title: Overcoming recalcitrant transformation and gene manipulation in Pucciniomycotina yeasts.
abstract: The red yeasts of the Pucciniomycotina have rarely been transformed with DNA molecules. Transformation methods were recently developed for a species of Sporobolomyces, based on selection using uracil auxotrophs and plasmids carrying the wild-type copies of the URA3 and URA5 genes. However, these plasmids were ineffective in the transformation of closely related species. Using the genome-sequenced strain of Rhodotorula graminis as a starting point, the URA3 and URA5 genes were cloned and tested for the transformation ability into different Pucciniomycotina species by biolistic and Agrobacterium-mediated transformations. Transformation success depended on the red yeast species and the origin of the URA3 or URA5 genes, which may be related to the high G + C DNA content found in several species. A new vector was generated to confer resistance to nourseothricin, using a native promoter from R. graminis and the naturally high G + C nourseothricin acetyltransferease gene. This provides a second selectable marker in these species. Targeted gene disruption was tested in Sporobolomyces sp. IAM 13481 using different lengths of homologous DNA with biolistic and Agrobacterium transformation methods. Both DNA delivery methods were effective for targeted replacement of a gene required for carotenoid pigment biosynthesis. The constructs also triggered transgene silencing. These developments open the way to identify and manipulate gene functions in a large group of basidiomycete fungi.
authors.affiliation: Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City, 5100 Rockhill Road, Kansas City, MO, 64110, USA.
authors.forename: Erika P
authors.initials: EP
authors.lastname: Abbott
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Ianiri
authors.forename: Raffaello
authors.initials: R
authors.lastname: Castoria
authors.forename: Alexander
authors.initials: A
authors.lastname: Idnurm
mesh_terms: D000935:Antifungal Agents / Q000633:toxicity; D001487:Basidiomycota / Q000235:genetics*; D053898:Biosynthetic Pathways / Q000235:genetics; D025141:Drug Resistance, Fungal; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D005827:Genetics, Microbial / Q000379:methods*; D059765:Homologous Recombination; D016254:Mutagenesis, Insertional; D010957:Plasmids; D012641:Selection, Genetic; D013309:Streptothricins / Q000633:toxicity; D014170:Transformation, Genetic; D014498:Uracil / Q000096:biosynthesis
chemical_list: D000935:Antifungal Agents; D013309:Streptothricins; D014498:Uracil