The use of FLP-mediated recombination for the functional analysis of an effector gene family in the biotrophic smut fungus Ustilago maydis.
*In the Ustilago maydis genome, several novel secreted effector proteins are encoded by gene families. Because of the limited number of selectable markers, the ability to carry out sequential gene deletions has limited the analysis of effector gene families that may have redundant functions. *Here, we established an inducible FLP-mediated recombination system in U. maydis that allows repeated rounds of gene deletion using a single selectable marker (Hyg(R)). To avoid genome rearrangements via FRT sites remaining in the genome after excision, different mutated FRT sites were introduced. *The FLP-mediated selectable marker-removal technique was successfully applied to delete a family of 11 effector genes (eff1) using five sequential rounds of recombination. We showed that expression of all 11 genes is up-regulated during the biotrophic phase. Strains carrying deletions of 9 or all 11 genes showed a significant reduction in virulence, and this phenotype could be partially complemented by the introduction of different members from the gene family, demonstrating redundancy. *The establishment of the FLP/FRT system in a plant pathogenic fungus paves the way for analyzing multigene families with redundant functions.
Khrunyk
Yuliya
Y
Department of Organismic Interactions, Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Strasse 10, D-35043 Marburg, Germany.
Münch
Karin
K
Department of Organismic Interactions, Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Strasse 10, D-35043 Marburg, Germany.
Schipper
Kerstin
K
Department of Organismic Interactions, Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Strasse 10, D-35043 Marburg, Germany.
Lupas
Andrei N
AN
Department of Protein Evolution, Max Planck Institute for Developmental Biology, Spemannstr. 35, D-72076 Tübingen, Germany.
Kahmann
Regine
R
Department of Organismic Interactions, Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Strasse 10, D-35043 Marburg, Germany.
D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D017353:Gene Deletion; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D005838:Genotype; D005810:Multigene Family; D010641:Phenotype; D010935:Plant Diseases / Q000382:microbiology*; D011995:Recombination, Genetic; D014170:Transformation, Genetic; D014588:Ustilago / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity*; D003313:Zea mays / Q000382:microbiology
D005656:Fungal Proteins