Genetic Transformation System for the Fungal Soybean Pathogen Cercospora kikuchii
Abstract
Genetic Transformation System for the Fungal Soybean Pathogen Cercospora kikuchii R. G. Upchurch 1 * , M. Ehrenshaft 1 , D. C. Walker 2 † and L. A. Sanders 2 ‡ 1 Agricultural Research Service, U.S. Department of Agriculture, and Department of Plant Pathology, 2 North Carolina State University, Raleigh, North Carolina 27695-7616 ABSTRACT An altered β-tubulin gene that confers resistance to the fungicide benomyl was isolated from a genomic library of a UV-induced mutant of Cercospora kikuchii and used as a selectable marker for transformation. The level of benomyl resistance conferred to the transformants was at least 150-fold greater than the intrinsic resistance of the C. kikuchii recipient protoplasts. In the majority of cases, the tubulin fragment was integrated at the native β-tubulin locus, apparently by gene replacement or gene conversion. The frequency of transformation ranged from 0.2 to 6 transformants per μg of DNA, depending on the recipient strain. Transformation with linearized plasmid resulted in a higher frequency, without changing the type of integration event. Transformants were phenotypically stable after eight consecutive transfers on medium without benomyl. This is the first report of a genetic transformation system for a Cercospora species.