Studies On Functional Of Transcription Factor STEA In Beauveria Bassiana

Beauveria bassiana,a insect pathogenic fungus,is widely used in biological control ofagricultural pests.As a fungal insecticide,its large-scale application is limited to a certain extent in pest control,which is due to killed insects slowly and easily affected by the environment（including light,humidity,heat,and ultraviolet light）.Transcription factor STE12 of filamentous fungi,as the main regulator in the MAPK pathway,is closely related to the growth and pathogenicity of pathogenic fungi,however,few reports about the function of transcription factor STEA in B.bassiana.In the present study,the ste A was deleted by homologous recombination technology,andthe phenotypic changes of the mutant strains in terms of growth and development,stress resistance and pathogenic process were analyzed.Our results will expand the understand of the biological function of transcription factor STEA and provide an important basis for the development of genetically engineered strains of B.bassiana with high toxicity and stress resistance.The specific results are as follows:The results of bioinformatics analysis and positioning experiments:the sequence of STEA transcription factor can be found in B.bassiana in NCBI,the gene sequence number is BBA＿01078,and the full length is 2110bp,encoding 685 amino acids.Conserved domain analysis shows that it contains a STE12 domain and two C₂H₂ zinc finger domain.Subcellular localization analysis revealed that the protein is localized in the nucleus.Through homologous recombination and random insertion techniques,a knockout strain（△Bbste A）and a complment strain（Comp）was obtained.Compared with the wild type,the growth rate of colonies and conidial yields of knockout strains are decreased,which suggested that the transcription factor STEA is involved in growth and development of B.bassiana.Under the conditions of UV and heat shock,compared with the wild type,there was no significant difference in the germination rate after 24h,although the germination of the mutant spores was advanced.These results indicate that the STEA is not involved in the response of UV and heat stress.Moreover,deletion of STEA lead to increased tolerance to Congo red,hydrogen peroxide,and menadione,compared with the wild type.However,disrupted of STEA resulted in almost lost tolerance to sodium chloride.These results suggest that the transcription factor STEA is involved in regulating the cell wall integrity,oxidative stress and salt stress in B.bassiana.The results of bioassay:the results of cuticle infection showed that the half-lethal time(LT₅₀)of wild type,mutant and complement strains were 6.09,0,6.12（d）,respectively.No blackening occurred was observed on the surface of the cuticle.These results suggest that the virulence of knockout strain was completely lost during cuticle infection.The results of blood cavity injection showed that the LT₅₀of wild-type,knockout and complement strains were 3.72,3.66,3.69（d）,respectively,and there was no significant difference among them.Furhtermore,the hyphae of the knockout strain cannot penetrate the surface of the cadaver.These results indicate that ste A plays an important role in the stage of cuticle infection.Moreover,the results of penetration experiments using cicada wings showed that the knockout strains completely lost their ability to penetrate cicada wings.At the same time,the results of RT-q PCR analysis show that the expression level of 8 genes related to cuticle-degrading were no change or significantly down-regulated.These results suggest the transcription factor STEA plays an essential role in the process of cuticle penetration.In summary,STEA of B.bassiana is a transcription factor and located in the nucleus.Disrupted of STEA resulted in a decrease in spore production and loss of salt stress resistance,an increase in tolerance to oxidative stress.More importantly,there are the complete loss of virulence during the infection,which may be due to the fact the mycelium cannot penetrate the cuticle of the insect and the expression levels of cuticle-degrading genes were significantly downregulated.