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  • Title: Agrobacterium tumefaciens-mediated transformation as a tool for insertional mutagenesis in the symbiotic ectomycorrhizal fungus Hebeloma cylindrosporum.
    Author: Combier JP, Melayah D, Raffier C, Gay G, Marmeisse R.
    Journal: FEMS Microbiol Lett; 2003 Mar 14; 220(1):141-8. PubMed ID: 12644240.
    Abstract:
    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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