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An autonomously replicating plasmid transforms Botrytis cinerea to phleomycin resistance
M Santos1, I Vallejo, L Rebordinos
1Laboratorio de Genética y Microbiología, Facultad de Ciencias del Mar, Universidad de Cádiz, Polígono del Río San Pedro, Puerto Real, Spain.
FEMS Microbiology Letters
|April 1, 1996
Summary
A new transformation system for the fungus Botrytis cinerea was developed using the pUT737 plasmid. This system enables stable genetic modification of the fungus, paving the way for further research into this important plant pathogen.
Area of Science:
- * Molecular Biology
- * Mycology
- * Plant Pathology
Background:
- * Botrytis cinerea is a significant fungal pathogen affecting numerous plant species.
- * Genetic manipulation of B. cinerea is crucial for understanding its pathogenicity and developing control strategies.
- * Existing transformation methods for B. cinerea have limitations.
Purpose of the Study:
- * To develop and characterize a novel transformation system for the phytopathogenic fungus Botrytis cinerea.
- * To utilize the wide-host plasmid pUT737, conferring phleomycin resistance via the Sh ble gene.
- * To assess the stability and replication of the introduced plasmid in transformed fungal strains.
Main Methods:
- * Development of a transformation protocol using Botrytis cinerea protoplasts.
- * Selection of transformants on media containing varying concentrations of phleomycin.
- * Southern hybridization analysis to confirm plasmid presence and structure.
- * Reisolation and characterization of the plasmid from transformed fungal strains.
- * Assessment of plasmid stability over multiple generations under non-selective conditions.
Main Results:
- * Successful transformation of Botrytis cinerea protoplasts was achieved using the pUT737 plasmid.
- * Transformants exhibited resistance to phleomycin up to 50 µg/ml.
- * Southern hybridization confirmed the presence of autonomously replicating circular pUT737 plasmid in transformants.
- * Reisolation and transformation of E. coli confirmed the integrity of the rescued plasmid as pUT737.
- * Plasmids remained detectable for at least four generations under non-selective conditions.
- * Modified plasmid forms, stabilized in size and configuration, were observed in all transformants.
Conclusions:
- * A robust transformation system for Botrytis cinerea has been established using the pUT737 plasmid.
- * The developed system allows for stable genetic modification of B. cinerea.
- * Evidence suggests a selection process favoring stabilized plasmid forms within the fungal host.
- * This transformation system provides a valuable tool for future research on Botrytis cinerea.