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Restriction Enzyme-Mediated Integration Used to Produce Pathogenicity Mutants of Colletotrichum graminicola

2000; American Phytopathological Society; Volume: 13; Issue: 12 Linguagem: Inglês

10.1094/mpmi.2000.13.12.1356

1943-7706

Michael R. Thon, Etta M. Nuckles, Lisa J. Vaillancourt,

Chromosomal and Genetic Variations

We have developed a restriction enzyme-mediated insertional mutagenesis (REMI) system for the maize pathogen Colletotrichum graminicola. In this report, we demonstrate the utility of a REMI-based mutagenesis approach to identify novel pathogenicity genes. Use of REMI increased transformation efficiency by as much as 27-fold over transformations with linearized plasmid alone. Ninety-nine transformants were examined by Southern analysis, and 51% contained simple integrations consisting of one copy of the vector integrated at a single site in the genome. All appeared to have a plasmid integration at a unique site. Sequencing across the integration sites of six transformants demonstrated that in all cases the plasmid integration occurred at the corresponding restriction enzyme-recognition site. We used an in vitro bioassay to identify two pathogenicity mutants among 660 transformants. Genomic DNA flanking the plasmid integration sites was used to identify corresponding cosmids in a wild-type genomic library. The pathogenicity of one of the mutants was restored when it was transformed with the cosmids.