Fig. 3

Asp-melanin formation and subcellular protein localisation from polycistronic gene expression in the ATNT system. a Schematic presentation of polycistronic expression constructs separated by P2A sequences. tyrP:tdTom denotes an in frame fusion of the tyrP gene with the gene coding for the red fluorescent protein tdTomato. b Colonies in top and bottom view of the parental strain ATNT16 and strains carrying the expression construct with one or two P2A separations grown in the absence and presence of doxycycline. Addition of doxycycline induces the formation of Asp-melanin, which is indicated by brown colouration of mycelium in the bottom view. c Liquid cultures of ATNT16 strains carrying the expression construct with one or two P2A sequences. Mycelium was harvested after 24 h of incubation. Cultures were grown without doxycycline (Doxy 0 h) or were induced with doxycycline for the last 6 h of total incubation time (Doxy 6 h) or for the whole 24 h (Doxy 24 h). A stronger colouration of mycelium is observed when TyrP and tdTomato are separated by an additional P2A peptide. d Scheme of the polycistronic P2A_P2A mRNA. Localisation of the individual gene sequences are indicated above and localisation and size of PCR products for verification of transcription are shown below the transcript. e Semiquantitative RT-PCR on cDNA derived from cultures in c. The actin gene was used for normalisation of cDNA. Amplification from genomic DNA (gDNA) is shown as a control with a decrease in fragment size of the actin gene due to intron splicing. Full length-transcription of the polycistronic messenger is confirmed by PCR products from all genes when grown in the presence of doxycycline. f Fluorescence analysis for subcellular localisation of proteins produced from the two polycistronic expression constructs. Nuclei are shown in blue by DAPI staining. Red fluorescence indicates localisation of tdTomato. In the P2A construct the fusion of TyrP with tdTomato reflects a punctuated fluorescence consistent with ER and Golgi. When tdTomato is separated by P2A in the P2A_P2A construct, tdTomato localises to the cytoplasm