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J Vis Exp. 2016 Sep 30;(115). doi: 10.3791/54522.

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions.

Author information

1
Institute for Microbiology, Heinrich-Heine University Düsseldorf; Bioeconomy Science Center (BioSC).
2
Institute for Microbiology, Heinrich-Heine University Düsseldorf.
3
Department of Genetics, Institute of Applied Biosciences, Karlsruhe Institute of Technology.
4
Institute for Microbiology, Heinrich-Heine University Düsseldorf; Bioeconomy Science Center (BioSC); Cluster of Excellence in Plant Sciences (CEPLAS), Heinrich-Heine University Düsseldorf; vera.goehre@hhu.de.

Abstract

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the replacement of the entire gene with a selectable marker via homologous recombination. During homologous recombination, exchange of DNA takes place between sequences with high similarity. Therefore, linear genomic sequences flanking a target gene can be used to specifically direct a selectable marker to the desired integration site. Blunt ends of the deletion construct activate the cell's DNA repair systems and thereby promote integration of the construct either via homologous recombination or by non-homologous-end-joining. In organisms with efficient homologous recombination, the rate of successful gene deletion can reach more than 50% making this strategy a valuable gene disruption system. The smut fungus Ustilago maydis is a eukaryotic model microorganism showing such efficient homologous recombination. Out of its about 6,900 genes, many have been functionally characterized with the help of deletion mutants, and repeated failure of gene replacement attempts points at essential function of the gene. Subsequent characterization of the gene function by tagging with fluorescent markers or mutations of predicted domains also relies on DNA exchange via homologous recombination. Here, we present the U. maydis strain generation strategy in detail using the simplest example, the gene deletion.

PMID:
27768088
DOI:
10.3791/54522
[Indexed for MEDLINE]

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