Involvement Of PH Signaling Pathway In Regulation Of Toxin Biosynthesis And Pathogenicity In Fusarium Graminearum

Fusarium head blight(FHB)caused by Fusarium graminearum has a serious impact on wheat yield and grain quality.In addition to the economic losses,,Fusarium head blight have serious impacts on human and animal health by contaminating wheat with trichothecenes.It is of great significance to prevent the epidemic of Fusarium head blight for the food safety production and food safety in China.Therefore,the biological function of the key components of the pH signal pathway in regulation of toxin biosynthesis and pathogenicity in Fusarium graminearum were studied.1.The pH signal pathway key elements knockout mutants were successfully obtained.Through PEG-mediated protoplast transformation method,the mutations of seven key components of the Fusarium graminearum pH signal pathway were obtained(△FgPalA、△FgPalB、△FgPalC、△FgPalF、△FgPalH、△FgPacC and AFgPall).These results laid the foundation for the study of its biological function.2.The pH signal pathway is positively related to the ability of Fusarium graminearum to resist external stress.It regulates the resistance of Fusarium graminearum to the alkaline,salt,osmotic pressure,oxidative stress and membrane stress.The phenotypic assays results showed that the aerial hyphae of the mutants △FgPalA,△FgPalB,△FgPalC,△FgPalF,△FgPalH and △FgPacC in Fusarium graminearum were significantly decreased in PDA,CM and MM medium,and showed significant growth defects under alkaline(pH=8.0)conditions.These knockout mutants became more sensitive to salt(Nacl,Kcl,Licl)stress and were sensitive to osmotic pressure,oxidative stress and membrane stress.Western blot assay found that the key element FgPacC in the pH signal pathway was able to be cleaved to the active state FgPacC27 after receiving the basic signal and the salt(Nacl)signal.Through the immunoprecipitation technique(CHIP)and gel migration assay(EMSA),the active FgPacC27 was able to bind to the promoter region of the ENA5 gene of the sodium ion pump to regulate the expression of ENA5,which regulated the tolerance of Fusarium graminearum to salt stress.3.Interaction analysis of the pH signal pathway key elements.Through yeast two-hybrid experiment,FgPalA and FgPalB were interacted with FgPalB and FgPalC,respectively.FgPalA and FgPalC interacted with FgPacC,respectively.The FgPalA and FgPalC interacted with FgPalB and FgPalC respectively.And found that FgPacC is located downstream of other elements of the pH signal pathway.4.The pH signaling pathway negatively regulate the expression of DON toxins and ZEN toxins.Through the fluorescence microscopy of the toxic structure,we found that the expression of the Tril gene in the mutant was advanced and the number of rounded toxins structure was also increased.The content of DON toxin in mutants △FgPalA,△FgPalB,△FgPalH and AFgPacC was significantly higher than that of wild type PH-1 by high performance liquid chromatography.And found that zearalenone toxin(ZEN)content in the mutant △FgPalA,△FgPalB,△FgPalC,△FgPalF,△FgPalH,△FgPacC and △FgPalI were significantly higher than those in the wild type PH-1.Gel electrophoresis was used to analyze the binding of FgPacC27 to the gene promoter region.It was found that FgPacC27 was not only able to bind to the promoter region of Tri6,Tri5,Tri1 gene,but can also bind to the promoter region of PKS4,PKS12,which encodes the zearalenone toxin(ZEN).Thus,it is speculated that the pH signaling pathway may negatively regulate the expression of DON toxins and ZEN toxins.5.Under alkaline conditions,the absence of pH signaling pathway components significantly reduced the pathogenicity of Fusarium graminearum.In the cellophane penetration test,the mutants △FgPalA,△FgPalB,△FgPalC,△FgPalF;△FgPalH,△FgPacC can not penetrate under alkaline conditions.The pathogenicity experiments on maize stalks also showed that the pathogenicity of each mutant(except the mutant AFgPalI)was weakened under alkaline conditions.And at the same time,the pathogenicity of each mutant was not significantly different from that of wild type under neutral conditions.It also suggests that there may be multiple pathways to control the pathogenicity of Fusarium graminearum,and PacC-mediated pH signaling pathways dominate the pathogenicity in alkalinity,which also fully demonstrates the importance of studying pH signaling pathway in regulation of pathogenicity in Fusarium graminearum.