Functional Characterization Of The Hypoxia-induced Dehydrogenase In Fusarium Graminearum

Fusarium head blight（FHB）severely harms the production of cereal crops.Fusarium graminearum is the main pathogen of Fusarium head blight.The infection of F.graminearum will reduce the yield and quality of crops,and producing monotoxins such as deoxynivalenol（DON）,which is seriously harmful to health.The hypoxia-induced dehydrogenase（Hor A）is involved in the biosynthesis of coenzyme Q,which mediates the activity of the electron transport chain.As a fungal specific protein,the hypoxia-induced dehydrogenase is involved in maintaining the redox homeostasis of cells and plays an important role in the function of mitochondrial.But it is poorly known about Hor A in plant pathogenic fungi.To this end,we studied the function of the hypoxia-induced dehydrogenase in F.graminearum.In this study,one Fg Hor A gene（FGSG＿06738）in F.graminearum was retrieved by Blastp with the Aspergillus fumigatus Hor A（AFUA＿4G09810）as a query.SMART-PFAM analysis revealed that Fg Hor A consists of a typical short-chain dehydrogenases/reductases（SDR）family（GGXGXXG,where X is any amino acid）.A phylogenetic analysis of fungal Hor A displayed that Fg Hor A is most closely to Hor A from Fusarium verticillioides and Fusarium oxysporum.Knock-out fragments were obtained using split-marker technology,and threeΔHor A mutants verified by PCR and southern blot were finally obtained using protoplast transformation methods.In addition,the Fg Hor A reverting strainsΔHor A-C and the localized strains Fg Hor A-GFP were constructed.Then,we further investigated the related phenotypic changes of theΔHor A mutants.Analysis of the related phenotype of theΔHor A mutants revealed that the colony growth rates of theΔHor A mutants were significantly reduced compared to the wild type.Although the morphology of conidia did not change significantly,the formation rates and number of conidia of theΔHor A mutants slowed down and the germination rates were significantly delayed,indicating that Fg Hor A had delayed mycelial growth and conidial germination.At the same time,the sexual reproduction of theΔHor A mutants was severely affected,and it failed to produce normal perithecia and ascospores consistent with the wild type.As a result of coleoptile inoculation,it was found that theΔHor A mutants only occurred at the inoculation site and could not spread to the surroundings,and the pathogenicity was significantly reduced.Correspondingly,the DON content of the mutants also decreased significantly.Through quantitative PCR analysis,it was found that the expression levels of two key genes related to toxin production,TRI5 and TRI6,were also significantly down-regulated.Stress experiments showed that theΔHor A mutants showed significantly improved resistance to osmotic stresses such as Na Cl,KCl and sorbitol,and showed reduced resistance to oxygen stress such as 1 m M H₂O₂ and 15μM menadione.In addition,the resistance to CR and SDS did not change significantly.It is indicated that Fg Hor A participates in osmotic and oxygen stresses of Fusarium graminearum,but has no obvious regulation effect on the integrity of cell membrane and cell wall.Colocalization experiments were performed using dual markers of Fg Hor A-GFP and the mitochondrial indicator Mito Tracker.The results indicated that Fg Hor A was localized to the mitochondria in F.graminearum.In addition,the study also demonstrated that Fg Hor A affected the biosynthesis of endogenous ROS in F.graminearum.Correspondingly,the expression levels of four genes related to ROS synthesis were also significantly down-regulated,indicating that Fg Hor A was involved in the ROS synthesis process.In summary,this study proves that Fg Hor A plays an important role in the development of mycelia,sexual and asexual reproduction,pathogenicity,toxin production and the biosynthesis of ROS.