Functional Characterization Of Endocytic Protein Syp1 In Fusarium Graminearum

Fusarium head blight(FHB)is a fungal disease that damages wheat,barley and other cereal crops,and it occurs severely in major wheat producing areas in China.The dominant pathogen of FHB is Fusarium graminearum in Shandong Province.The pandemic of FHB in the field caused crop yield loss and quality decline,and even no harvest in severe cases.The mycotoxins produced in infected wheat kernels are extremely harmful to the health of humans and animals.Endocytosis is a process by which external substances are transported into cell through the invagination of the plasma membrane to form vesicles.It is critical to maintaining cell homeostasis,regulating signal transduction and nutrient absorption.Syp1 is an endocytic adaptor protein located on the plasma membrane of the cell,which plays a role in the formation and polarization of the endocytic site.The main results obtained from the study are as follows: After identifying a Fg Syp1 gene in Fusarium graminearum,SMART-Pfam analysis shows that Fg Syp1 has a F-BAR domain and a mu HD domain.The analysis of phylogenetic tree construction shows that Fg Syp1 is closely related to Syp1 in Fusarium verticillium and Fusarium oxysporum.Interaction between Fg Syp1 and Fg Ede1 in Fusarium graminearum was verified by the yeast two-hybrid method.The Fg Syp1 and Fg Syp1/Ede1 knockout vectors were constructed by Split-marker PCR technology.According to the principle of homologous recombination,the target gene was knocked out using PEG-mediated protoplast method.After resistance screening,PCR verification and Southern blot hybridization verification,we obtained ΔSyp1 mutant,ΔΔSyp1/Ede1 mutant.In order to confirm the function of Fg Syp1,the ΔSyp1-C replenishment strain was obtained by the method of in vitro cloning.The growth rate of the ΔSyp1 mutant on the PDA plate was decreased by 50.4% compared with the wild type,and the growth rate of the ΔΔSyp1/Ede1 mutant on the PDA plate was 58.2%lower than that of the wild type.The conidia production of the ΔSyp1 mutant decreased by 21.4%compared with the wild type,and the conidia production of the ΔΔSyp1/Ede1 double knockout mutant decreased by 58.5% compared with the wild type,which was lower than the spore production of knocking out Fg Syp1 and Fg Ede1 respectively.The conidia germination rate of the mutant was lower than that of the wild-type strain,and the germ tube produced by the spore germination was shorter than that of the wild-type strain in YEPD mediam.The ΔSyp1 mutant produced ascospores and ascospores through sexual reproduction,and the ΔΔSyp1/Ede1 double knockout mutant produced ascoscapes but not ascos and ascospores,and the number of ascus shells was less than that of the wild type.The pathogenicity of mutant strains was significantly lower than that of wild type.The pathogenicity of ΔSyp1 mutant was decreased by 65.5%compare with wild type.The pathogenicity of ΔΔSyp1/Ede1 double knockout mutant strain was decreased by 92% compared with wild type.The determination of DON toxin content showed that the DON toxin content of ΔSyp1 and ΔΔSyp1/Ede1 mutant strains decreased by 84.8% and91.3% respectively compared with the wild type.The expression of toxin producing genes TRI5,TRI6,and TRI10 in the ΔSyp1 mutant decreased by 39%,51.3%,and 89.3%,respectively,compared with the wild-type.99.8%,99.4%,99.7%.The ΔSyp1 mutant also reduced the endocytosis of the hyphae,and the ΔΔSyp1/Ede1 double knockout mutant inhibited the endocytosis more significantly.In summary,Fg Syp1 plays an important role in the growth,sporulation,pathogenicity,and endocytosis of Fusarium graminearum,while Fg Ede1 and Fg Syp1 have a synergistic effect in sporulation,pathogenicity and endocytosis.