The Molecular Characterization Of T-DNA Integration Of The Ustilaginoidea Virens Mutant B2510 And Preliminary Study On Pathogenicity Related-gene Uvt3277

Rice false sumt disease,caused by Ustilaginoidea.virens(U.virens),has become one of the most devastating rice grain diseases worldwide.U.virens infects rice spikelets and converts grains into smut balls which results in quantitative and qualitative losses in rice.Green false smut balls with numerous chlamydospores contain mycotoxins(ustilaginoidins and ustiloxins),which are poisonous to humans and animals.Therefore we need to gain more insights into the pathogenic mechanism of U.virens and provide a potential molecular target for disease control.1?The decresed virulence mutant strain B2510 was screened from a T-DNA insertion library of U.virens.This study could provide a method and theoretical basis for researching the molecular pathogenesis of U.virens.We analysed the biological characteristics and T-DNA flanking gene in mutant strain B2510.Compared with wild-type P1,mutant strain B2510 produced few conidiophores and the growth rate was significantly lower on MM medium,but was no obvious different on PSA and TB3 medium which that are nutritionally endowed.Southern blot analysis confirmed double-T-DNA events inserted in mutant strain B2510.HiTAIL-PCR was used to amplify T-DNA flanking sequences and two genes,UVC6TF and UVRASG.AP were cloned.Comparative analysis found that T-DNA were inserted in the promoter region of UVC6TF and downstream 3' end of UVRASGAP,respectively.The flanking U.virens sequences of T-DNA were adjacent in the wild type and with no sequences lost.Semi-quantitative RT-PCR analysis showed that the expression levels of two genes in the mutant strain B2510 were significantly decreased compared with the P1.The two genes may be associated with the pathogenicity and participate in the regulation of pathogenic process of U.virens in rice.To gain more insights in the function of genes,a UVC6TF silencing vector was constructed and introduced into U.virens by ATMT.Two silencing transformants were obtained and the expression of UVC6TF were 50%and 90%inhibited,respectively.The biological characteristics(i.e.,sporulation,growth rate and pathogenicity)of silencing transformants were analysed and found that this gene did not affect normal growth and development of U.virens,but had regulatory role in the process of sporulation.The less conidiospore had decreased effects on pathogenicity of silencing transformants.In addition,T-DNA insertion also disrupted the expression of gene UVRASGAP.To know more about the pathogenic mechanism,a series of experiments like knockouting a single gene or two genes would be done.2,Fungi has special mechanisms for iron transportion and iron transport proteins are involved in intracellular iron ions transporting through membranes.The transport proteins sometimes function as a secondary regulatory that influences gene expression and metabolism.It's important to keep the body Fe homeostasis and production of virulence factors.Uvt3277 is a low-affinity iron transport protein,and may be associated with the pathogenesis of U.virens.In this study,the function of gene Uvt3277 was further verified.Hairpin-RNAi vector was constructed and introduced into U.virens by ATMT.Phenotypes analysis(i.e.,growth,pathogenicity and ferrous iron stress experiment)of silencing transformants were showed that compared with the wild-type strain P1,the growth rate was a little slower than that of P1 without obvious linear relationship between the silencing efficiency and growth rate.We speculate that Uvt3277 regulates the growth and development of the strain with other regulators.Ferrous iron stress experiments indicated that silencing strains could utilize Fe2+ to grow normally under certain Fe2+ concentration.More virulence of silencing transformants were detected than that of P1.The gene Uvt3277 was verified to affect the pathogenic negative regulatory pathways.Meanwhile,yeast two-hybrid system was used to screen interacting proteins of low iron transporter protein.We only got two suspected proteins,ATP/ADP carrier(AAC)protein and SH3 domain-containing protein and needed to verify the two proteins or screen candidate proteins again by yeast two-hybrid system to provide a theoretical basis for the study of pathogenesis of U.virens.