Function Analysis Of Five Hydrophobins From Fusarium Verticillioide

Hydrophobins are small proteins of about10kDa that produced by filamentous fungi, itwas noted that one main unifying feature was the presence of eight Cys residues, and the eightCys formed four disulfide bonds in the amino acid chain. Hydrophobins could beself-assembled on the hydrophobic surface of the medium and change the surface of thehydrophobic or hydrophilic characteristics.They played an important role in the growth anddevelopment of the fungal hyphae, spores dispersed and pathogens on plant pathogenic,preparatory studies showed that the fungal hydrophobin had the nature of the lectin, lectin hasan important role in the molecular recognition. In order to verifying the agglutination activityand probing into the mechanism of biological recognition between the parasitic fungi O.tenellum and its host F.verticillioide, five Hydrophobin genes of Fusarium verticillioide wereselected as the main research object in this article.The primers were designed on the sequence of GenBank，and three cDNA fragmentswere obtained through RT-PCR. The three genes and expression vector pPIC9K weredigested ligated in vitro to construct eukaryocyte expression plasmid pPIC9K/HYD3,pPIC9K/HYD4and pPIC9K/HYD5, the plasmid pPIC9K/HYD3-eg1which contained thefusion gene that contained cellulase gene eg1from T.aurantiacus var.levisporus and FvHYD3was also constructed. The recombinant vectors were transformed to P.pastoris GS115competent cell, the recombinant P.pastori G-HYD3, G-HYD4, G-HYD5and G-HYD3-eg1were obtained and also induced the engineering yeast strains G-HydI and the G-HydII whichcan express the produts of FvHYD1gene and FvHYD2gene. The purified yeast expressionproducts were named Hydrophobin3, Hydrophobin4, Hydrophobin5, fusion protein RH,Hydrophobin1and Hydrophobin2. The purified products were hydrophobins by theWestern-blot verification.We used purified hydrophobins to verify the agglutination activity by adding them to thered blood cells of rabbit, the spores of F.verticillioide, the spores of F.kyushuense and thespores of O. tenellum, the reseach have shown that all the hydrophobins have the nature of thelectin,but the agglutination activity of five hydrophobins is not the same, the sugar solutionand the metal ions had influnce on the agglutination activity of five hydrophobins.The fusiongene expression product could adsorb in the surface of yeast cell wall and hydrophobicpolyvinyl chloride(PVC), the experiment indicated that hyrophobins had a strong adhesionproperty, possibly due to a lectin-like activity.In order to study initial recognition mechanismbetween the parasitic fungi O. tenellum and its host F.verticillioide, the recombinant plasmid vector of five hydrophobin genes were used for disrupting the genes by PEG-mediatedmethod, after hygromycin B resistance screening, we get the FvHYD5gene deletion mutantsuccessfully, but we didn’t got good resuilt by the interaction with each other.Maize leaves were inocubated with the purified protein Hydrophobin3, Hydrophobin4andHydrophobin5, and leaves was inocubated with dialysis buffer as a control.The result showedthat class Ⅱhydrophobin were able to cause the expression of the maize defense relatedenzymes, especially Hyrophobin4could increase enzyme activity significantly, but the effectcaused by Hydrophobin5is not significant. The completely dialyzed Hyrophobin4wasinjected into corn pith tissue to verify the fuction of Hydrophobin4, results showed thatHyrophobin4could significantly improve the activity of pith tissues defense related enzymes,especially the POD enzyme activity.Through the research, the results showed that five hydrophobins have the nature of thelectin and four hydrophobins can agglutinate the spores of parasitic fungi O. tenellum,so weput forward the new idear that hydrophobin played an important role in the initial molecularrecognition between the parasitic fungi O.tenellum and its host F.verticillioide. Pathogenesisof class II hydrophobin had not been clearly explained, and the research had shown that classII Hyrophobin4could significantly improve the expression and activity of mazi defensiveenzymes, our research provided new evidence and a new way to explain the pathogenesis ofthe class II hydrophobin.