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A dual selection based, targeted gene replacement tool for Magnaporthe grisea and Fusarium oxysporum
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 Authours
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Chang Hyun Khang, Sook-Young Park, Yong-Hwan Lee, Seogchan Kang |
| Title |
A dual selection based, targeted gene replacement tool for Magnaporthe grisea and Fusarium oxysporum |
| Journal |
Fungal Genetics and Biology, 2005 (42) ~ |
Abstract
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Rapid progress in fungal genome sequencing presents many new opportunities for functional genomic analysis of fungal biology through the systematic mutagenesis of the genes identiWed through sequencing. However, the lack of eYcient tools for targeted gene replacement is a limiting factor for fungal functional genomics, as it often necessitates the screening of a large number of transformants to identify the desired mutant. We developed an eYcient method of gene replacement and evaluated factors affecting the effciency of this method using two plant pathogenic fungi, Magnaporthe grisea and Fusarium oxysporum. This method is based on Agrobacterium tumefaciens-mediated transformation with a mutant allele of the target gene flanked by the herpes simplex virus thymidine kinase (HSVtk) gene as a conditional negative selection marker against ectopic transformants. The HSVtk gene product converts 5-Xuoro-2-deoxyuridine to a compound toxic to diverse fungi. Because ectopic transformants express HSVtk, while gene replacement mutants lack HSVtk, growing transformants on a medium amended with 5-Xuoro-2-deoxyuridine facilitates the identification of targeted mutants by counter-selecting against ectopic transformants. In addition to M. grisea and F. oxysporum, the method and associated vectors are likely to be applicable to manipulating genes in a broad spectrum of fungi, thus potentially serving as an effcient, universal functional genomic tool for harnessing the growing body of fungal genome sequence data to study fungal biology. |
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