The Mechanism Of Aflatoxins Biosynthesis Regulated By Protein Post-translational Modification

Aspergillus flavus is a kind of conmon pathogenic fungus of crops.It could produce toxic and carcinogenic secondary metabolites aflatoxins,contaminating crops and agricultural products.A.flavus and aflatoxins are dangerous to human health and cause huge economic losses.Hence,studies on of morphogenesis and regulating mechanism of aflatoxins in A.flavus become more and more important.Protein phosphorylation is an important and reversible posttranslational modification and regulates many life actions.A study of Aspergillus parasiticus reported that phosphorylated status of calmodulin-dependent protein regulated aflatoxin production.Therefore,the phosphoproteome method was used to study the relationship between phosphorylation and the formation mechanism of aflatoxins in A flavus.Firstly,the hypha proteins of A.flavus were extracted from the growth stages of the lowest and highest aflatoxins production.By high-throughput LC-MS/MS,544 phosphorylated peptides and 283 phosphorylated proteins were identified,containing 598 high confidence phosphorylated sites.From these identified phosphorylated proteins,one phosphorylated protein Stell was selected for functional study.The homologous protein of Ste11 in Saccharomyces cerevisiae and Aspergillus is a mitogen-activated protein kinase kinase kinase,located in mitogen-activated protein kinase pathway(MAPK pathway)to stimulate the phosphorylation of mitogen-activated protein kinase kinase.To study the effect of phosphorylation of Ste11 on aflatoxins production in A.flavus,stell deletion strain(?ste11)and ste11 complementation strain(?ste11:ste11)were constructed.Compared to wild type,it was found that the colony growth,conidia production,sclerotial formation and host infection were suppressed in ?ste11 mutant.Especially,aflatoxins biosynthesis was suppressed seriously after stell was deleted.To prove the relationship between the function of Ste11 and phosphorylation,the point mutants,S187A and S187D,were constructed.The results of phenotypic analyses showed that the phenotypic characteristics of S187A were similar to that of Astell.The results demonstrated that Ste11 played significant roles in morphogenesis and aflatoxins biosynthesis and the phosphorylation of 187th S in Ste11 was closely related to the function of Ste11.Except for phosphorylation,succinylation was another research content,to study the effect of this important posttranslational modification on morphogenesis and aflatoxins biosynthesis.The change of carbon sources has been proved to influence the level of succinylation proteins in Escherichia coli and the production of aflatoxins in A.flavus.So the method of succinylome was used to investage the relationship between succinylation and aflatoxins biosynthesis.Totally,349 succinylated proteins were identified,and 986 high confidence succinylation sites were identified in 1013 succinylation peptides.One succinylation protein AflE was selected for further study.Its homologue in A.parasiticus is the norsolorinic acid reductase locating in upstream of aflatoxins biosynthesis pathway.To know the effect of succinylation on aflatoxins production in A.flavus,aflE deletion strain(?aflE)and complementation strain(?aflE::aflE)were construced for phenotype observation,cultured with synthetic media.Compared with those of wild type,sclerotial and aflatoxins formation were decraesed in ?aflE.For the experiments of peanuts infection,the infecting ability,spores formation and aflatoxins production declined in AaflE.With the purpose of proving that the function of AaflE was decided by succinylation modification,point mutation strains of succinylation sites in ?flE,K370A and K370R,were constructed to observe the phenotypic changes of A.flavus.The formation of sclerotia and aflatoxins in both K370A and K370R were less than those of wild type,similar to the phenotypic characteristics of AaflE.These results demonstrated that the succinylated site(370th K)in succinylated protein ?flE played vital roles in the function of AflE.Acetylation of lysine has already been proved as a crucial protein post-translational modification to regulate metabolism.Hence,the method of acetylome was used to study the effect of acetylation on morphogenesis and aflatoxins biosynthesis in A.flavus.After identified with high throughput LC-MS/MS,727 acetylation proteins were obtained,and 1313 high confidence acetylation sites were identified in 1293 acetylation peptides.Acetylation protein AflK was used to further study.Its homologue in A.parasiticus is an emzyme of aflatoxins pathway.alfK deletion strain(?aflK)and complementation strain(?aflK::aflK)were constructed and the phenotypes of these two strains were compared with those of wild type.It was found that less sclerotia and aflatoxins were producted in AaflK.The host infection of AaflK was natural,but aflatoxins biosynthesis was decreased.The results suggested that the roles of AflK were noticeable in the production of sclerotia and aflatoxins.Based on these analyses and results,the post-translational modifications have been proved to regulate the morphogenesis and aflatoxins biosynthesis in A.flavus.