title: Tetracycline-regulated gene expression in the pathogen Ustilago maydis.
abstract: A powerful approach to explore gene function is the use of tetracycline-regulated expression. Here, we report the establishment of this titratable gene expression system for Ustilago maydis. Obstacles of premature polyadenylation of the native tetR gene, high basal activity of the tetracycline-responsive promoter, and toxicity of the viral activation domain were overcome by designing a synthetic tetR* gene according to context-dependent codon usage, removing cryptic enhancer elements from the promoter, and using an acidic minimal activation domain, respectively. We verified tetracycline-dependent dose-response using optimised components and applied a straightforward single-step promoter replacement cassette to regulate expression of pheromone response factor, a key transcription factor regulating mating. Pheromone response in liquid culture and mating on solid media was abolished in the presence of tetracycline and doxycycline. Thus, functionality of this versatile new tool for the plant pathogen was proven in a biological context.
authors.affiliation: Max-Planck Institute for Terrestrial Microbiology, Department of Organismic Interactions, Karl-von-Frisch-Strasse, 35043 Marburg, Germany.
authors.forename: Kathi
authors.initials: K
authors.lastname: Zarnack
authors.forename: Sebastian
authors.initials: S
authors.lastname: Maurer
authors.forename: Florian
authors.initials: F
authors.lastname: Kaffarnik
authors.forename: Oliver
authors.initials: O
authors.lastname: Ladendorf
authors.forename: Andreas
authors.initials: A
authors.lastname: Brachmann
authors.forename: Jörg
authors.initials: J
authors.lastname: Kämper
authors.forename: Michael
authors.initials: M
authors.lastname: Feldbrügge
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001483:Base Sequence / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D004742:Enhancer Elements, Genetic / Q000235:genetics; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects / Q000235:genetics*; D005813:Genes, Synthetic / Q000235:genetics; D010675:Pheromones / Q000494:pharmacology; D010935:Plant Diseases / Q000382:microbiology; D026723:Polyadenylation / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D017434:Protein Structure, Tertiary / Q000235:genetics; D017384:Sequence Deletion / Q000235:genetics; D013752:Tetracycline / Q000494:pharmacology; D013753:Tetracycline Resistance / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics*; D019076:Transgenes / Q000235:genetics*; D014588:Ustilago / Q000235:genetics* / Q000472:pathogenicity; D003313:Zea mays / Q000382:microbiology
chemical_list: D000900:Anti-Bacterial Agents; D010675:Pheromones; D014157:Transcription Factors; D013752:Tetracycline