Functional Study And Application Of Tryptophan Metabolism In Fungal Growth,development,Toxin Production And Pathgenesis Of Fusarium Graminearum

Fusarium graminearum can cause Fusarium head blight(FHB),resulting in wheat yield reduction,in the process of its infection,fungi could produce a variety of toxic and harmful secondary metabolites,known as Fusarium toxins,such as Deoxynivalenol(DON),Nivalenol(NIV),etc.,threatening food security and human and animal health.In this thesis,we investigated the role of tryptophan(Trp)metabolism(primary metabolism)in fungal growth and development of Fusarium graminearum and the biological functions and differentiation of the homologous genes FgIDOA/B/C,which encode Indoleamine-2,3-dioxygenase(Ido),the first rate-limiting enzyme of Trp metabolism.The Trp metabolic branch includes the Kynurenine Pathway(KP),the Melatonin Pathway(MP)and the Indole Pathway(IP).KP provides the substrate nicotinic acid mononucleotide for the synthesis of the essential cofactor NAD~+,MP consumes Trp to provide the substrate melatonnin for the synthesis of the neuroactive substance 5-hydroxytryptamine,and the Indole Pathway metabolized Trp to synthesize the active substances indole and indole-3-aldehyde acid.The main research results of this thesis are as follows:(1)Genetic evolutionary analysis shows that Ido is widely distributed among species,and FgIdo A and FgIdo B in F.graminearum have high homology with Fusarium Fujikuroi,Fusarium verticillioides,Aspergillus spp.,Saccharomyces cerevisiae and some bacteria,while FgIdo C is more distantly related to the above species,and is more closely related to a few Basidiomycota fungi and invertebrates.(2)Single,double and triple deletion mutants of three homologous genes FgIDOA/B/C,complementary mutants of FgIDOA gene and overexpression mutants of FgIDOB/C gene in F.graminearum were constructed by double-junction PCR or yeast gap repair strategy,and the series growth phenotypes of the above mutants were determined.Compared with the wild-type strain PH-1,the FgIDOB/C gene deletion mutants showed no significant difference in growth and virulence.The FgIDOA gene deletion mutant showed different levels of NAD~+defects in mycelial nutritional growth and other phenotypic changes including altered conidial morphology,significantly reduced conidial germination rate,DON production and pathogenicity on flowering wheat heads.Exogenous addition of NAD~+and other intermediates and re-introduction of the full-length FgIDOA gene rescued the phenotypic defects of the FgIDOA deletion mutant.(3)By using targeted and untargeted metabolome analysis,we compared the differences in Trpmetabolic profiling in wild-type strains with that in FgIDOA/B/C mutants.Trp metabolic pathway was altered after FgIDOs were functionally defective,and the synthesis of KP-related metabolites was significantly decreased after FgIDOA gene deletion,while the content of metabolites related to IP and MP was significantly up-regulated after the deletion of FgIDOB gene.(4)The expression levels of FgIDOA/B/C genes in wild-type strain PH-1 and different mutants before and after Trp treatment were determined by RT-PCR.Results showed that the expression levels of partner FgIDO genes inΔFgIDOA,ΔFgIDOAB andΔFgIDOAC were increased.In addition,the NAD~+growth defect of the overexpression mutants OE::FgIDOBΔFgIDOA and OE::FgIDOCΔFgIDOA was restored.Results indicates that there might be a functional complementarity at the transcriptional level between the three homologous FgIDOA/B/C in F.graminearum in the reaction of Trp metabolism via the kynurenine pathway to provide feedstock for NAD~+biosynthesis.In summary,this thesis analyzed the biological functions and differentiation of three paralogous FgIDOA/B/C genes in F.graminearum involved in Trp metabolism,growth and development and virulence production,and elucidated the regulatory role of primary metabolic process(Trp metabolism)on the growth and development of pathogenic bacteria and secondary metabolism(toxin synthesis,etc.).We identified important virulence production-related genes.Results of this thesis provide a potential target for the development of new antifungals and a theoretical basis for the sustainable prevention and control of FHB and the integrated management of toxin contamination.