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 |
| Published: |
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 |
| Summary: | 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. |
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| ISSN: | 0894-0282 1943-7706 |