Molecular Basis Of A Sterol Regulatory Element-Binding Protein,BbSre1,Regulating Oxidative Stress Response In The Insect Fungal Pathogen,Beauveria Bassiana

Pathogens would inevitably encounter stress of host immune response-generated excessive reactive oxygen species（ROS）during infection,which is harmful to either pathogens or host cells.Correspondingly,pathogens have evolved a series of strategies detoxify ROS,termed oxidative stress response,which are complex and diverse in organisms.Many molecules are involved in stress response,which include transcription factors that directly or indirectly control stressresponse-associated effector genes.A sterol regulatory element binding protein（SREBP）present in eukaryote,which has been demonstrated to be responsible for the dynamic balance of steroid levels,and other similar biological processes such as fatty acid and cholesterol synthesis.Besides the conserved roles,SREBP is involved in hypoxia adaptation of some fungal species.Our previous study showed that SREBP protein BbSre1,not only control hypoxia adaption,but was also associated with oxidative stress response in an insect fungal pathogen,Beauveria bassiana.Here,role of BbSre1 in oxidative stress response and pathogenicity was verification by generation of gene knockout,recovery complementation and overexpression strains.Furthermore,chromatin immunoprecipitation and RNA-seq methods were used to reveal the molecular basis of BbSre1 in regulating of oxidative stress response.The main results are as followings:1.Oxidative stress promotes accumulation of BbSre1 in the nucleusBbSre1 encodes 989 amino acids,in which a highly conserved alkaline helix loop helix（bHLH）leucine zinc finger DNA binding domain present in the N-terminal contains a tyrosine residue that can bind to the steroid regulatory element.Observation of subcellular localization by fusion of GFP tag revealed that the GFP signal distributed in the cytoplasm and nucleus.Whereas,GFP signal was increasly accumulated in nucleus when fungal cells were stressed by oxidative stress.These results suggested that BbSre1 might be involved in the transcriptional regulation of oxidative stress-associated genes.2.BbSre1 participates in fungal growth and conidia germinationIn order to investigate role of BbSre1 in fungal growth and development,the gene complementary strain（Comp）,and the overexpression strain(BbSre1^OE)were constructed based on the knockout strain（ΔBbSre1）generated previously.The fungal growth,sporulation and germination were examined on the basic medium（CZA）and the nutrient-rich medium（1/4 SDY and PDA）.The results showed that the disruption of the gene resulted in reduced growth and lowered conidial germination,but no effect on conidiation.Whereas,no obvious difference was examied in growth,conidiation and conidial germination between the overexpression strain and control strains(BbSre1^OEand WT).These data suggested that BbSre1 was involved in control of the growth and germination.3.BbSre1 mediates oxidative stress responseIn order to detect function of BbSre1 in oxidative stress response,the sensitivities of WT,ΔBbSre1,BbSre1^OEand Comp strains to oxidative stress were determined.Disruption of the gene led to a significant increased in sensitivity to oxidants H₂O₂and Menadione.However,no obvious change in oxidative stress tolerance was examied between the overespression and wild type strains.To understand the physiological basis of increased oxidative sensitivity inΔBbSre1,some antioxidant enzyme activities,as well as their gene transcription patterns,were examined in theΔBbSre1 cells.The antioxidant enzyme activities inΔBbSre1 cells were significantly lower than those of WT and Comp,accompanying down regulated some antioxidant enzyme genes.The results suggested that BbSre1 mediated antioxidant-related gene and antioxidant enzyme activities.4.The molecular basis of BbSre1 regulating the oxidative stress responseTo reveal the molecular basis of BbSre1 regulating the oxidative stress response,target genes of BbSre1 were identified by adoption of chromatin immunoprecipitation and comparative transcriptomics.There were 108 target genes of BbSre1 under normal conditions,including 60 up-regulated genes and 48 down-regulated genes.Eighteen target genes of BbSre1 were identified under H₂O₂stress,including 11 up-regulated genes and 7down-regulated genes respectively.Under MND stress,305 target genes of BbSre1 were identified,which included 137 up-regulated genes and 168 down-regulated genes.GO enrichment analysis indicated that the target genes regulated by BbSre1 under normal conditions were mainly enriched in alcohol dehydrogenase activity,oxidoreductase activity and phosphotransferase activity.Under oxidative stress,the target genes were mainly enriched in oxidoreductase activity,intrinsic component of membrane and cinnamate reductase activity.These data suggested that BbSre1 regulates the oxidative stress response by direct control of transcription of fungal development and differentiation,membrane components,redox enzymes and detoxification-involved genes.5.BbSre1 is involved in fungal virulenceSince BbSre1 mediated the growth,germination and oxidative stress response,effect of gene disruption and overexpression on fungal virulence to insect was detected with bioassay by inoculation of conidia suspension either cuticle infection or injection bypass the cuticle.The results indicated that the deletion of BbSre1 led to a decrease in virulence and a significant decrease in mortality of the insect.Moreover,ΔBbSre1 cells were easy to be encapsulated by hematocytes in the insect hemocoel with serious melanization rection.The proliferation of the mutant was significantly lower than that of WT cells,while the number of blood cells were higher than that of WT treated insect.There was no significant difference in virulence between the overexprssion and WT strains.These results suggested that BbSre1mediated virulence and evasion of immune response.Overall,our results demonstrated that BbSre1 mediated oxidative stress response,fungal virulence and evasion of host immunue response via regulation of redox process,membrane components,and detoxification-involved genes in B.bassiana.