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

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. Keyword: P. lilacinum, Subtilisin like serine proteases, Phylogenetic analysis, gene tree, species tree, conserved motif analysis, natural selection, type I functional divergence, type II functional divergence, protein structure modeling.真菌中的类枯草杆菌丝氨酸蛋白酶（Subtilisin-like serine proteases，Subtilases）对于宿主侵染与定殖至关重要。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若干特性。然而，其在昆虫致病过程中承担特定功能的现象，或许由进化历程中获得的独特生化与结构特征所调控。受此类功能转变的影响，肉座菌目不同科属类群编码的枯草杆菌蛋白酶类，会根据各自的宿主与生活方式呈现出差异化特征。 本研究开展了系统发育与DIVERGE分析，鉴定出可赋予肉座菌目真菌各分类群枯草杆菌蛋白酶类功能特异性的关键蛋白残基。 结果：本研究共纳入肉座菌目5个科、10个物种编码的161个枯草杆菌蛋白酶类序列。基于保守结构域的存在情况，这类基因被划分为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）。我们针对上述亚家族开展了系统发育关联分析、正选择作用下的蛋白残基分析以及旁系同源进化枝间的功能分化分析，并将观测结果与对应真菌类群/物种的生活方式进行关联分析。 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与功能分化分析显示，旁系同源进化枝清晰反映了蛋白序列的科系起源。本研究观测到植物病原真菌丛赤壳科（Nectriaceae）、昆虫病原真菌虫草科（Cordycipitaceae）/麦角菌科（Clavicipitaceae）与食线虫真菌线虫草科（Ophiocordycipitaceae）的旁系同源进化枝间存在显著分化。此外，寄生线虫的真菌淡紫紫孢菌（Purpureocillium lilacinum）的枯草杆菌蛋白酶类基因形成了独特的进化簇，这暗示该真菌可能演化出了专属的线虫致病机制。 本研究的进化遗传学分析发现，肉座菌目虫草科、麦角菌科与线虫草科的昆虫病原及食线虫物种的枯草杆菌蛋白酶（S08.005）与蛋白酶K（S08.054）序列存在正选择作用的证据。 结论：本研究为肉座菌目（一类主要由生物防治物种组成的真菌类群）中类枯草杆菌丝氨酸蛋白酶的演化提供了全新视角。枯草杆菌蛋白酶（S08.005）与蛋白酶K（S08.054）蛋白似乎在肉座菌目不同类群与物种的生活方式转变过程中发挥了关键作用。本研究中经功能分化分析鉴定出的显著蛋白残基，可为未来的蛋白质工程研究提供重要支撑。 关键词：淡紫紫孢菌（P. lilacinum）、类枯草杆菌丝氨酸蛋白酶、系统发育分析、基因树、物种树、保守基序分析、自然选择、I型功能分化、II型功能分化、蛋白质结构建模。