Abstract
Red yeasts belong to the subphylum Pucciniomycotina, one of the three major lineages in the Basidiomycete fungi. These yeasts, commonly found as saprophytes in the environment, have unique features that make them important for academia, industry, agriculture, and occasionally human health. To understand the genetic basis behind biological processes of interest, the availability of molecular tools for gene mutation plays a crucial role. Here we outline how to perform recently developed protocols of Agrobacterium tumefaciens-mediated transformation to Pucciniomycotina red yeasts. This method can be applied to generate libraries of T-DNA insertional mutants that can be screened for phenotypes of interest, or for targeted gene replacement.
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Notes
- 1.
Six different binary vectors are available for transformation of Pucciniomycotina red yeasts. Four vectors, pAIS3, pAIS4, pPZPWU3, and pPZPWU5, were generated using the wild type copies of the URA3 and URA5 from Sporobolomyces sp. IAM 13481 (see Ianiri et al. 2011 for details), and URA3 and URA5 of R. graminis WP1 (see Abbott et al. 2013 for details) as selection markers. The plasmid pGI3 contains as a selective marker the high G + C content nourseothricin acetyltransferase (NAT) gene obtained from Streptomyces noursei, placed under the promoter and terminator of the TUB2 gene of R. graminis WP1 (see Abbott et al. 2013 for details). For selection and maintenance of these vectors in bacteria, 50 μg/ml of kanamycin is added to Luria-Bertani medium. The vector pRH2031 features a codon-optimized enhanced green fluorescent protein gene (RtGFP) and hygromycin phosphotransferase gene (hpt-3) placed under the control of the promoter and terminator of the glyceraldehyde-3-phosphate dehydrogenase gene (GPD1) of Rhodosporidium toruloides (see Liu et al. 2013 for details). For selection and maintenance of the vectors in bacteria, 50 μg/ml of rifampicin and 50 μg/ml of spectinomycin are added to the medium.
- 2.
Make a 2.5x salt solution stock of 2 L H2O containing: KH2PO4 (7.25 g), K2HPO4 (10.25 g), NaCl (0.75 g), MgSO4 · 7H2O (2.5 g), CaCl2 · 2H2O (0.33 g), FeSO4 · 7H2O (12.4 mg), (NH4)2SO4 (2.5 g). Store at room temperature. To make 1 L of IM, combine 400 ml salt solution, 540 ml H2O, 5 ml glycerol, and 0.9 g glucose. Agar can be added to make plates (20 g/L). Autoclave, and cool the solution (to 55–58 °C if it contains agar). While cooling, dissolve 7.7 g 2-(N-morpholino)ethanesulfonic acid (MES) in 50 ml H2O. Adjust the pH to 5.3 with 5 M KOH. Add to this solution 19 mg acetosyringone dissolved in 250 μl DMSO. Filter sterilize the MES-AS solution, add to the cooled solution, and pour plates if agar is present. For transformation of uracil auxotrophs, 20 mg of uracil can also be supplemented into the induction medium.
- 3.
The presence of a physical substrate (e.g. membranes made of cellulose, nitrocellulose, or nylon) on the plates for the co-incubation step was tested, but found to be unnecessary for the success of transformation.
- 4.
The medium YNB + 2 % glucose is used to select prototrophic strains when the transformation is performed with the URA genes as markers. When the gene markers NAT and hpt-3 are used, YPD medium supplemented with 200 μg/ml nourseothricin or 150 μg/ml hygromycin B is used for selection. To prevent A. tumefaciens cell growth, 100 or 200 μg/ml of cefotaxime is added to the medium.
References
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Acknowledgements
Our research on the Pucciniomycotina fungi has been supported by the University of Missouri Research Board, the US National Science Foundation, and the National Institutes of Health.
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Ianiri, G., Idnurm, A. (2015). Agrobacterium tumefaciens- Mediated Transformation of Pucciniomycotina Red Yeasts. In: van den Berg, M., Maruthachalam, K. (eds) Genetic Transformation Systems in Fungi, Volume 1. Fungal Biology. Springer, Cham. https://doi.org/10.1007/978-3-319-10142-2_15
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DOI: https://doi.org/10.1007/978-3-319-10142-2_15
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