title: Photo-convertible tagging for localization and dynamic analyses of low-expression proteins in filamentous fungi.
abstract: Photo-convertible fluorescent proteins (PCFPs) undergo a dramatic change in their excitation and emission spectra upon irradiation at specific wavelengths, thus rendering a different color. Dendra2 is a commercially available PCFP used to track the redistribution of proteins within cellular compartments, their life-time or interactions. Before photo-conversion Dendra2 exhibits green fluorescence, which becomes red after irradiation with either UV or blue lights. Multiple studies including Dendra2 as a molecular tool have been described in eukaryotes but not in filamentous fungi. Here we present a method to tag low-expression proteins from the filamentous fungus Aspergillus nidulans with Dendra2 and track their cellular dynamics. The regulator of asexual development FlbB was selected as control, a transcription factor that is expressed at low levels and can be used as a marker for the tip and nuclei of vegetative hyphae. This control provided us with a visual way to confirm the functionality of our genomic and plasmid constructs, since a non-functional FlbB protein renders a block in development and a characteristic aconidial phenotype. Our protocol combines standardized cloning and transformation procedures with the use of a mercury lamp microscope to convert and follow Dendra2 within cells. Hence, we present a rapid, simple and inexpensive method that makes tracking analysis of proteins that present technical difficulties to be followed feasible in filamentous fungi.
authors.affiliation: Biochemistry II Laboratory, Department of Applied Chemistry, Faculty of Chemistry, University of the Basque Country, 20018 San Sebastian, Spain.
authors.forename: Elixabet
authors.initials: E
authors.lastname: Perez-de-Nanclares-Arregi
authors.affiliation: Biochemistry II Laboratory, Department of Applied Chemistry, Faculty of Chemistry, University of the Basque Country, 20018 San Sebastian, Spain. Electronic address: oier.echeveste@ehu.es.
authors.forename: Oier
authors.initials: O
authors.lastname: Etxebeste
mesh_terms: D001233:Aspergillus nidulans / Q000254:growth & development / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D025301:Hyphae / Q000254:growth & development / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: C583169:Dendra2 protein, Dendronephthya; D005656:Fungal Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors
keywords: Asexual development; Dendra2; Filamentous fungi; Growth; Photo-convertible tagging; Protein dynamics