Imaging the last stages of protein secretion in the filamentous fungus Trichoderma reesei

Available data suggest that the basic principles of secretion obtained from studies on S. cerevisiae and animal cells also apply to filamentous fungi. Although considerable research has been performed on the secretion process in filamentous fungi, little is known about their secretory machinery. The transport of proteins between the cellular compartments occurs in membrane-bounded vesicles that bud from the donor and fuse with the acceptor compartment. Membrane bound proteins called SNAREs (Soluble N-ethylaleimide-sensitive factor Attachment protein REceptors) play a key role in membrane fusion. SNAREs have been found to be involved in most membrane fusion events in the cell. This study describes the cloning and characterization of a putative exocytotic t-SNARE (sso1) from Trichoderma reesei. We cloned the T. reesei homologue of Neurospora crassa nsyn2. This protein was labelled with the Cerulean fluorescent protein mCer and the fusion protein expressed in T. reesei. Transformants expressing the fusion protein exhibit a clear labelling of the plasma membrane. The fusion protein is uniformly distributed along the hyphae with no tip-focused gradient. A v-SNARE SNCI was labelled with mRFP to analyze its interaction with the SSOI protein using fluorescence resonance energy
transfer (FRET) imaging.