title: Development of an efficient gene targeting system in Colletotrichum higginsianum using a non-homologous end-joining mutant and Agrobacterium tumefaciens-mediated gene transfer.
abstract: The hemibiotrophic ascomycete Colletotrichum higginsianum is the casual agent of anthracnose disease of cruciferous plants. High efficiency transformation by Agrobacterium tumefaciens-mediated gene transfer has been established for this fungus. However, targeted gene mutagenesis through homologous recombination rarely occurs in C. higginsianum. We have identified and disrupted the C. higginsianum homologue of the human Ku70 gene, ChKU70, which encodes a protein that plays a role in non-homologous end-joining for repair of DNA breaks. chku70 mutants showed a dramatic increase in the frequency of integration of introduced exogenous DNA fragments by homologous recombination without any detectable phenotypic defects. This result demonstrates that the chku70 mutant is an efficient recipient for targeted gene mutagenesis in C. higginsianum. We have also developed a novel approach [named direct repeat recombination-mediated gene targeting (DRGT)] for targeted gene disruption through Agrobacterium tumefaciens-mediated gene transfer. DRGT utilizes homologous recombination between repeated sequences on the T-DNA flanking a partial fragment of the target gene. Our results suggest that DRGT in the chku70 mutant background could be a useful tool for rapid isolation of targeted gene disruptants in C. higginsianum.
authors.affiliation: Laboratory of Plant Pathology, Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Kyoto, 606-8522, Japan.
authors.forename: Takuma
authors.initials: T
authors.lastname: Ushimaru
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Terada
authors.forename: Kie
authors.initials: K
authors.lastname: Tsuboi
authors.forename: Yuki
authors.initials: Y
authors.lastname: Kogou
authors.forename: Ayumu
authors.initials: A
authors.lastname: Sakaguchi
authors.forename: Gento
authors.initials: G
authors.lastname: Tsuji
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Kubo
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics*; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D020231:Colletotrichum / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018014:Gene Transfer Techniques; D009154:Mutation
chemical_list: D004268:DNA-Binding Proteins