Study On The Bio-function Of Histone Methyltransferase Ash1 In Aspergillus Flavus

Aspergillus flavus is an opportunistic pathogen,which is one of the major pathogens causing human and animal hazards worldwide.Growth and development of A.flavus and its secondary metabolites are affected by a variety of factors.Among them,histone methylation modification is one of the main influencing factors.Histone modification is an important epigenetic modification,including methylation,acetylation,phosphorylation,and AP glycosylation.Histone methylation has been reported in saccharomyces.cerevisiae and other species,but it is unclear that whether the histone methylation modification is involved in the synthesis of AFs and pathogenicity of A.flavus.Therefore,this study is an important histone methyltransferase Ashl in A.flavus.In this study,we used the principle of homologous recombination to construct ?awh1 knockout strain and complementary strain.The study revealed that the ash1 gene deletion strain(?ash1)significantly increased conidiation on YES medium when compared to wild type flavus strain(WT)and ash1 deletion and complementation strains(com-ash1).It was found that ash1 gene significantly up-regulated the development of radiation growth of mycelium and sclerotia,which was confirmed by qRT-PCR on sclerotia regulator genes,nsdC and nsdD.TLC and HPLC analysis showed that aflatoxin B1(AFB1)production was dramatically down-regulated when ash1 gene was deleted.The expression level of AFs transcriptional regulator genes(aflR and aflS)and AFs bio-synthesis catalyzing genes(aflC,aflD,aflK,and aflQ)were further analyzed by qRT-PCR.The results showed that all these AFs bio-synthesis enzymes and regualtor genes were down-regulated in ?ash1 strain compared to WT and com-ash1 stains.Pathogenicity analysis showed that ash1 gene played an important role in crop infections and animal aspergillosis in A.flavus.On the peanut and corn surface,the conidiation and AFs bio-synthesis capacity were significantly down-regulated when ash1 gene was deleted,which suggested that ash1 gene was required in offspring transmission and in the process of mycotoxin synthesis to A.flavus in the process of colonizing on crops.In silkworm models,it was found that Ash1 significantly improved the lethality of A.flavus invaded silkworm,and increased the bio-synthesis of AFB1 and conidiation of A.flavus in silkworm.Further exploration in the study found that Ash1 might up-regulat the virulence and pathogenicity of A.flavus to plants and animals by increasing the activity of digestive enzymes,including lipase,protease and amylase.mCherry mediated Ash1 subcellular location analysis revealed that Ash1 was stably accumulated inside nucleus from germination to hyphae growth stage,as well as under H2O2 stress.Western-blotting assay revealed that Ash1 regulated H3K36me2,H3K9me2,H4K20me2 and H4K20me3 in A.flavus nucleus.All the results in this study showed that histone lysine methylation transferase Ash1 played a critical role in morphogenesis,AFB1 bio-synthesis and pathogenicity in A.flavus.