Preliminary Identification Of Zn(?)2Cys6 Transcription Factor Of Valsa Mali

Apple valsa canker,caused by the filamentous fungus Valsa mali(V.mali),was first found in Japan in 1903.It was difficult to prevent and has been a serious threat to apple production across China.No effective control strategy was made because of the complex infection process and little is known of pathogenic molecular mechanism for this disease.Therefore,the discovery of pathogenesis of V.mali is urgent and necessary to the disease control.The Zn(?)2Cys6 binuclear cluster DNA-binding proteins are transcriptional activators exclusive to fungi,which participate in basal metabolism,secondary metabolism,the infection process and so on.This experiment predicted some of the Zn(?)2Cys6transcription factors by analysis based on homologous sequence acquired from genome sequencing data of V.mali.After that,qRT-PCR,genes knockout and complement were conducted to analyze the function of 10 Zn(?)2Cys6 transcription factors Vmtf28?Vmtf36?Vmtf81?Vmtf239?Vmtf315?Vmtf544?Vmtf585?Vmtf898?Vmtf1034?Vmtf1049 during V.mali growth,development and pathogenicity.The main results are as follows:1.The expression of seven of the ten Zn(?)2Cys6 transcription factors in 72 hpi(hours post inoculation)V.mali-apple branch interaction samples was up-regulated compared with72 hpi mycelia samples,and Vmtf36?Vmtf81?Vmtf239?Vmtf1034?Vmtf1049 up-regulated more than two times.2.Knockout cassetes were constructed by Double-joint PCR.Then,using the PEG-mediated transformation,Vmtf28?Vmtf36?Vmtf81?Vmtf239?Vmtf315?Vmtf544?Vmtf585?Vmtf898?Vmtf1034?Vmtf1049 got 7?4?8?1?6?4?1?6?1?3 mutants respectively after PCR.At last,15 single copy mutants were successfully got by Southern blot analysis.3.Phenotype,Na~+and K~+stress and pathogenicity were examined between the deletion mutants and the wild type 03-8.The results showed that,?Vmtf36-59,?Vmtf315-1 and?Vmtf1034-71 grew slowly and had less propagulum amount than 03-8.?Vmtf1034-71 displayed sensitivity to Na~+and ?Vmtf28-19??Vmtf81-11??Vmtf315-1??Vmtf544-70??Vmtf898-30??Vmtf1034-71??Vmtf1049-33 displayed sensitivity to K~+.Compared with the wild type 03-8,the ?Vmtf81-11 exhibited significantly reduced virulence on apple twigs.Furthermore,the phenotype or pathogenicity of complementation strains ?Vmtf36-C,?Vmtf1034-C and ?Vmtf81-C could recover approximately to that of the wild type 03-8,proving that the gene knockout is the reason for the phenotype or pathogenicity defects of the deletion mutants.All these prove that Vmtf36 ? Vmtf315 ? Vmtf1034 are related with growth and development of V.mali.,with Vmtf81 related to pathogenicity.Besides,Vmtf28?Vmtf81?Vmtf315?Vmtf544?Vmtf898?Vmtf1034?Vmtf1049 may be invoved in stress reponse of V.mali.