U. maydis Protoplast Generation and Transformation.

Ustilago maydis is a dimorphic basidiomycete that exists as a yeast-like haploid form and a filamentous dikaryotic form. The yeast-like morphology is exploited for protoplast-based, PEG (polyethylene glycol)-mediated transformation, which is a simple and reliable means for introducing DNA into protoplasts. In U. maydis, this approach has been widely used for gene deletion/insertion, complementation, and transgene expression. In short, U. maydis yeast-like cells are grown to mid-log phase, and their cell walls are enzymatically digested in the presence of an osmotic stabilizer to generate stable protoplasts. Exogenous naked DNA, usually a linear integration construct with antibiotic resistance cassette or any other selection marker, is incubated with protoplasts, followed by the addition of high-molecular-weight PEG to enable DNA uptake. In this chapter, we point out the theoretical basis and outline of the procedure.