Functional Analysis Of FpHam-2/3/4 Complex In Fusarium Pseudograminearum

Wheat crown rot caused by Fusarium pseudograminearum is a serious soil-borne disease that occurs in wheat growing areas around the world.Especially in the Huanghuai wheat area of China,wheat crown rot is rapidly spreading and increasing year by year.The distribution,genetic diversity and pathogenicity differentiation of F.pseudograminearum were systematically analyzed in previous studies,but the molecular mechanism of its pathogenicity was widely unknown.This study mainly analyzed the function of FpHam-2/3/4 complex of F.pseudograminearum,and basically clarified that FpHam-2/3/4 complex has an effect on the morphology,growth rate,sporulation,hyphae fusion and pathogenicity of F.pseudograminearum,which results might provide scientific foundation for development of disease control strategies.Through research,the following main results are obtained:1、A mutant F28 with slower mycelial growth and reduced pathogenicity was selected from the T-DNA random insertion mutant library of F.pseudograminearum.The hiTAIL-PCR technique was used to amplify the flanking sequences of the T-DNA,and the T-DNA insertion region was located in the coding region of the mycelial fusion homologous gene FpHam-2（FPSE₁1215）.In N.crassa,Ham-2 interacts with Ham-3 and Ham-4 to form the core element of the STRIPAK complex.The Blastp method was used to identify the elements of the STRIPAK complex in F.pseudograminearum.The yeast two-hybrid analysis revealed that FpHam-2 interacts with FpHam-3 and FpHam-4,indicating the FpHam-2/3/4 complex is very conserved in fungi.2、In order to analyze the biological function of FpHam-2/3/4 complex in F.pseudograminearum,FpHam-2,FpHam-3 and FpHam-4 were knocked out in F.pseudograminearum by PEG-mediated protoplast transformation method.After hygromycin resistance screening and four pairs of PCR primer detection,three FpHam-2 gene deletion mutants,three FpHam-3 gene deletion mutants and three FpHam-4 gene deletion mutants.By constructing the fragments with the target gene and its promoter region on the pKNTG vector,using protoplast transformation,complementary strains 2-cp,3-cp and 4-cp were obtained.The results of phenotypic determination showed that compared with wild-type and Complementary strains of F.pseudograminearum,the phenotypes of FpHam-2,FpHam-3 and FpHam-4 gene deletion mutants were similar,and the hyphae fusion was blocked and the hyphae grew.The speed slows down,the conidia cannot be produced,and the pathogenicity to wheat coleoptile and barley leaves is significantly reduced.3、qRT-PCR analysis found that FpHam-2,FpHam-3 and FpHam-4 have complex regulatory pathways for the expression of STRIPAK complex elements.The above results indicate that the regulatory mechanism of FpHam-2/3/4 complex of F.pseudograminearum on STRIPAK complex is complicated.