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

Mol Genet Genomics. 2004 Sep;272(2):216-26. doi: 10.1007/s00438-004-1047-z. Epub 2004 Aug 17.


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 SfiI 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Fungal Proteins / genetics
  • Fungal Proteins / metabolism
  • Gene Expression
  • Genes, Fungal*
  • Genes, Reporter
  • Genetic Engineering
  • Genetic Techniques*
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Mutagenesis, Insertional
  • Mutation
  • Pheromones / genetics
  • Pheromones / metabolism
  • Plants / microbiology
  • Plasmids / genetics
  • Polymerase Chain Reaction
  • Promoter Regions, Genetic
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Recombination, Genetic
  • Ustilago / genetics*
  • Ustilago / pathogenicity


  • Fungal Proteins
  • Pheromones
  • Recombinant Proteins
  • Green Fluorescent Proteins