Agrobacterium tumefaciens-mediated transformation as a tool for insertional mutagenesis in the symbiotic ectomycorrhizal fungus Hebeloma cylindrosporum.
We transformed haploid mycelium of Hebeloma cylindrosporum via Agrobacterium tumefaciens and optimised the procedure to develop a new tool for insertional mutagenesis in this fungus. Southern blot analysis of 83 randomly selected transformants showed that they all contained plasmid inserts. Each of them showed a unique hybridisation pattern, suggesting that integration was random in the fungal genome. Sixty percent of transformants obtained in the presence of bacteria pre-treated with acetosyringone integrated a single transferred DNA copy. Thermal asymmetric interlaced polymerase chain reaction allowed us to recover the left border and the right border junctions in 85% and 15% of transformants analysed, respectively. Results show that A. tumefaciens-mediated transformation may be a powerful tool for insertional mutagenesis in H. cylindrosporum.
Combier
Jean-Philippe
JP
Université Lyon 1, UMR CNRS d'Ecologie Microbienne, Bât. A. Lwoff, 43 boulevard du 11 Novembre 1918, 69622 Villeurbanne Cedex, France.
Melayah
Delphine
D
Raffier
Colette
C
Gay
Gilles
G
Marmeisse
Roland
R
D000098:Acetophenones / Q000494:pharmacology; D000363:Agaricales / Q000235:genetics* / Q000502:physiology; D016960:Agrobacterium tumefaciens / Q000235:genetics*; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000379:methods*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D012641:Selection, Genetic; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D013559:Symbiosis; D014170:Transformation, Genetic
D000098:Acetophenones; D004269:DNA, Bacterial; D004271:DNA, Fungal; C036483:T-DNA; C051667:acetosyringone; D017853:Phosphotransferases (Alcohol Group Acceptor); C063242:hygromycin-B kinase