Application Of PEG-mediated Genetic Transformation In Functional Analysis Of Three Types Transcription Factors Of Fusarium Pseudograminearum

As one of the most important food crops in China,wheat production safety is very important.Fusarium crown rot caused by Fusarium pseudograminearum is a worldwide soil-borne disease.Especially in the Huanghuai wheat region of China,it is rapidly spreading and increasing year by year.Moreover,the lack of effective disease-resistant wheat varieties in production leads to difficulties in disease control.The distribution,genetic diversity and pathogenicity differentiation of F.pseudograminearum were systematically analyzed in previous studies,but the molecular mechanism of its growth,development and pathogenicity was widely unknown.Genetic transformation is a main strategie to analyze the molecular regulation mechanism of fungal pathogenicity.Agrobacterium-mediated and PEGmediated genetic transformation are very common genetic transformation methods.In this study,we established and improved the PEG-mediated genetic transformation for gene knockout and complementin F.pseudograminearum,and analyzed the functions of some transcription factors in the growth,conidiation and pathogenesis of F.pseudograminearum.The main results are shown as follows:Biological functions of APSES transcription factors in F.pseudograminearum were analyzed using the PEG-mediated genetic transformation.Four APSES candidates were identified in F.pseudograminearum based on the known APSES transcription factors in other species,which all contained the conserved DNA binding domain HTH.By the phylogenetic tree,FpAPSES1,FpAPSESl and FpAPSES4 were divided into A group,while FpAPSES3 belonged to the C group.Gene expression analysis revealed that FpStuA was highly expressed and all FpAPSESs were up-regulated at infection stages,which suggested that FpAPSESs might play important roles in infection.We obtained FpStuA,FpAPSESl and FpAPSES4 for further analysis.The deletion mutants were obtained by polyethylene glycol(PEG)-mediated protoplast stable transformation.According to the analysis results,compared with the wild-type strain,△fpstua,△fpapses1 and △fpapses4 mutants grew relatively slowly and had less aerial hyphae,and and conidia production was reduced.Furthermore,conidia of △fpstua,△fpapsesl and△fpapses4 mutants were smaller and less separation.Pathogenicity assays showed that all △fpstua,△fpapsesl and △fpapses4 mutants were dramatically decreased in virulence on barley leaves,wheat coleoptiles and wheat roots,and deoxynivalenol production was also significantly reduced in all these mutants.Taken together,these results indicated that FpStuA,FpAPSES1 and FpAPSES4 were important for growth,conidiation and virulence in F.pseudograminearum.Phenotypes of these mutants obtained through PEG-mediated genetic transformation were stable.However,no conplementary transformant of FpAPSESs was obtained by the same genetic transformation methods.Biological functions of C2H2 zinc finger transcription factor FpCzf14 in F.pseudograminearum were analyzed using the PEG-mediated genetic transformation.C2H2 zinc finger transcription factor is abundant and plays a wide regulatory role in fungal growth and conidiation.In the present study,we cloned and characterized a C2H2 zinc finger transcription factor gene,FpCzfl 4,in F.pseudograminearum.The transcript level of FpCzf14 increased to a maximum in conidia,suggesting a potential role of this gene in conidiation of F.pseudograminearum.The FpCzf14 deletion mutants and the FpCzfl 4 gene complement strain were obtained by PEG-mediated genetic transformation method.FpCzf14 localized in nucleus.According to the phenotypic analysis results,compared with the wild-type strain,△fpczf14 mutants grew relatively slowly and can’t produce conidia.Pathogenicity assays showed that △fpczzf14 mutants were dramatically decreased in virulence on barley leaves,wheat coleoptiles and wheat roots.The △fpczfl4 mutant produced very few invasive hyphae inside wheat epidermis.Additionally,results of qRT-PCR showed that FpCzf14 regulated expression of several conidiation-related genes in F.pseudograminearum,suggesting that FpCzf14 might contribute to conidiation.Biological functions of bZIP transcription factor Fpkapc in F.pseudograminearum were analyzed using the PEG-mediated genetic transformation.The basic leucine zipper(bZIP)transcription factors family are widely distributed in eukaryotes,that involved in regulating various biological processes such as growth,development and stress resistance.In the present study,we characterized a bZIP transcription factor gene Fpkapc in F.pseudograminearum.The Fpkapc deletion mutants and the Fpkapc gene complement strain were obtained by PEG-mediated genetic transformation method.Fpkapc localized in nucleus.According to the phenotypic analysis results,compared with the wild-type strain,△fpkapc mutants grew relatively slowly,and conidia production of △fpkapc was reduced and the conidiawere shorter.However,conidia germination of △fpkapc was faster.Pathogenicity assays showed that △fpkapc mutants were decreased in virulence on barley leaves and wheat roots,and less invasive hyphae was observed in wheat epidermis cells.Taken together,these results indicated that Fpkapc played roles in growth,conidiation and virulence of F.pseudograminearum.In this study,PEG-mediated genetic transformation was used to analyze the roles of some transcription factors in the growth,development and pathogenicity of F.pseudograminearum,and the results might provide technical and theoretical support for understanding the pathogenicity mechanism in F.pseudograminearum.