A Study On The Mechanism Of The Interaction Proteins Of F-box Protein BbGrrA Mediate High Temperature Stress In Beauveria Bassiana

Beauveria bassiana is an insect pathogen which has advantages of long-lasting,broad host range,no environmental pollution and et al.However,it is easy to be inactive in the field when it is subjected to high temperature,which extremely limits its application.Hence it is of great significance to identify the genes involved in response to high temperature,and reveal the regulatory mechanism,which would provide clues to construct high temperature tolerant strains and expand its application.In previous study,we found that BbGrrA functions in regulating the response to high temperature in B.bassiana.Its interacting protein,BbCSN5(the fifth subunit of COP9 Signalosome protease complex),participates in the regulation of high temperature tolerance by maintaining the stability of BbGrrA protein.Nonetheless,the molecular mechanism of how BbCSN5 promotes the stability of BbGrrA is not clear,and the protein that promotes BbGrrA degradation is also needed to be unveiled.In this study,the fusion gene of BbGrrA::Myc was constructed and transformed into wild type and ΔBbcsn5,respectively.The ubiquitination level of BbGrrA::Myc protein in WT and ΔBbcsn5 were detected.The results showed that the ubiquitination level of BbGrrA in ΔBbcsn5 was significantly higher than that in WT,suggesting that BbCSN5 could maintain the stability of BbGrrA protein by affecting the ubiquitination level of BbGrrA.In addition,BbMHK1(a histidine kinase),a protein interacting with BbGrrA,was isolated from the cDNA expression library by yeast two-hybrid.The knockout mutant of BbMHK1 showed high temperature sensitivity.Further studies showed that deletion of BbGrrA had no visible effect on the protein level of BbMHK1,while knockout of BbMHK1 resulted in the accumulation of BbGrrA protein.To sum up,this study revealed the molecular mechanism of the interacting proteins of BbGrrA,BbCSN5 and BbMHK1,in response to high temperature in B.bassiana.1.BbCSN5 maintains the stability of BbGrrA protein by affecting the ubiquitination level of BbGrrA BbCSN5 Positively regulates BbGrrA stability:In order to detect the protein level of BbGrrA in WT and ΔBbcsn5,we constructed the vector harboring the fusion gene of BbGrrA::Myc and tranferred it into WT and ΔBbcsn5 mutant,respectively.Then the fusion protein of BbGrrA::Myc in WT and ΔBbcsn5 were detected.It was found that the protein level of BbGrrA in ΔBbcsn5 was obviously less than that in WT,which indicates that the degradation of BbGrrA in ΔBbcsn5 was faster compared with WT.The effect of high temperature and BbCSN5 on the modification of BbGrrA: When we detected the fusion protein of BbGrrA::Myc in WT and ΔBbcsn5 after high temperature treatment,we found that the modification of BbGrrA in ΔBbcsn5 was significantly more than that in WT,and was increased under high temperature stress.BbCSN5 affects the stability of BbGrrA by mediating its ubiquitination level: In order to explore the reason for the accelerated degradation of BbGrrA in ΔBbcsn5,BbGrrA::Myc protein in WT and ΔBbcsn5 were purified by IP and the ubiquitination level were tested.The results showed that ubiquitination level of ΔBbcsn5 was significantly higher than that of wild type.Overexpression of BbGrrA in ΔBbcsn5 restores its high temperature sensitive phenotype: The sensitive phenotype of high temperature of ΔBbcsn5 was restored when BbGrrA was overexpressed in it.This data indicates that BbCSN5 functions in response to high temperature via regulating BbGrrA degradation.2.BbMHK1 promotes BbGrrA degradation BbGrrA interacts with BbMHK1: BbMHK1 was identified by Yeast Two Hybrid with BbGrrA as bait to screen fr.The BbMHK1 encoding gene is 3956 bp,including two exons and one intron,which encodes 277 amino acids,and its protein molecular weight is about 140.47 kDa.BbMHK1 expression is induced by high temperature: In order to investigate whether the BbMHK1 expression is induced by high temperature,the BbGrrA::Myc fusion gene was constructed was transformed into wild-type strain.After 32℃treatment for different time;BbMHK1 protein level was detected,the results showed that the expression of BbMHK1 was induced by high temperature.The BbMHK1 knockout strain was sensitive to high temperature stress: To investigate wehther BbMHK1 is involved in regulation of high temperature tolerance,we constructed BbMHK1 knockout-and complementary strains.The relative colony growth inhibition rate(RGI)of BbMHK1 knockout mutant is higher than that of WT,while that of complementary strain is comparable to that of WT.The results indicated that BbMHK1 also do some contribution to the response of high temperature stress.BbMHK1 negatively regulates the stability of BbGrrA: The fusion protein of BbGrrA::Myc in WT and ΔBbMHK1 were detected after the vector containing the fusion gene of BbGrrA::Myc was transformed into ΔBbMHK1.It was found that the protein level of BbGrrA in ΔBbMHK1 was obviously more than that in WT,which indicates that the degradation of BbGrrA in ΔBbMHK1 was reduced in contrast to WT.BbMHK1 indirectly affects the phosphorylation modification of BbGrrA: Because BbMHK1 is a histidine kinase,we firstly thought that it could promote the phosphorylation level of BbGrrA,and deletion of BbMHK1 would result in descrease of phosphorylation level of BbGrrA.On the contrary,the phosphorylation level of BbGrrA in ΔBbMHK1 was significantly higher than that in wild-type,this data indicates that BbMHK1 indirectly affects the phosphorylation modification of BbGrrA.