Molecular Genetics and Genomics

, Volume 272, Issue 2, pp 216–226 | Cite as

A reverse genetic approach for generating gene replacement mutants in Ustilago maydis

  • A. Brachmann
  • J. König
  • C. Julius
  • M. Feldbrügge
Original Paper
First Online:
Received:
Accepted:

Abstract

We describe a versatile strategy for generating gene replacement mutants in the phytopathogenic fungus Ustilago maydis. The system includes the choice of 32 different insertion cassettes for genetic engineering purposes, such as gene disruption and more sophisticated insertions of reporter genes, heterologous promoters or combinations of the two. PCR-amplified flanking sequences needed for homologous recombination are ligated to the respective insertion cassettes via Sfi I sites. As proof of principle we generated two replacement mutants in which the endogenous promoter of the pheromone gene mfa1 drives expression of the Green Fluorescent Protein gene (gfp). Simultaneously, expression of the mfa1 ORF is controlled either by the carbon source-regulated crg1 promoter or the nitrogen source-regulated nar1 promoter. In both cases gfp expression was pheromone-inducible and pheromone expression was only detected when the heterologous promoters were active.

Keywords

Ustilago maydis Gene replacement Knock out/knock in Mating factor Regulated gene expression 
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Notes

Acknowledgements

We acknowledge Dr. R. Kahmann for critically reading the manuscript, and thank J. Hohenner, S. Hester and P. Roth for excellent technical assistance. This work was supported by the DFG through Sonderforschungsbereich 369 and by funding from Bayer CropScience

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Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • A. Brachmann
    • 1
    • 2
  • J. König
    • 1
  • C. Julius
    • 1
  • M. Feldbrügge
    • 1
  1. 1.Department of Organismic InteractionsMax Planck Institute for Terrestrial MicrobiologyMarburgGermany
  2. 2.National Institute for Diabetes and Digestive and Kidney DiseasesNIHBethesdaUSA

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