title: The dominant Hc.Sdh (R) carboxin-resistance gene of the ectomycorrhizal fungus Hebeloma cylindrosporum as a selectable marker for transformation.
abstract: In an attempt to get a marker gene suitable for genetical transformation of the ectomycorrhizal fungus Hebeloma cylindrosporum, the gene Hc.Sdh (R) that confers carboxin-resistance was isolated from a UV mutant of this fungus. It encodes a mutant allele of the Fe-S subunit of the succinate dehydrogenase gene that carries a single amino acid substitution known to confer carboxin-resistance. This gene was successfully used as the selective marker to transform, via Agrobacterium tumefaciens, monokaryotic and dikaryotic strains of H. cylindrosporum. We also successfully transformed hygromycin-resistant insertional mutants. Transformation yielded mitotically stable carboxin-resistant mycelia. This procedure produced transformants, the growth of which was not affected by 2 microg l(-1) carboxin, whereas wild-type strains were unable to grow in the presence of 0.1 microg l(-1) of this fungicide. This makes the carboxin-resistance cassette much more discriminating than the hygromycin-resistance one. PCR amplification and Southern blot hybridisation indicated that more than 90% of the tested carboxin-resistant mycelia contained the Hc.Sdh (R) cassette, usually as a single copy. The AGL-1 strain of A. tumefaciens was a much less efficient donor than LBA 1126; the former yielded ca. 0-30% transformation frequency, depending on fungal strain and resistance cassette used, whereas the latter yielded ca. 60-95%.
authors.affiliation: Université de Lyon, France.
authors.forename: Chrisse
authors.initials: C
authors.lastname: Ngari
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Combier
authors.forename: Jeanne
authors.initials: J
authors.lastname: Doré
authors.forename: Roland
authors.initials: R
authors.lastname: Marmeisse
authors.forename: Gilles
authors.initials: G
authors.lastname: Gay
authors.forename: Delphine
authors.initials: D
authors.lastname: Melayah
mesh_terms: D002261:Carboxin; D055364:Hebeloma / Q000187:drug effects / Q000235:genetics*; D009154:Mutation; D013385:Succinate Dehydrogenase / Q000235:genetics; D014170:Transformation, Genetic
chemical_list: D002261:Carboxin; D013385:Succinate Dehydrogenase