Function Of Arginine Methyltransferase BcHMT2 In Growth,Development And Pathogenicity Of Botrytis Cinerea

Arginine methylation is a post translational modification catalyzed by arginine methyltransferase(PRMTs).Arginine methyltransferase HMT2 is an important epigenetic modifier that participates in the growth and pathogenesis of pathogens by regulating the level of arginine methylation.However,studies on the arginine methyltransferase from Botrytis cinerea have not been reported.In this study,the arginine methyltransferase HMT2 of Botrytis cinerea was analyzed by bioinformatics methods,and the coding gene of arginine methyltransferase HMT2 of Botrytis cinerea was identified as BcHMT2.The BcHMT2 gene knockout vector of Botrytis cinerea was constructed by homologous recombination technology.The transformants of the BcHMT2 gene of Botrytis cinerea were obtained through protoplast transformation technology.Through protoplast transformation,we obtained the knockout mutants of BcHMT2 gene of Botrytis cinerea.By analyzing the phenotype and pathogenicity of BcHMT2 knockout mutants,the function of the BcHMT2 gene in the growth,development,and pathogenicity of Botrytis cinerea was determined.The cell wall degrading enzyme activity and acid production ability of ΔBcHMT2 mutants were analyzed to explore the mechanism of BcHMT2 gene regulating the pathogenicity of pathogens.The results laid an important foundation for clarifying the molecular mechanism of BcHMT2 gene regulating the growth,development,and pathogenicity of Botrytis cinerea.The research results are as follows:1.Bioinformatics identification of arginine methyltransferase HMT2 from Botrytis cinerea.Using bioinformatics methods,sequence alignment,phylogenetic analysis,and conservative domain analysis were performed on the arginine methyltransferase HMT2 of eight fungi,including Botrytis cinerea,Saccharomyces cerevisiae,Fusarium graminearum,Magnaporthe grisea,Neurospora rough,Aspergillus flavus,Aspergillus nesting,and Candida albicans.It was found that the arginine methyltransferase HMT2 is highly conserved among these 8 fungis,all of which contain the Methyltransf domain and have a close homologous relationship.The coding gene for the arginine methyltransferase from Botrytis cinerea was determined to be BcHMT2.2.Construction of a knockout mutants of the BcHMT2 gene from Botrytis cinerea.In this experiment,the BcHMT2 gene knockout vector of Botrytis cinerea was constructed by homologous recombination technology;Using protoplast transformation technology,a BcHMT2 gene knockout transformant containing hygromycin resistance was obtained.Through purification,screening,and molecular identification of the transformants,the BcHMT2 gene knockout mutants ΔBcHMT2-1,ΔBcHMT2-2 andΔBcHMT2-3.3.Study on the function of BcHMT2 gene of Botrytis cinerea during the growth,development,and pathogenicity of the pathogen.This experiment analyzed the phenotype and pathogenicity of the BcHMT2 gene knockout mutants of Botrytis cinerea,and found that compared with the wild type B05.10,the growth rate of BcHMT2 gene knockout mutants were significantly slowed down,sclerotia yield were reduced,mycelial cells became smaller,altered spore morphology,spore yield were decreased,and the pathogenicity to tomato and tobacco leaves were significantly reduced.The results showed that the BcHMT2 gene had a positive regulatory effect on the growth,development,and pathogenicity of Botrytis cinerea.4.Analysis of pathogenic factors of BcHMT2 gene knockout mutants of Botrytis cinerea.Analysis of the cell wall degrading enzyme activity,cell wall degrading enzyme gene expression and acid production ability of BcHMT2 gene knockout mutants of Botrytis cinerea,the results showed that compared with wild-type B05.10,the activity of intracellular and extracellular wall degrading enzymes in the mutants was decreased,the genes of wall degrading enzymes were significantly downregulated,and the acid production ability was lowered.This indicates that BcHMT2 gene is regulating the activity of cell wall degrading enzyme and acid production ability of Botrytis cinerea.5.Functional analysis of BcHMT2 gene of Botrytis cinerea in response to stress.The sensitivity of the BcHMT2 mutants of Botrytis cinerea to osmotic stress,oxidative stress,and cell integrity was determined,and it was found that compared to the wild type B05.10,the sensitivity of BcHMT2 gene knockout mutants to osmotic stresses such as NaCl,KCl,glycerol,and sorbitol were decreased.There were no significant change on H2O2 and menadione media,and it was not involved in the influence of Botrytis cinerea on oxidative stress.The sensitivity of BcHMT2 gene knockout mutants to Congo Red and SDS stress were lower,the growth rate on fluconazole medium was partially lower than that of wild type,this indicates that the deletion of BcHMT2 gene may affect the integrity of Botrytis cinerea cells.The results showed that BcHMT2 gene was involved in the regulation of osmotic stress and cell integrity of Botrytis cinerea,and then affected the growth,development and pathogenicity of the pathogen.