| Aflatoxins(AFs)contaminate agricultural products,nuts,feed and related processed products due to the widespread infestation of Aspergillus flavus,posing a serious threat to human and animal health and causing serious damage to the quality and economic value of agricultural products.To address the hazards of AFs,it is important to study and reveal the biosynthetic mechanisms of the AFs to provide a theoretical basis for the prevention and control of the AFs at source.The pheromone Fus3-MAPK signaling pathway is essential for the physiological activities of fungi,as it significantly regulates growth,sexual development,conidia formation,secondary metabolism and pathogenicity.However,very little research has been conducted on the downstream targets of the pheromone Fus3-MAPK signaling pathway in recent years,and even less in A.flavus.In this work,we aimed to advance the regulatory mechanism of the Fus3-MAPK pathway in the regulation of AFs biosynthesis,and the results of the study on the mechanism of Fus3 and Gal83 regulating AFs biosynthesis were as follows:(1)Key phosphorylation function sites for Fus3 were found,and Fus3 performed a phosphorylation function.The T182 and Y184 sites of the Fus3 protein were chosen using a combination of phosphoproteomic identification and prediction analysis of phosphorylation functional sites.The successful construction and analysis of mutant strains for the Fus3 protein phosphorylation sites showed that the Fus3 point mutant strains were similar to those of the fus3 gene deletion strain.This means phosphorylation function of Fus3 is a major function and that the T182 and Y184 phosphorylation sites are important phosphorylation function sites.(2)Fus3 controls aflatoxin production by controlling the concentrations of aflatoxin synthesis substrates: By using RT-qPCR,transcriptomic,and phosphoproteomic analysis,it was shown that neither a significant down-regulation of the aflatoxin gene cluster’s transcript levels in the Δfus3 strain nor a change in the gene cluster’s phosphate levels had occurred.GO and KEGG analyses revealed that down-regulated expression of genes associated with primary metabolism.Additionally,the transcription and phosphorylation levels of some important enzyme genes in the primary metabolic pathways,such as pyruvate dehydrogenase and Acetyl-CoA carboxylase,were down-regulated.Acetyl-CoA and Malonyl-CoA were significantly down-regulated in the Δfus3 strain,according to ELISA.This suggests that Fus3 affects primary metabolism,especially the precursors to the synthesis of the aflatoxins Acetyl-CoA and Malonyl-CoA.(3)The Gal83 positively regulated the growth,sexual development,secondary metabolism and pathogenicity of A.flavus,and its mechanism of regulating aflatoxin synthesis is through the regulation of aflatoxin synthesis substrate levels: The functions of Gal83 were characterized by constructingΔgal83 and complementary strains.The deletion of gal83 resulted in reduced growth,significantly reduced conidia formation,no sclerotia formation,significantly reduced aflatoxin biosynthesis and reduced pathogenicity in A.flavus,while the complementary strain was similar to the wild-type phenotype,indicating that the gene positively regulates the growth and development,aflatoxin biosynthesis and pathogenicity of A.flavus.Transcriptome and RT-qPCR revealed no significant down-regulation of aflatoxin synthesis genes.The transcript levels of some key enzyme genes of the primary metabolic pathway were significantly down-regulated,and some genes were similar to the results of the Fus3 deletion strain,including pyruvate dehydrogenase,glucose dehydrogenase6-phosphate,hexokinase and other key enzyme genes.The levels of Acetyl-CoA and Malonyl-CoA in the Δgal83 strain were significantly reduced by ELISA.In summary,this study identified a new target protein of Fus3,Gal83,and demonstrated that Fus3 stably interacts with Gal83 and regulates the primary metabolic processes of A.flavus,especially the levels of Acetyl-CoA and Malonyl-CoA,which in turn regulate the biosynthesis of aflatoxin.The results reveal a new mechanism for the regulation of aflatoxin synthesis substrate levels by the protein kinase Fus3 in the pheromone Fus3-MAPK signaling pathway,which advances the research progress of the Fus3-MAPK regulatory network in fungi and provides new data support for the precise prevention and control of aflatoxins. |
