Molecular Genetics and Genomics

, Volume 271, Issue 1, pp 103–110 | Cite as

A PCR-based system for highly efficient generation of gene replacement mutants in Ustilago maydis

Original Paper

Abstract

Ustilago maydis, the causative agent of corn smut disease, is one of the most versatile model systems for the study of plant pathogenic fungi. With the availability of the complete genomic sequence there is an increasing need to improve techniques for the generation of deletion mutants in order to elucidate the functions of unknown genes. Here a method is presented which allows one to generate constructs for gene replacement without the need for cloning. The 5′ and 3′-regions of the target gene are first amplified by PCR, and subsequently ligated directionally to a marker cassette via two distinct Sfi I sites, providing the flanking homologies needed for homologous recombination in U. maydis. Then the ligation product is used as a template for the amplification of the deletion construct, which can be used directly for transformation of U. maydis. The use of the fragments generated by PCR drastically increases the frequency of homologous recombination when compared to the linearized plasmids routinely used for gene replacement in U. maydis.

Keywords

Ustilago maydis Gene replacement Homologous recombination Ligation-mediated PCR 

Notes

Acknowledgments

I would like to thank R. Kahmann and U. Kämper for critically reading the manuscript and for helpful suggestions, and A. Waldschmidt and V. Vincon for excellent technical assistance. This work was supported by funding from Bayer CropScience AG

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Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  1. 1.Department of Organismic InteractionsMax Planck Institute for Terrestrial MicrobiologyMarburgGermany

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