Involvement of Xyr1 and Are1 for Trichodermapepsin Gene Expression in Response to Cellulose and Galactose in Trichoderma reesei.

Abstract

The secretome of Trichoderma reesei contains a mixture of cellulases, hemicellulases, amylases, proteases, and lipases that synergistically degrade plant biomass. Trichodermapepsin (TrAsP), the most prominent protease of T. reesei, affects the stability of cellulases. Similar to cellulase production, TrAsP production also depends on carbon and nitrogen sources. Unlike the cellulase mechanism, the regulatory mechanism of TrAsP remains unknown. Therefore, this study aimed to determine the effect of the main cellulase regulator Xyr1 and nitrogen regulator Are1 on trasp regulation. Cellulase inducer Avicel and TrAsP inducer galactose were used as carbon sources. qRT-PCR analysis revealed that Xyr1 and Are1 acted as a repressor and an activator for trasp expression, respectively. Compared to Avicel, relative expression was higher in galactose. The binding motifs of Xyr1 and Are1 were located in upstream of the trasp promoter. From promoter deletant analysis using the β-glucuronidase reporter gene, the area from - 870bp to - 670bp was identified as the only region for positive regulation and there were both binding motifs of Xyr1 and Are1. Reporter assay of mutants confirmed functions of downregulation of Xyr1 and upregulation of Are1. Electrophoretic mobility shift assay demonstrated the binding ability of Xyr1 and Are1 to the particular binding motifs and their functionality was confirmed. Further, this study demonstrated that Cre1, Xpp1, and Pac1 downregulate trasp expression similar to that in cellulase regulation mechanism. These results demonstrate that transcriptional regulators of cellulase control trasp expression and suggest the possibility of the existence of specific protease regulators in T. reesei.