| Stemphylium species are a kind of filamentous fungi,which are widely distributed and cause leaf spot disease in economic crops.Stemphylium eturmiunum belongs to saprophytic fungi and optimistic pathogen.It can form conidia and ascospores during the growth process.S.eturmiunum is one of the few species with complete life history in Stemphylium.It is a homothallic ascomycete,which is able to form pseudothecia without a mating partner.In addition,fungal pathogens transmit mainly through spores,sexual fruiting body and spores can aid survival in adverse environmental conditions.S.eturmiunum was used as experimental materials in the subject.The effects of 5-azacytidine(5-AC),an inhibitor of DNA methylase,on the S.eturmiunum development and metabolities were studied.The expression levels of mating type genes,key genes related to G-protein signal transduction pathway,arachidonic acid biosynthesis genes and melanin biosynthesis genes were tested by fluorescence quantitative PCR and RNA-seq.Moreover,histone methylation modification in prompter regions of the target genes were analyzed by ChIP-qPCR.At the same time,we employed methylation sensitive and none sensitive enzymes to test the DNA methylation in the promoter or ORF of the target genes.The metabolic accumulation of fatty acids,melanin,and the formation number of pseudothecia were tested and compared among different strains.Furthermore,the relationship between melanin synthesis and nitric oxide content,and the signal transduction of nitric oxide on sexual development of S.eturmiunum were researched,combined with the effect of epigenetic modification on the key genes of its metabolic pathway.We studied the effects of DNA methyltransferase inhibitor on morphological,and the content of melanin and nitric oxide,together with the epigenetic modification influence on key genes relate to the different pathways,to determine the mechanism of the pseudothecia formation in filamentous fungi.Incubation of S.eturmiunum with 5-AC,an DNA methytransferase inhibitor,changed obviously.5-AC induced high-frequency conversion of S.eturmiunum to a fluffy and whitevariant.The conidiation morphology of S.eturmiunum was changed.There was no formation of conidia and pseudothecia in 5-AC treated S.eturmiunum.In addition,the changed phenotype can inherit stably.The morphology and accumulation of metabolites in all tested strains(S.solani,S.sarciniforme,S.botryosum,S.nabarii)were changed by 5-AC.The effects of 5-AC on the development and metabolities of Stemphylium species has global function.The fatty acid content of S.eturmiunum was analyzed by GC-MS.It was found that the content of linoleic acid increased,the content of olieic acid decreased,and the content of arachidonic acid decreased obviously in 5-AC treated S.eturmiunum.Furthermore,there was no accumulation of melanin and the content of nitric oxide decreased significantly in 5-AC treated S.eturmiunum.The expression level of mating type genes,and genes related to Gprotein signal transduction pathway and melanin synthesis were decreased after the treatment of 5-AC.The modification of H3K4Me3 and H3K9Me3 in the promoter of the mating type genes and key genes,which related to G-protein signal transduction pathways,arachidonic acid systhetase,and melanin synthesis had obvious changes by 5-AC.Except the DNA methylation modification in the peomoter region of VeA was inhibited by 5-AC,the DNA methylation modification of other tested genes did not change.The results of RNA-Seq indicated that there was no expression of magl in 5-AC treated S.eturmiunum.After comparison of magl sequence between 5-AC treated and untreated S.eturmiunum,we found that there had three base mutation in 5-AC treated strain and formed 6 termination codons.At the same time,the modification of H3K9Me3 in the promoter of magl was much highter than in the untreated S.eturmiunum.Therefore,we concluded that these two resaons lead to the loss of MAGL activity.After added arachidonic acid to the medium of 5-AC treated and untreated strains,we found that there were conidia and pseudothecia formation in 5-AC treated strains,and there was accumulation of melanin,too.We can get a conclution that magl can influence the development and melanin accumulation in S.eturmiunum.Furthermore,we found that the sexual development of S.eturmiunum regulated by G-protein signaling system,where AA acts as ligand binding to GPCR,thereby activates signal transduction,and consequently incurs transcription of mating genes,leading to the formation of pseudothecia.In order to further study the effect of melanin on the growth and development in S.eturmiunum,we found there are two pathyways to synthesis melanin by arbutin and tricyclazole,and they are L-DOPA and DNH.In fungi,the main biosynthesis gene related to DNH pathway is polyketide synthase.We transferred the genes which encode polyketide synthase in S.eturmiunum that related to melanin synthesis to Aspergillus nidulans,which confirmed the function of polyketide synthase genes in the synthesis of melanin.Moreover,the melanin from the wild type of S.eturmiunum and heterologous expression in A.nidulans had similar antioxidant and anti-ultraviolet activities.In addition,tyrosinase is the key enzyme in the L-DOPA pathway.There was no accumulation of melanin in 5-AC induced S.eturmiunum strain,so we compared the expression and activity of tyrosinase between 5-AC treated and untreated strains,we found the the activity in 5-AC treated S.eturmiunumw as very lower.In addition,the content of nitric oxide can regulate the accumulation of melanin.The contents of nitric oxide and melanin positively correlated to each other.Difference dose of nitric oxide has different influence in the formation of pseudothecia in S.eturmiunum.In this study,we found that low concentration of nitric oxide could promote the formation number of pseudothecia,and accelerate the formation of pseudothecia,furthermore increase the accumulation of melanin.However,high concentration of nitric oxide lead to mycelium to the condition of nitrogen stress,and the number of conidia and pseudothecia were significantly lower than in the control medium.In addition,exogenous nitric oxide can activate the expression of genes related to G-protein signal transduction pathway and the mating type genes by regulating the level and types of epigenetic modification in the promoter region of the target genes,thus regulating the formation of sexual types.We also found that the nitric oxide content in the 5-AC treated strain was significantly lower than that of the wild type S.eturmiunum,and there was no melanin accumulation in 5-AC treated S.eturmiunum.The disruption of the balance of nitric oxide content in S.eturmiunumo affect the sexual development.The results proved that,the sexual development of S.eturmiunum could influenced by epigenetic modification,the content of arachidonic acid,melanin and nitric oxide.Histone modification can influence the state of chromatin,then impacte the formation of pseudothecia and the expression of genes related to mating type gene,G-prptein signaling pathyway,arachidonic acid synthesis,nitric oxide signaling pathyway and so on.The balance of arachidonic acid or nitric oxide play importment role in the pseudothecia formation,and arachidonic acid in signal transduction via a putative G protein-coupled receptor 1.During the process,magl is the main regulator to influence the accumulation of arachidonic acid,and it affects the accumulation of melanin.The results of the factors to regulate the formation of pseudothecia in S.eturmiunum can provide important reference information for the study of fungal biological evolution.Further study on the mechanism of sexual development can help us to understand the biological and ecological significance of the species in the environment,and provide a reference for the inhibition of the transmission of pathogenic fungi. |
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