| Glycoside hydrolase 16 family（GH16）is a family of polysaccharide-hydrolzing enzymes,which are involved in the degradation or remodeling of cell wall polysaccharides,and are associated with control of fungal growth and development,cell wall stability,and pathogenicity.However,roles of the GH16 family glycoside hydrolases in development,differentiation and pathogenicity in animal pathogenic fungi remains unclear.Here,the GH16 family proteins were identified from the genome of an important insect fungal pathogen Beauveria bassiana.Analysis of the gene expression patterns was performed to predict their involved cellular processes.To reveal their roles in the fungal pathogen,gene disruption and overexpression strains of the representative proteins were characterized in fungal development,differentiation,and virulence.The main results are as follows:1.Analysis of the GH16 family protein features and their gene expression patternsTwenty-three GH16 family proteins were identified from B.bassiana via search the genome with the conserved domain of GH16 family proteins as probe.Bioinformatic analysis revealed that 14 members of those proteins contain typical N-terminal signal peptide sequence.The theoretical molecular weights of this family proteins are between 31.38-130.67 k Da,with isoelectric points between 4.44-8.49.The isoelectric points of 3 members of those proteins are higher than 7,suggesting basic proteins,the remaining other 20 members are acidic proteins.The grand average of hydropathy（GRAVY）values of all proteins are negative,ranging from-0.786 to-0.016,suggesting hydrophilic proteins.In order to predict the GH16 family proteins-involved cellular processes,the expression patterns of their genes were analysed by RT-q PCR.Those 23 genes showed different expression patterns in different morphological cells,that 14,7 and 2 genes were significantly upredulated in aerial hyphae,liquid hyphae and hyphal bodies,respectively.Different expression patterns were also examined during conidial germination,in which only 3 genes were significantly upregulated in the resting conidia（0 h）,and 17,15,14 and 16 genes were up-regulated at 4 h,8 h,12 h and 16 h during germination,respectively.Those genes also displayed diverse expression patterns in response to different cell wall（membrane）stressors.These data suggested that different GH16 family proteins might play distinct roles in maintainance of fungal morphology,development and cell wall integrity.2.GH16 family proteins BbGlcA and BbBglA participate in fungal development and differentiationTo further reveal roles of the GH16 family proteins in the fungal development,differentiation and virulence,two protein-coding genes,BBA＿00046 and BBA＿05825（named BbGlcA and BbBglA）,which displayed different expression patterns during germination but are all up-regulated in response to cell wall stressors,were characterized through invesitigation of the generated gene disruption and overexpression strains.Disruption of either BbGlcA or BbBglA resulted in a decreased ability to produce conidia,but no obvious effect on conidia yield was examined by overexpression of the two genes.The two proteins exerted distinct functions on spore morphology and germination.Disruption of BbGlcA did not affect the spore morphology,but reduced germination rate,whereas the overexpressing BbGlcA resulted in producing enlarged conidia and accelerating germination.However,BbBglA mutant produced a large number of enlarged oval conidia with rapid germination as compatred to the wild type,but the BbBglAOEstrain displayed the opposite phenotypes.Moreover,ΔBbGlcA hyphae developed slowly with sparse septa,while the BbGlcA overexpressing strain displayed accelerated hyphae with increased septum.However,disruption of BbBglA promoted hyphae development with increased septa,but the opposite phenotypes were examined in the BbBglA overexpression strain.These results demonstrated that BbGlcA and BbBglA played distinct roles in fungal development and differentiation in B.bassiana.3.BbGlcA and BbBglA display distinct functions in maintainance of cell wall stabilityTo investigate functions of BbGlcA and BbBglA in cell wall stabilty,sensitivieis of the genetically modificated fungal strains to different cell wall（membrance）stressors was examined.BbGlcA mutant did not affect sencitivities to different stressors,while disruption of BbBglA resulted in increased sentivitys to Congo red.Overexpression of both genes significantly increased tolerance to Congo red.Transmission electron microscopy revealed that disruption of BbGlcA did not affact cell wall thickness,but the electron density layer was significantly changed,while the cell wall was thickened in BbGlcA overexpressing strain.However,reduced electron density layer was seen in cell wall of the BbBglA mutant,but increased electron density layer（with increased cell wall thickness）present in cell wall of the BbBglA overexpressing strain.Assay for cell wall composition showed that the content of glucan was decreased in theΔBbGlcA strain,but increased in the BbGlcAOEstrain with increased chitin levels.However,both glucan and chitin levels were examined in cell wall of theΔBbBglA strain,but decreased in the BbBglAOEstrain.These results demonstrated that BbGlcA and BbBglA also have distinct functions in maintainance of of cell wall stability.4.BbGlcA and BbBglA play different roles in the pathogenesisTo understand the relationship between BbGlcA and BbBglA and fungal virulence,fungal conidia were inoculated in insect using two methods:normal cuticle infection（conidial spray onto cuticle）and intrahemocoel injection inoculation.The two assays indicated that BbGlcA mutation led to reduced virulence,while increased virulence was examined for the BbGlcA overexpression strain.In contrast,disruption of BbBglA increased fungal virulence,however the gene overexpression resulted in decreased virulence.Among them,the changes in virulence were correlated with fungal proliferation rate in the infected insects,ability to penetrate the cuticle and evade immune response.The results demonstrated that BbGlcA and BbBglA played distinct roles in fungal virulence.In summary,23 GH16 family proteins were identified from B.bassiana in this study.Our results suggested that different members of those GH16 family proteins play distinct roles in fungal development,differentiation,maintenance of cell wall stability and virulence,indicating that these proteins act synergistically to precisely control the development,differentiation and pathogenicity-related phenotypes of fungi. |
