title: Establishing a versatile Golden Gate cloning system for genetic engineering in fungi.
abstract: The corn pathogen Ustilago maydis is a well-studied fungal model organism. Along with a broad set of experimental tools, versatile strategies for the generation of gene replacement mutants by homologous recombination in U. maydis have been developed. Nevertheless, the production of corresponding linear DNA constructs still constitutes a time-limiting step. To overcome this bottleneck, various resistance cassette modules were adopted for use with the so-called Golden Gate cloning strategy. These modules allow not only simple gene deletions but also more sophisticated genetic manipulations like inserting sequences for C-terminal protein tagging. The type IIs restriction enzyme BsaI was selected for this novel approach as its recognition sites are comparatively rare in the U. maydis genome. To test the efficiency of the new strategy it was used to test the influence of varying flank lengths as well as the effect of non-homologous flank ends on homologous recombination. Importantly, to proof a broad applicability in other fungi the same strategy was used to generate mutants in the filamentous ascomycete Aspergillus nidulans. Hence, we present a highly efficient and economic cloning strategy that speeds up reverse genetic approaches in fungi.
authors.affiliation: Institute for Microbiology, Center of Excellence on Plant Sciences (CEPLAS), Department of Biology, Heinrich-Heine University Düsseldorf, 40204 Düsseldorf, Germany.
authors.forename: Marius
authors.initials: M
authors.lastname: Terfrüchte
authors.affiliation: Institute of Microbiology and Genetics, Department of Molecular Microbiology and Genetics, Georg-August-University, 37077 Göttingen, Germany.
authors.forename: Bastian
authors.initials: B
authors.lastname: Joehnk
authors.affiliation: Departamento de Microbiología, Centro de Investigación Científica y de Educación Superior de Ensenada CICESE, 22860 Baja California, Mexico.
authors.forename: Rosa
authors.initials: R
authors.lastname: Fajardo-Somera
authors.affiliation: Institute of Microbiology and Genetics, Department of Molecular Microbiology and Genetics, Georg-August-University, 37077 Göttingen, Germany.
authors.forename: Gerhard H
authors.initials: GH
authors.lastname: Braus
authors.affiliation: Departamento de Microbiología, Centro de Investigación Científica y de Educación Superior de Ensenada CICESE, 22860 Baja California, Mexico.
authors.forename: Meritxell
authors.initials: M
authors.lastname: Riquelme
authors.affiliation: Institute for Microbiology, Center of Excellence on Plant Sciences (CEPLAS), Department of Biology, Heinrich-Heine University Düsseldorf, 40204 Düsseldorf, Germany.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Schipper
authors.affiliation: Institute for Microbiology, Center of Excellence on Plant Sciences (CEPLAS), Department of Biology, Heinrich-Heine University Düsseldorf, 40204 Düsseldorf, Germany. Electronic address: feldbrue@hhu.de.
authors.forename: Michael
authors.initials: M
authors.lastname: Feldbrügge
mesh_terms: D001233:Aspergillus nidulans / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D017353:Gene Deletion; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors; D059765:Homologous Recombination; D009154:Mutation; D014588:Ustilago / Q000235:genetics*
keywords: Filamentous fungi; Golden Gate cloning; Homologous recombination; Resistance-marker recycling