Functional Characterization Of Rabring7 Genes In Fusarium Graminearum

Fusarium head blight?FHB?is a common disease in the world.It can cause wheat yield reduction and quality decline.The main pathogen of FHB is Fusarium graminearum,and the deoxynivalenol?DON?produced by it has a great threat to the health of humans and animals.The synthesis of DON toxin is influenced by physical,chemical and biological macromolecules.Rab7 has an effect on the synthesis of DON toxin,and the function of Rab7requires the action of upstream regulators and downstream effectors.The function of Rab7needs the action of upstream regulator and downstream effector.In mammals,Rabring7 is an effector of Rab7,but the function of Rabring7 is unknown in F.graminearum.In this study,the interaction between Rabring7 and FgRab7 was analyzed.The function of Rabring7 was preliminarily characterised.The main results are as follows:By homologous alignment,we found two genes homologous to Rabring7 in the F.graminearum database,like FGSG-00316 and FGSG-01047.Bioinformatics analysis showed that FGSG-00316 protein contains RING-H2 domain,subcellular localization in the plasma membrane.FGSG-01047 protein contains Ring U-box domain,subcellular localization in the nucleus.Yeast two-hybrid technique was used to study the interaction between Rabring7 and FgRab7.The results showed that there was no interaction between them.To clarify the functions of Rabring7,we performed gene knockout,complementation and overexpression of FGSG-00316 and FGSG-01047 by homologous recombination,and phenotypic of the mutants was analyzed.The results showed that the deletion and overexpression of FGSG-00316 and FGSG-01047 genes had no effect on the growth of mycelia,the morphology of mycelia and spores was normal,and the spore yield was not significantly different from that of the wild type.However,the DON toxin synthesis of the knockout mutant was significantly reduced,indicating that Rabring7 has a regulatory effect on the biosynthesis of DON toxin.Stress analysis showed that the growth rate of wild-type and mutant under stress was slower than that without stress.The three knockout mutants grew more slowly on the medium containing SDS than the wild type,growth in the medium containing Congo red was faster than the wild type.On the CM medium containing 1.0 M NaCl and 0.05%H₂O₂,the growth rate of the knockout mutant was not significantly different from that of the wild type,suggesting the regulation of Rabring7 in cell wall and cell membrane sensitivity.Pathogenicity analysis showed that the double knockout mutants of the two genes were less pathogenic and the over-expressed mutants of the two genes were more pathogenic,speculating that Rabring7 gene was associated with pathogenicity.To investigate the relationship between Rabring7 and FgRab7,we further analyzed the expression of Rabring7 and FgRab7 in each other's deletion mutants.Fluorescence quantitative analysis showed that the relative expression of FGSG-00316 and FGSG-01047 genes was down-regulated after FgRab7 gene deletion.After the deletion of Rabring7 gene,the relative expression of FgRab7 gene was down-regulated too,indicating a certain correlation between Rabring7 gene and FgRab7 gene.The results showed that Rabring7 gene had regulatory effects on the synthesis of DON toxin,stress sensitivity and pathogenicity of F.graminearum,which laid a foundation for revealing the pathogenic mechanism and the molecular regulatory mechanism of DON toxin synthesis in F.graminearum,and provided a theoretical basis for the prevention and control of wheat scab.