Functional Characterization Of The Dynamin-like GTPase FgSey1 In Fusarium Graminearum

Fusarium head blight(FHB)is getting worse and worse with climate and farming systems changed in China.In the past decade,approximately 30%wheat planting area,84 million mu,is disease area.FHB is seriously affecting the quality and yield of wheat and produces vomiting toxins and secondary metabolites which is harmful to humans and animals.Fusarium graminearum is the main pathogen causing FHB in wheat and one of the main model fungi for studying plant pathogenic fungi.Deoxynivalenol produced by it is its main pathogenic factorIn eukaryotic cells,the endoplasmic reticulum(Endoplasmic reticulum,ER)is in a highly dynamic process.It is a closed reticular duct system composed of the inner membrane and has a high degree of polymorphism.The morphology,structure and stability of the endoplasmic reticulum play an important role in the normal function of its biological function.Studies have shown that the endoplasmic reticulum is an important organelle in eukaryotes and is responsible for the production of many metabolites,including DON toxins and lipid droplets.Toxin synthesis of F.graminearum needs to first form a type of organelle called toxisome on the endoplasmic reticulum.This organelle is thought to be formed by endoplasmic reticulum remodeling.The normal formation of lipid droplets also affects the formation of toxinsSeylp and its homologous proteins(called Atlastinl/2/3 in animals and Rhd3 in Arabidopsis thaliana)belong to the class of dynamin-like GTPases,and play a important role in homologous membranes fusion and structure shaping of endoplasmic reticulum.In saccharomyces cerevisiae,Seyl is involved in endoplasmic reticulum homologous membrane fusion and nucleus fusion during yeast mating.In mammals,Atlastins are involved in homologous membrane fusion of endoplasmic reticulum and tubular endoplasmic reticulum formation.In Arabidopsis,the knockout of Rhd3(Root Hair Defective 3)causes the plant to produce short,wavy Root hairs.Seyl is essential for the normal physiological function of the endoplasmic reticulum.However,research on the function of Seyl in pathogenic fungi is still blankIn this study,a dynamin-like GTPase FgSeyl was identified from the genome of F.graminearum PH-1 species.SMART domain analysis showed that the FgSey 1 has five domains,GTPase domain,Helix bundles domain,Transmembrane segments,and Cytosolic tail domains,which contains two transmembrane domains.At the same time,consistent with the eukaryote Seyl/Atlastin,FgSeyl is aslo localized on the endoplasmic reticulum in F.graminearum.To further explore the biological function of FgSey 1,a knockout fragment was constructed using Split-marker PCR technology,and FgSEYl gene knockout mutants was obtained by PEG-mediated protoplast transformation.Based on the knockout mutants,a AFgseyl/FgSEYl complement strain was further constructed.The main results obtained are as follows:(1)Compared with the wild-type PH-1 strain,the my celia of the ?Fgseyl knockout mutant became dense,the colony size was about 25.5%of the wild-type PH-1 colony size,the height of the aerial my celia became lower,the conidium production decreased 30%,and the pigment production significantly decreased.The conidium morphology of ?Fgseyl mutant is abnormal,most of the conidium diaphragms are 3,which is significantly less than the wild-type PH-1.The pathogenicity of AFgseyl mutant decreased by approximately 95%compared to the wild-type ph-1 strain,and an extremely significant decrease in the production of the mutant DON toxin showed the same trend as the pathogenicity defect.(3)Compared with the wild-type PH-1 strain,the TRI gene expression of ?Fgseyl mutant was significantly down-regulated,and the expressions of TRI1,TRI4,TRI5,TRI6 and TRI10 were decreased by about 72%,49%,51%,91%and 50%,respectively.(4)Compared with wild-type PH-1,AFgseyl mutant is more sensitive to salt stress and oxidative stress,but its tolerance to cell wall integrity stress is improved,and it has obvious defects in the synthesis of toxisome and lipid dropletsIn order to explore the function of FgSey1 domains,functional domains were mutated one by one.The results showed that,after all the domains were deleted,they were consistent with the physiological phenotypes of the ?Fgseyl mutant;the GTPase domain had amino acid mutations at positions 63 and 87,the mutant had the same phenotype as the ?Fgseyl mutant;the 644th amino acid mutation in the Helix bundles domain did not have any effect on the physiological process of the strainIn summary,these results indicate that F.graminearum FgSeyl is essential for mycelial development,asexual reproduction,stress response,pigment synthesis,toxisome formation,and lipid droplet synthesis.