title: Identification of pathogenicity-related genes in the vascular wilt fungus Verticillium dahliae by Agrobacterium tumefaciens-mediated T-DNA insertional mutagenesis.
abstract: Verticillium dahliae is the causal agent of vascular wilt in many economically important crops worldwide. Identification of genes that control pathogenicity or virulence may suggest targets for alternative control methods for this fungus. In this study, Agrobacterium                         tumefaciens-mediated transformation (ATMT) was applied for insertional mutagenesis of V. dahliae conidia. Southern blot analysis indicated that T-DNAs were inserted randomly into the V. dahliae genome and that 69% of the transformants were the result of single copy T-DNA insertion. DNA sequences flanking T-DNA insertion were isolated through inverse PCR (iPCR), and these sequences were aligned to the genome sequence to identify the genomic position of insertion. V. dahliae mutants of particular interest selected based on culture phenotypes included those that had lost the ability to form microsclerotia and subsequently used for virulence assay. Based on the virulence assay of 181 transformants, we identified several mutant strains of V. dahliae that did not cause symptoms on lettuce plants. Among these mutants, T-DNA was inserted in genes encoding an endoglucanase 1 (VdEg-1), a hydroxyl-methyl glutaryl-CoA synthase (VdHMGS), a major facilitator superfamily 1 (VdMFS1), and a glycosylphosphatidylinositol (GPI) mannosyltransferase 3 (VdGPIM3). These results suggest that ATMT can effectively be used to identify genes associated with pathogenicity and other functions in V. dahliae.
authors.affiliation: Department of Plant Pathology, University of California-Davis, CA 93905, USA.
authors.forename: K
authors.initials: K
authors.lastname: Maruthachalam
authors.forename: S J
authors.initials: SJ
authors.lastname: Klosterman
authors.forename: S
authors.initials: S
authors.lastname: Kang
authors.forename: R J
authors.initials: RJ
authors.lastname: Hayes
authors.forename: K V
authors.initials: KV
authors.lastname: Subbarao
mesh_terms: D016960:Agrobacterium tumefaciens / Q000378:metabolism*; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics*; D004271:DNA, Fungal / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D018628:Gene Dosage / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal / Q000235:genetics*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000379:methods*; D010935:Plant Diseases / Q000382:microbiology*; D058526:Plant Vascular Bundle / Q000382:microbiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014170:Transformation, Genetic; D020172:Verticillium / Q000235:genetics* / Q000472:pathogenicity*
chemical_list: D004269:DNA, Bacterial; D004271:DNA, Fungal; D005656:Fungal Proteins; C036483:T-DNA