Tracking localization and secretion of cellulase spatiotemporally and directly in living Trichoderma reesei

Filamentous fungi secret hydrolytic enzymes like cellulase and hemicellulase outside the cells, serving as important scavengers of plant biomass in nature and workhorses in the enzyme industry. Unlike the extensive study on the mechanism of cellulase production in fungi, research on spatiotemporal distribution and secretion of cellulase in fungi is lacking, retarding the deeper understanding of the molecular mechanism behind the fungal cellulase production. Recombinant Trichoderma reesei strains RBGL, RCBH, and RCMC were successfully constructed from T. reesei RUT-C30, expressing red fluorescent protein DsRed-tagged versions of β-glucosidase (BGL), cellobiohydrolase (CBH), and endoglucanase (CMC), respectively. With the assistance of these strains, we found that all three cellulase components BGL, CBH, and CMC diffused throughout the whole fungal mycelium with major accumulation at the hyphal apexes. These enzymes located in ER, Golgi, vacuoles and cell membrane/wall, but not septum, and secreted abundantly into the culture medium. Moreover, the major secretion of CBH and CMC started more early than that of BGL. Brefeldin A (BFA) completely blocked cellulase expression and secretion in T. reesei. Based on recombinant T. reesei RBGL, RCBH, and RCMC expressing DsRed-fused versions of BGL, CBH, and CMC, respectively, the distribution and secretion of cellulase production in T. reesei were first visualized directly in a dynamic way, preliminarily mapping the location and secretion of T. reesei cellulase and providing evidence for revealing the secretion pathways of cellulase in T. reesei . The obtained results suggest that cellulase excretion majorly occurs via the conventional ER–Golgi secretory pathway, and might be assisted through unconventional protein secretion pathways.