蛋白酶K（S08.054）似乎发挥了重要作用。本研究中经功能趋异分析鉴定得到的关键蛋白质残基，可为未来的蛋白质工程研究提供理论支撑。