Dissecting Cellular Function and Distribution of β-Glucosidases in Trichoderma reesei.

has 11 putative β-glucosidases in its genome, playing key parts in the induction and production of cellulase. Nevertheless, the reason why the genome encodes so many β-glucosidases and the distinct role each β-glucosidase plays in cellulase production remain unknown. In the present study, the cellular function and distribution of 10 known β-glucosidases (CEL3B, CEL3E, CEL3F, CEL3H, CEL3J, CEL1A, CEL3C, CEL1B, CEL3G, and CEL3D) were explored in , leaving out BGL1 (CEL3A), which has been well investigated. We found that the overexpression of or significantly enhanced extracellular β-glucosidase production, whereas the overexpression of severely inhibited cellulase production by cellulose, resulting in nearly no growth of Four types of cellular distribution patterns were observed for β-glucosidases in : (i) CEL3B, CEL3E, CEL3F, and CEL3G forming clearly separated protein secretion vesicles in the cytoplasm; (ii) CEL3H and CEL3J diffusing the whole endomembrane as well as the cell membrane with protein aggregation, like a reticular network; (iii) CEL1A and CEL3D in vacuoles; (iv) and CEL3C in the nucleus. β-glucosidases CEL1A, CEL3B, CEL3E, CEL3F, CEL3G, CEL3H, and CEL3J were identified as extracellular, CEL3C and CEL3D as intracellular, and CEL1B as unknown. The extracellular β-glucosidases CEL3B, CEL3E, CEL3F, CEL3H, and CEL3G were secreted through a tip-directed conventional secretion pathway, and CEL1A, via a vacuole-mediated pathway that was achieved without any signal peptide, while CEL3J was secreted via an unconventional protein pathway bypassing the endoplasmic reticulum (ER) and Golgi. Although β-glucosidases play an important role in fungal cellulase induction and production, our current understanding does not provide a global perspective on β-glucosidase function. This work comprehensively studies all the β-glucosidases regarding their effect on cellulase production and their cellular distribution and secretion. Overexpression of or significantly enhanced β-glucosidase production, whereas overexpression of severely inhibited cellulase production on cellulose. In addition, overexpression of , , , , , , or delayed endoglucanase (EG) production. We first identified four cellular distribution patterns of β-glucosidases in Specially, CEL3C was located in the nucleus. CEL3J was secreted through the nonclassical protein secretion pathway bypassing endoplasmic reticulum (ER) and Golgi. CEL1A was secreted via a vacuole-mediated conventional secretion route without a signal peptide. These findings advance our understanding of β-glucosidase properties and secretory pathways in filamentous fungi, holding key clues for future study.