Agrobacterium-mediated transformation of Botrytis cinerea, simple purification of monokaryotic transformants and rapid conidia-based identification of the transfer-DNA host genomic DNA flanking sequences.
The Agrobacterium tumefaciens-mediated transfer of foreign DNA to the phytopathogenic fungus Botrytis cinerea was investigated. Fifteen stable transformants per 10(6) conidia were consistently produced. Monokaryons were purified in a single step and their molecular analysis demonstrated the random integration of predominantly single or tandem copies of the foreign DNA into their genome. Thermal asymmetric interlaced PCR performed directly on conidia led to the rapid identification of the genomic DNA sequences that flanked the integration sites of the transfer-DNA. Transcriptional fusions of green fluorescent protein and beta-glucuronidase-encoding genes to the promoter of the secreted proteolytic enzyme ACP1 were realised to validate the system. We provide herein observations of B. cinerea hyphae producing green fluorescent protein or beta-glucuronidase under growth conditions similar to those known to induce transcription of the acp1 gene.
Rolland
Stéphane
S
Laboratoire de Biologie Cellulaire Fongique, UMR 5122 CNRS INSA UCB, Université Claude Bernard Lyon 1, 10 Rue Dubois, 69622 Villeurbanne cedex, France.
Jobic
Cécile
C
Fèvre
Michel
M
Bruel
Christophe
C
D016960:Agrobacterium tumefaciens / Q000235:genetics*; D001203:Ascomycota / Q000235:genetics*; D015139:Blotting, Southern; D015153:Blotting, Western; D020171:Botrytis / Q000235:genetics*; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D005966:Glucuronidase; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D014170:Transformation, Genetic / Q000235:genetics*
D017931:DNA Primers; D004269:DNA, Bacterial; D008164:Luminescent Proteins; C036483:T-DNA; D049452:Green Fluorescent Proteins; D005966:Glucuronidase