title: Agrobacterium tumefaciens-mediated transformation for investigating pathogenicity genes of the phytopathogenic fungus Colletotrichum sansevieriae.
abstract: Agrobacterium tumefaciens-mediated transformation (AtMT) has become a common technique for DNA transformation of yeast and filamentous fungi. In this study, we first established a protocol of AtMT for the phytopathogenic fungus Colletotrichum sansevieriae. Binary T-DNA vector containing the hygromycin B phosphotransferase gene controlled by the Aspergillus nidulans gpdA promoter and the trpC terminator was constructed with pCAMBIA0380 and used with three different strains LBA4404, GV3101, and GV2260 of A. tumefaciens. Transformants were most effectively obtained when GV2260 and C. sansevieriae Sa-1-2 were co-cultivated; there were about 320 transformants per 10(6) spores. When 1,048 transformants were inoculated on Sansevieria trifasciata, three transformants were found to have completely lost their pathogenicity and two transformants displayed reduced pathogenicity. All of the five transformants had a single copy of T-DNA in their genomes. The three pathogenicity-deficient transformants were subjected to thermal asymmetric interlaced polymerase chain reaction and the reaction allowed us to amplify the sequences flanking the left and/or right borders. The flanking sequences of the two transformants, M154 and M875, showed no homology to any sequences in databases, but the sequences of M678 contained motifs of alpha-1,3-glucan synthase, suggesting that the gene might contribute to the pathogenicity of C. sansevieriae. This study describes a useful method for investigating pathogenicity genes in C. sansevieriae.
authors.affiliation: Faculty of Agriculture, Kagoshima University, Japan. masa@agri.kagoshima-u.ac.jp
authors.forename: Masayuki
authors.initials: M
authors.lastname: Nakamura
authors.forename: Hideto
authors.initials: H
authors.lastname: Kuwahara
authors.forename: Keisuke
authors.initials: K
authors.lastname: Onoyama
authors.forename: Hisashi
authors.initials: H
authors.lastname: Iwai
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics* / Q000254:growth & development*; D000935:Antifungal Agents / Q000378:metabolism; D020231:Colletotrichum / Q000235:genetics* / Q000472:pathogenicity*; D004269:DNA, Bacterial / Q000235:genetics; D005800:Genes, Fungal; D005822:Genetic Vectors; D005827:Genetics, Microbial / Q000379:methods; D006921:Hygromycin B / Q000378:metabolism; D012641:Selection, Genetic; D014170:Transformation, Genetic; D014774:Virulence; D037521:Virulence Factors / Q000235:genetics* / Q000378:metabolism*
chemical_list: D000935:Antifungal Agents; D004269:DNA, Bacterial; C036483:T-DNA; D037521:Virulence Factors; D006921:Hygromycin B