Broadening the spectrum of fluorescent protein tools for use in the encapsulated human fungal pathogen Cryptococcus neoformans

•Fluorescent proteins are valuable molecular biology research tools, however, only handful are available in C. neoformans.•Eight fluorescent proteins were identified and expressed, with five highly visible under the microscope.•Heterologous expression of these five bright fluorescent proteins within C. neoformans does not influence virulence.•Developed six fluoroblasters for seamless tagging. Green fluorescent protein (GFP) and its counterparts are modern molecular biology research tools indispensable in many experimental systems. Within fungi, researchers studying Saccharomyces cerevisiae and other model ascomycetes have access to a wide variety of fluorescent proteins. Unfortunately, many of these tools have not crossed the phylum divide into the Basidiomycota, where only GFP S65T, Venus, Ds-Red, and mCherry are currently available. To address this, we searched the literature for potential candidates to be expressed in the human fungal pathogen Cryptococcus neoformans and identified a suite of eight more modern fluorescent proteins that span the visible spectrum. A single copy of each fluorophore was heterologously expressed in Safe Haven 1 and their fluorescence intensities compared in this encapsulated yeast. mTurquoise2, mTFP1, Clover, mNeonGreen, mRuby3, and Citrine were highly visible under the microscope, whereas Superfolder GFP and mMaroon1 were not. Expressed fluorophores did not impact growth or virulence as demonstrated by an in vitro spotting assay and murine inhalation model, respectively.