Visualisation of the ER in the industrially-exploited filamentous fungus Trichoderma reesei using a BiP-Venus fusion protein

Tracking the progress of proteins through the secretory pathway requires reliable markers for cell organelles participating in secretion. Endoplasmic reticulum (ER) is the cytoplasmic organelle where the early steps of the protein secretory pathway are localised. Chemical markers such as DIOC6(3) and ER tracker series have an inherent problem of non-specificity and therefore are not optimal for detailed subcellular localisation studies. Fluorescent proteins on the other hand, provide better specificity by means of genetic tagging of organelles of interest. Here we report the fusion of the coding sequence for the VenusYFP to the fungal gene encoding Bip, a molecular chaperone located in the ER. Visualisation of the ER in a T. reesei transformant was carried out by fluorescence using confocal microscopy and the optical properties of the Venus fluorescence were investigated in detail. The accuracy of ER-labelling by the fusion protein approach was confirmed by EM studies. The expression of the venusbip1 fusion construct as a function of time was further examined by Northern blotting and Western analysis. Our success in targeting a relatively stable fluorescent protein to the ER in the filamentous fungus T. reesei provides a powerful tool for studies into the function of the ER in protein secretion in living fungal hyphae.