A dual selection based, targeted gene replacement tool for Magnaporthe grisea and Fusarium oxysporum

Hyun Khang Chang, Sook Young Park, Yong Hwan Lee, Seogchan Kang

Research output: Contribution to journalArticle

89 Citations (Scopus)

Abstract

Rapid progress in fungal genome sequencing presents many new opportunities for functional genomic analysis of fungal biology through the systematic mutagenesis of the genes identified through sequencing. However, the lack of efficient 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 efficient method of gene replacement and evaluated factors affecting the efficiency 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-fluoro-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-fluoro-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 efficient, universal functional genomic tool for harnessing the growing body of fungal genome sequence data to study fungal biology.

Original languageEnglish (US)
Pages (from-to)483-492
Number of pages10
JournalFungal Genetics and Biology
Volume42
Issue number6
DOIs
StatePublished - Jun 1 2005

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Magnaporthe
Fusarium
Thymidine Kinase
Simplexvirus
Genes
Fungal Genome
Fungi
Agrobacterium tumefaciens
Poisons
Genomics
Mutagenesis
Alleles

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Genetics

Cite this

Chang, Hyun Khang ; Park, Sook Young ; Lee, Yong Hwan ; Kang, Seogchan. / A dual selection based, targeted gene replacement tool for Magnaporthe grisea and Fusarium oxysporum. In: Fungal Genetics and Biology. 2005 ; Vol. 42, No. 6. pp. 483-492.
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A dual selection based, targeted gene replacement tool for Magnaporthe grisea and Fusarium oxysporum. / Chang, Hyun Khang; Park, Sook Young; Lee, Yong Hwan; Kang, Seogchan.

In: Fungal Genetics and Biology, Vol. 42, No. 6, 01.06.2005, p. 483-492.

Research output: Contribution to journalArticle

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