Expression and Detection of Heterologous Proteins in Ustilago maydis.

Expression of transgenes of both bacterial and eukaryotic origin is critical for the versatility of model species. A range of heterologous genes has been successfully transplanted to and cognate proteins functionally synthesized in Ustilago maydis, ranging from cytosolic reporters like the enhanced green fluorescent protein and its derivatives to cytoplasmic or secreted enzymes from bacterial, plant, or filamentous ascomycete sources. Here, a brief summary of the most important considerations for expression of heterologous genes, including expression strain selection, codon optimization, the choice of insertion loci, as well as potential protein fusions for targeting, localization, purification, and detection, is provided. A detailed protocol for the prevalent constitutive expression of heterologous proteins and subsequent verification via Western blot analysis is provided. All steps are described in detail using the example of the secreted endo-glucuronoxylanase Xyn30A, a carbohydrate-active enzyme from Thermothelomyces thermophilus. In essence, recombinant protein synthesis is central for the broad exploitation of U. maydis as a fungal model, and applications range from fundamental research to biotechnological applications.