Overcoming recalcitrant transformation and gene manipulation in Pucciniomycotina yeasts
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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.
KeywordsBasidiomycete Rhodotorula slooffiae Rhodosporidium kratochvilovae β-Carotene RNAi T-DNA
We thank Joseph Heitman, James Fraser, and the FGSC for providing strains. This research was supported by grants from the United States National Science Foundation (MCB-0920581), the Italian Ministry of Education, University and Scientific Research (PRIN 2008, 2008JKH2MM), and the Italian Ministry of Foreign Affairs (joint research project LS-7, within the executive programme of cooperation in the field of science and technology between Italy and USA 2008–2010). G. I. was also supported by a scholarship from the Department of Agricultural, Environmental and Food Sciences, University of Molise, Italy.
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