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

December 2000 , Volume 13 , Number  12
Pages  1,356 - 1,365

M. R. Thon , E. M. Nuckles , and L. J. Vaillancourt

Department of Plant Pathology, S-305 Agricultural Sciences Center-North, University of Kentucky, Lexington 40546-0091, U.S.A.

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Accepted 29 August 2000.

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.

Additional keywords: anthracnose leaf blight, anthracnose stalk rot, corn, Glomerella graminicola.

© 2000 The American Phytopathological Society