Open AccessAgrobacterium tumefaciens ‐mediated transformation as a tool for insertional mutagenesis in the symbiotic ectomycorrhizal fungus Hebeloma cylindrosporum
Author(s) -
Combier JeanPhilippe,
Melayah Delphine,
Raffier Colette,
Gay Gilles,
Marmeisse Roland
Publication year - 2003
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1016/s0378-1097(03)00089-2
Subject(s) - insertional mutagenesis , agrobacterium tumefaciens , acetosyringone , biology , transformation (genetics) , agrobacterium , plasmid , mutagenesis , southern blot , transfer dna , microbiology and biotechnology , genetics , gene , genome , mutation
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 .