Figure 1
Fluorescence microscopy of K. petricola. (a) Fluorescence-based assays distinguish viable and dead cells. The fluorescent dyes fluorescein diacetate (FDA) and propidium iodide (PI) stain viable cells (green) and dead cells (red), respectively. WT:A95 cells were resuspended in PBS and incubated for 15, 120 and 360 min at the indicated temperatures prior to staining. Numbers of green- and red-stained cells were determined in three independent experiments. Mean values and standard deviations are shown. PC (positive control)—incubation for 360 min at 25 °C, NC (negative control)—incubation for 360 min in isopropanol. (b) Genetically encoded fluorescent (fusion) proteins localise in specific cellular compartments. WT:A95 protoplasts were transformed with linearised plasmids containing expression cassettes (constitutive promoter::reporter gene::terminator) fused to hygromycin (hygR) or nourseothricin (natR) resistance cassettes. Fluorescent proteins: GFP—mammalian codon-optimised enhanced GFP, DsRED—mammalian codon-optimised DsRED-Express, H2B-GFP—K. petricola histone 2B fused to GFP, Lifeact-GFP—first 17 amino acids of Saccharomyces cerevisiae actin-binding protein Abp140 fused to GFP (codon-optimised for B. cinerea), Mito-DsRED—first 60 amino acids of Sordaria macrospora CAS2 fused to DsRED, DsREDSKL—peroxisomal targeting motif ‘SKL’ added to the C-terminus of DsRED, H2B-TOM—S. macrospora H2B fused to tandem dimer tomato (DsRED variant). For details see Tables S1 and S2. Fluorescent dyes: CFW—calcofluor white (cell walls), MTG—MitoTracker Green (mitochondria), DAPI—4′,6-diamidino-2-phenylindole (nuclei), FM4-64—N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino) phenyl) hexatrienyl) pyridinium dibromide (membranes). BF—bright field.
