title: Nonmycorrhizal (myc-) mutants of Hebeloma cylindrosporum obtained through insertional mutagenesis.
abstract: Polyethylene glycol-mediated transformation of protoplasts was used as a method for insertional mutagenesis to obtain mutants of the ectomycorrhizal fungus Hebeloma cylindrosporum impaired in symbiotic ability. Following restriction enzyme-mediated integration or conventional plasmid insertion, a library of 1,725 hygromycin-resistant monokaryotic transformants was generated and screened for the symbiotic defect, using Pinus pinaster seedlings as host plants. A total of 51 transformants displaying a dramatically reduced mycorrhizal ability were identified. Among them, 29 were nonmycorrhizal (myc-), but only 10 of them had integrated one or several copies of the transforming plasmid in their genome. Light and scanning electron microscopy observations of pine roots inoculated with myc- mutants suggested that we selected mutants blocked at early stages of interaction between partners or at the stage of Hartig net formation. Myc- mutants with plasmid insertions were crossed with a compatible wild-type monokaryon and allowed to fruit. Monokaryotic progenies were obtained in three independent crosses and were analyzed for symbiotic activity and plasmid insertion. In all three progenies, a 1:1 myc-:myc+ segregation ratio was observed, suggesting that each myc- phenotype resulted from a single gene mutation. However, for none of the three mutants, the myc- phenotype segregated with any of the plasmid insertions. Our results support the idea that master genes, the products of which are essential for symbiosis establishment, do exist in ectomycorrhizal fungi.
authors.affiliation: Université Lyon 1, UMR CNRS 5557 d'Ecologie Microbienne Bât. A. Lwoff, 43 boulevard du 11 Novembre 1918, 69622 Villeurbanne Cedex, France.
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Combier
authors.forename: Delphine
authors.initials: D
authors.lastname: Melayah
authors.forename: Colette
authors.initials: C
authors.lastname: Raffier
authors.forename: Régis
authors.initials: R
authors.lastname: Pépin
authors.forename: Roland
authors.initials: R
authors.lastname: Marmeisse
authors.forename: Gilles
authors.initials: G
authors.lastname: Gay
mesh_terms: D001487:Basidiomycota / Q000187:drug effects / Q000235:genetics*; D003433:Crosses, Genetic; D008855:Microscopy, Electron, Scanning; D016254:Mutagenesis, Insertional; D028223:Pinus / Q000235:genetics / Q000382:microbiology* / Q000648:ultrastructure; D010935:Plant Diseases / Q000382:microbiology; D010957:Plasmids / Q000235:genetics; D011092:Polyethylene Glycols / Q000494:pharmacology; D014170:Transformation, Genetic
chemical_list: D011092:Polyethylene Glycols