Agrobacterium tumefaciens-mediated transformation as a tool for insertional mutagenesis in the symbiotic ectomycorrhizal fungus Hebeloma cylindrosporum
Jean-Philippe Combier
Université Lyon 1, UMR CNRS d'Ecologie Microbienne, Bât. A. Lwoff, 43 boulevard du 11 Novembre 1918, 69622 Villeurbanne Cedex, France
Search for more papers by this authorDelphine Melayah
Université Lyon 1, UMR CNRS d'Ecologie Microbienne, Bât. A. Lwoff, 43 boulevard du 11 Novembre 1918, 69622 Villeurbanne Cedex, France
Search for more papers by this authorColette Raffier
Université Lyon 1, UMR CNRS d'Ecologie Microbienne, Bât. A. Lwoff, 43 boulevard du 11 Novembre 1918, 69622 Villeurbanne Cedex, France
Search for more papers by this authorCorresponding Author
Gilles Gay
Université Lyon 1, UMR CNRS d'Ecologie Microbienne, Bât. A. Lwoff, 43 boulevard du 11 Novembre 1918, 69622 Villeurbanne Cedex, France
*Corresponding author. Fax: +33 (4) 72 43 16 43, E-mail address: [email protected]Search for more papers by this authorRoland Marmeisse
Université Lyon 1, UMR CNRS d'Ecologie Microbienne, Bât. A. Lwoff, 43 boulevard du 11 Novembre 1918, 69622 Villeurbanne Cedex, France
Search for more papers by this authorJean-Philippe Combier
Université Lyon 1, UMR CNRS d'Ecologie Microbienne, Bât. A. Lwoff, 43 boulevard du 11 Novembre 1918, 69622 Villeurbanne Cedex, France
Search for more papers by this authorDelphine Melayah
Université Lyon 1, UMR CNRS d'Ecologie Microbienne, Bât. A. Lwoff, 43 boulevard du 11 Novembre 1918, 69622 Villeurbanne Cedex, France
Search for more papers by this authorColette Raffier
Université Lyon 1, UMR CNRS d'Ecologie Microbienne, Bât. A. Lwoff, 43 boulevard du 11 Novembre 1918, 69622 Villeurbanne Cedex, France
Search for more papers by this authorCorresponding Author
Gilles Gay
Université Lyon 1, UMR CNRS d'Ecologie Microbienne, Bât. A. Lwoff, 43 boulevard du 11 Novembre 1918, 69622 Villeurbanne Cedex, France
*Corresponding author. Fax: +33 (4) 72 43 16 43, E-mail address: [email protected]Search for more papers by this authorRoland Marmeisse
Université Lyon 1, UMR CNRS d'Ecologie Microbienne, Bât. A. Lwoff, 43 boulevard du 11 Novembre 1918, 69622 Villeurbanne Cedex, France
Search for more papers by this authorAbstract
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.
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