Molecular Basis Of Bbmpk1 Negatively Mediating Oxidative And Osmotic Stress Response Of Beauveria Bassiana

Entomopathogenic fungi are a class of pathogenic microorganisms,which infect insects by direct penetration of host cuticle.And thus,penetration of cuticle is crucial for fungal pathogens to infect the target insects.Our previous study demonstrated that Fus3/Kss1 MAPK（Bbmpk1）and its potential downstream transcription factor,Bbste12,play crucial roles in regulating penetration of insect cuticle in the insect fungal pathogen,Beauveria bassiana.Either disruption Bbmpk1 or Bbste12 led to loss of ability to penetrate into insect cuticle.Comparative study of Bbmpk1 and Bbste12-mediated biological traits revealed that ?Bbmpk1displayed significantly increased tolerance to oxidative,osmotic stress and cell wall-perturbing stress,while no obvious difference was examined in sensitivity to those stressed demonstrated above between the ?Bbste12 WT strains.These results suggested that Bbmpk1 negatively mediated oxidative and osmotic stress and cell wall integrity,which seems independent of Bbste12.Based on those findings,here we forcused on Bbmpk1-mediated oxidative and osmotic stress response and investigated its biological or ecological significance,and physical response caused by Bbmpk1 mutation.Furthermore,molecular basis of Bbmpk1 negatively mediating oxidative and osmotic stress resonponses was investigated by identification of Bbmpk1-mediated genes and physically interacted proteins via comparative transcriptome analysis,Co-Immunoprecipitation and yeast-two hybrid methods,as well as gene disruption strategy.The main results are as follows:1.Bbmpk1 negatively mediated oxidative,osmotic stress and cell wall-perturbing stress response.Sensitivity of ΔBbmpk1 and ΔBbste12 strains to different stress was tested.ΔBbmpk1mutant was more tolerant to osmotic,oxidative stress and cell wall synthesis inhibitor Congo red than WT strain.However,no obvious difference was detected in sensitivity to the same stress between the ΔBbste12 mutant and WT strains.These results further suggested that Bbmpk1 negatively mediated osmotic,oxidative stress response and cell wall integrity independently of Bbste12 in B.bassiana.2.Knockout of Bbmpk1 improves the ability of the fungus to adapt to the insect hemocoel environment.To reveal biological and ecological significance of Bbmpk1 negatively mediating stress response,insects were innoculated with conidia by intrahemocoel injection to examied the mortality rate and fungal proliferation in insect hemocoel（oxidative and osmotic environment）.The results showed that the proliferation of ΔBbmpk1 in insect hemocoel and caused mortality rate of insects were significantly faster than WT.These data suggested thatΔBbmpk1 enhanced the adaption to insect hemocoel.3.Inactivation of Bbmpk1 increases antioxidant enzyme activities and altered expression patterns of antioxidant genes.To understand the physiological and molecular basis of increased tolerance to oxidative stress by Bbmpk1 mutation,several antioxidant enzyme activities,as well as expression patterns of antioxidant genes,were assayed under the normal and oxidative stress conditions.The results showed that the intracellular catalase（CAT）,peroxidase（POD）and superoxide dismutase（SOD）activities of ΔBbmpk1 strain were significantly increased,which were51.05%,16.36% and 8.99% higher than the WT strain,respectively.By analyzing the transcription patterns of 25 antioxidant-related genes,it was found that the expression levels of 19 genes were significantly up-regulated in ΔBbmpk1.Therefore,these results suggested that Bbmpk1 might control oxidative stress response of Beauveria bassiana by mediating the expression of antioxidant genes.4.Bbmpk1-mediated transcription profile under oxidative and osmotic stress.To further reveal the molecular basis of Bbmpk1-mediated oxidative and osmotic stress response,the gene expression profiles of ΔBbmpk1 and wild strains were analyzed by comparative transcriptome.Under oxidative stress,Bbmpk1 mutation caused 843 differentially expressed genes,including 362 up-regulated and 481 down-regulated genes.When fungal cells were cuntured in osmotic stressed-media,1300 differentially expressed genes were identified in the ΔBbmpk1 cells as compated the wild type strain,which included327 up-regulated and 973 down-regulated genes.Functional classification indicated that differential genes were involved in biological processes such as cell cycle,material metabolism,cell defense,and cell structure composition.KEGG enrichment revealed that differentially expressed genes were enriched into basal metabolism,energy metabolism,glycerophospholipid metabolism,secondary metabolism,and signal transduction（MAPK and c AMP）.The results suggesting that Bbmpk1 MAPK might participate in regulation of those metabolic processes or interact with other pathways to control the stress response.5.Identification of Bbmpk1-interacted proteins under oxidative and osmotic stressThe protein immunoprecipitation（IP）method was used to identify the proteins that interact with Bbmpk1 under oxidative and osmotic stress.Twenty-seven proteins that might interact with Bbmpk1 were identified via Protein-Protein Interaction Networks prediction.The yeast two-hybrid tests were used to secreen protein that physically interacted proteins with Bbmpk1 from IP pool.Only two of the 27 proteins were verified that directly interacted with Bbmpk1,named BBA₀5524（Vacuolar proton pump subunit,Bb Vma B）and BBA₀6096（Outer mitochondrial membrane protein porin,BbOmm1）.Bioinformatics analysis indicated that Bb Vma B and BbOmm1 were located on the vacuolar membrane and the outer mitochondrial membrane,respectively,which contained 9 and 5 putative serine phosphorylation sites,respectively.RT-q PCR analysis showed that that the expression of Bb Vma B was induced by the oxidant menadione,Na Cl and sorbitol-mediated osmotic stress and cell wall-perturbing agent（Congo Red）,but repressed by KCl-mediated osmotic stress.Expression of BbOmm1 was induced by oxidants menadione,T-BHP and Congo red.These results suggested that the Bbmpk1 migh physically interacted with two proteins via phosphorylation and control oxidative and osmitic stress responses.To verify role of the interacted proteinsin stress response,BbOmm1 gene knockout strain was constructed.The results indicated that disruption of BbOmm1 resulted in a significant decrease in fungal growth,with reduced spore production and decreased spore viability.However,ΔBbOmm1 strain displayed increased tolerance to oxidative stress,but decreased tolerance to osmotic stress.The bioassay revealed that BbOmm1 mutation led to a significantly reduced virulence.These results suggested that Bbmpk1 might regulate mitochondrial function by interacted with BbOmm1 and mediate oxidative stress response.