Cloning and characterization of multiple glycosyl hydrolase genes from Trichoderma virens

Abstract.

Trichoderma virens is a widely distributed soil fungus that is parasitic on other soil fungi. The mycoparasitic activity of T. virens is correlated with the production of numerous antifungal activities, including the secretion of a considerable repertoire of fungal cell wall-degrading enzymes. Here, we report the characterization of a diverse set of chitinase and glucanase genes from T. virens. In each case, full-length genomic clones were isolated and characterized, while sequencing of the corresponding cDNA clones and manual annotation provided a basis for establishing gene structure. Based on homology of the deduced amino acid sequences, we have identified three members of the 42Kd endochitinase gene family, two 33Kd exochitinases, two exochitinases with homology to N-acetylglucosaminidases, and three glucanase genes predicted to encode β-1,3- and β-1,6-proteins. The majority of these genes appear to encode signal peptides, suggesting an extracellular location for the corresponding proteins. Despite their overall similarity, the 42Kd class of chitinases can be subdivided, based on the presence of distinct N-terminal domains, suggesting that the proteins may have distinct cellular roles, while Northern blot analysis confirms that these genes possess distinct patterns of gene regulation. Similarly, one of the 33Kd chitinase genes is unique, because it is predicted to encode a protein C-terminus with high homology to the conserved family I cellulose-binding domain. The expression patterns of the chitinase genes were analyzed in both a wild-type strain and a strain disrupted for the major 42Kd chitinase gene of T. virens. The results of these transcript analyses, together with enzymatic assay of the extracellular proteins, suggest interdependent regulation of this important gene family in T. virens.