Spatially segregated snare protein interactions in filamentous fungus Trichoderma reesei

The machinery for trafficking proteins through the secretory pathway is well conserved in eukaryotes, but remains poorly characterized in filamentous fungi. Secretion is believed to be highly polarized in fungal hyphae and to mainly occur from hyphal tips. We describe the isolation of the snc1 and sso1 genes encoding exocytic SNARE proteins from Trichoderma reesei. The encoded SNCI protein can complement Snc protein depletion in S. cerevisiae whilst the T. reesei SSOI protein was unable to complement depletion of its yeast homologues. The localization and interactions of the T. reesei SNARE proteins were studied with advanced fluorescence imaging methods using fluorescent fusions of the SNARE proteins. The SSOI and SNCI proteins co-localized in sterol-independent clusters on the plasma membrane in sub-apical but not apical hyphal regions. The v-SNARE SNCI (but not the t-SNARE SSOI) localized to the apical vesicle cluster within the Spitzenkörper of the growing hyphal tips when expressed from the homologous T. reesei cbh1 promoter. Using fluorescence lifetime imaging microscopy (FLIM) and fluorescence energy transfer (FRET) analysis, we quantified the interactions between these proteins with high spatial resolution in living cells. Our data showed that the site of SNARE complex formation between these proteins is on the plasma membrane of non-growing hyphae in old sub-peripheral regions of the colony, but that there is no interaction between the proteins in growing hyphal tips in at the colony margin. These findings suggest spatially distinct sites of exocytosis within filamentous fungi and the existence of multiple exocytic SNAREs which are functionally and spatially segregated