title: Agrobacterium tumefaciens-mediated transformation in Colletotrichum falcatum and C. acutatum.
abstract: Agrobacterium tumefaciens-mediated transformation (ATMT) is becoming an effective system as an insertional mutagenesis tool in filamentous fungi. We developed and optimized ATMT for two Colletotrichum species, C. falcatum and C. acutatum, which are the causal agents of sugarcane red rot and pepper anthracnose, respectively. A. tumefaciens strain SK1044, carrying a hygromycin phosphotransferase gene (hph) and a green fluorescent protein (GFP) gene, was used to transform the conidia of these two Colletotrichum species. Transformation efficiency was correlated with cocultivation time and bacterial cell concentration and was higher in C. falcatum than in C. acutatum. Southern blot analysis indicated that about 65% of the transformants had a single copy of the T-DNA in both C. falcatum and C. acutatum and that T-DNA integrated randomly in both fungal genomes. T-DNA insertions were identified in transformants through thermal asymmetrical interlaced PCR (TAIL-PCR) followed by sequencing. Our results suggested that ATMT can be used as a molecular tool to identify and characterize pathogenicity-related genes in these two economically important Colletotrichum species.
authors.affiliation: Department of Agricultural Biotechnology, SeoulNational University, Seoul 151-921, Korea.
authors.forename: Karunakaran
authors.initials: K
authors.lastname: Maruthachalam
authors.forename: Vijayan
authors.initials: V
authors.lastname: Nair
authors.forename: Hee-Sool
authors.initials: HS
authors.lastname: Rho
authors.forename: Jaehyuk
authors.initials: J
authors.lastname: Choi
authors.forename: Soonok
authors.initials: S
authors.lastname: Kim
authors.forename: Yong-Hwan
authors.initials: YH
authors.lastname: Lee
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics*; D001483:Base Sequence; D015825:Chromosomes, Fungal / Q000737:chemistry / Q000235:genetics; D020231:Colletotrichum / Q000187:drug effects / Q000235:genetics*; D018628:Gene Dosage; D005822:Genetic Vectors / Q000235:genetics; D006921:Hygromycin B / Q000494:pharmacology; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000379:methods*; D016415:Sequence Alignment; D014170:Transformation, Genetic
chemical_list: D006921:Hygromycin B