Nonmycorrhizal (Myc¯) Mutants of Hebeloma cylindrosporum Obtained Through Insertional Mutagenesis
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 li...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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The American Phytopathological Society
2004-09-01
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| Series: | Molecular Plant-Microbe Interactions |
| Online Access: | https://apsjournals.apsnet.org/doi/10.1094/MPMI.2004.17.9.1029 |
| _version_ | 1819085337443434496 |
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| author | Jean-Philippe Combier Delphine Melayah Colette Raffier Régis Pépin Roland Marmeisse Gilles Gay |
| author_facet | Jean-Philippe Combier Delphine Melayah Colette Raffier Régis Pépin Roland Marmeisse Gilles Gay |
| author_sort | Jean-Philippe Combier |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-21T21:02:45Z |
| format | Article |
| id | doaj.art-1d55e943199a4ea692a7f89a77881728 |
| institution | Directory Open Access Journal |
| issn | 0894-0282 1943-7706 |
| language | English |
| last_indexed | 2024-12-21T21:02:45Z |
| publishDate | 2004-09-01 |
| publisher | The American Phytopathological Society |
| record_format | Article |
| series | Molecular Plant-Microbe Interactions |
| spelling | doaj.art-1d55e943199a4ea692a7f89a778817282022-12-21T18:50:23ZengThe American Phytopathological SocietyMolecular Plant-Microbe Interactions0894-02821943-77062004-09-011791029103810.1094/MPMI.2004.17.9.1029Nonmycorrhizal (Myc¯) Mutants of Hebeloma cylindrosporum Obtained Through Insertional MutagenesisJean-Philippe CombierDelphine MelayahColette RaffierRégis PépinRoland MarmeisseGilles GayPolyethylene 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.https://apsjournals.apsnet.org/doi/10.1094/MPMI.2004.17.9.1029 |
| spellingShingle | Jean-Philippe Combier Delphine Melayah Colette Raffier Régis Pépin Roland Marmeisse Gilles Gay Nonmycorrhizal (Myc¯) Mutants of Hebeloma cylindrosporum Obtained Through Insertional Mutagenesis Molecular Plant-Microbe Interactions |
| title | Nonmycorrhizal (Myc¯) Mutants of Hebeloma cylindrosporum Obtained Through Insertional Mutagenesis |
| title_full | Nonmycorrhizal (Myc¯) Mutants of Hebeloma cylindrosporum Obtained Through Insertional Mutagenesis |
| title_fullStr | Nonmycorrhizal (Myc¯) Mutants of Hebeloma cylindrosporum Obtained Through Insertional Mutagenesis |
| title_full_unstemmed | Nonmycorrhizal (Myc¯) Mutants of Hebeloma cylindrosporum Obtained Through Insertional Mutagenesis |
| title_short | Nonmycorrhizal (Myc¯) Mutants of Hebeloma cylindrosporum Obtained Through Insertional Mutagenesis |
| title_sort | nonmycorrhizal myc¯ mutants of hebeloma cylindrosporum obtained through insertional mutagenesis |
| url | https://apsjournals.apsnet.org/doi/10.1094/MPMI.2004.17.9.1029 |
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