title: Agrobacterium tumefaciens-mediated transformation of the plant pathogenic fungus Rosellinia necatrix.
abstract: Rosellinia necatrix is a soil-borne root pathogen affecting a wide range of commercially important plant species. The mycelium of R. necatrix was transformed to hygromycin B resistance by an Agrobacterium tumefaciens-mediated transformation system using a binary plasmid vector containing the hygromycin B phosphotransferase (hph) gene controlled by the heterologous fungal Aspergillus nidulans P-gpd (glyceraldehyde 3-phosphate dehydrogenase) promoter and the trpC terminator. Co-cultivation of R. necatrix strain W1015 and A. tumefaciens strain AGL-1 at 25 degrees C using the binary vector pAN26-CB1300, which contained the hygromycin B resistance cassette based on pAN26 and pCAMBIA1300, resulted in high frequencies of transformation. The presence of the hph gene in the transformants was detected by PCR, and single-copy integration of the marker gene was demonstrated by Southern blot analysis. This report of an Agrobacterium-mediated transformation method should allow the development of T-DNA tagging as a system f or insertional mutagenesis in R necatrix and provide a simple and reliable method for genetic manipulation.
authors.affiliation: Faculty of Life and Environmental Sciences, Prefectural University of Hiroshima, Nanatsuka-cho 562, Shobara-shi, Hiroshima, 727-0023, Japan.
authors.forename: Sanae
authors.initials: S
authors.lastname: Kano
authors.forename: Takuma
authors.initials: T
authors.lastname: Kurita
authors.forename: Satoko
authors.initials: S
authors.lastname: Kanematsu
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Morinaga
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics* / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001203:Ascomycota / Q000235:genetics* / Q000378:metabolism; D025141:Drug Resistance, Fungal / Q000235:genetics; D006921:Hygromycin B / Q000494:pharmacology; D016254:Mutagenesis, Insertional / Q000379:methods; D010935:Plant Diseases / Q000382:microbiology; D018517:Plant Roots / Q000382:microbiology; D010944:Plants / Q000382:microbiology; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism; D014170:Transformation, Genetic
chemical_list: D000900:Anti-Bacterial Agents; D006921:Hygromycin B