Agrobacterium-mediated transformation of Sclerotinia sclerotiorum.
Ascospores from the phytopathogenic fungus Sclerotinia sclerotiorum were transformed to hygromycin B resistance by co-cultivation with Agrobacterium tumefaciens. Transformed spores germinated and grew on PDA supplemented with 100 ug/ml hygromycin B. The presence of mitotically stable hph gene integration at random sites in the genome was confirmed by PCR and Southern blot analysis. A transformation frequency of 8 x 10(-5) was achieved in five separate experiments. This study is the first report of success co-cultivating A. tumefaciens with S. sclerotiorum. This report of a reproducible Agrobacterium-mediated transformation method should allow the development of T-DNA tagging as a system for insertional mutagenesis in S. sclerotiorum and provide a simple and reliable method for genetic manipulation.
Weld
Richard J
RJ
National Centre for Advanced Bio-Protection Technologies, PO Box 84, Lincoln University, Canterbury, New Zealand. weldr@lincoln.ac.nz
Eady
Colin C
CC
Ridgway
Hayley J
HJ
D016960:Agrobacterium tumefaciens / Q000235:genetics*; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001203:Ascomycota / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D015139:Blotting, Southern; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D004351:Drug Resistance; D016681:Genome, Fungal / Q000235:genetics / Q000502:physiology; D006921:Hygromycin B / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D014170:Transformation, Genetic / Q000235:genetics / Q000502:physiology*
D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; C036483:T-DNA; D006921:Hygromycin B