Regulation of chitinase 33 (chit33) gene expression in Trichoderma harzianum

We investigated the regulation of chit33 expression in Trichoderma harzianum CECT 2413. This gene encodes the Chit33 endochitinase, which is a major component of the fungus' chitinolytic enzyme system and is important for biocontrol. To this end, both Northern analysis and reporter gene fusions of a 1.4-kb fragment of the 5′-upstream sequences of chit33 to the Aspergillus niger goxA gene (encoding glucose oxidase) and the Aquorea victoria green fluorescent protein were used. Northern analysis and data obtained with the reporter systems were compatible, thus showing that the 1.4-kb fragment bears all necessary information for the regulation of chit33 gene expression. chit33 is weakly expressed during growth on chitin and Rhizoctonia solani cell walls. The addition of N-acetylglucosamine transiently induced chit33 expression in resting cells of the fungus. The addition of either glucose or glycerol prevented induction of chit33 gene expression by chitin or cell walls. Incubation of T. harzianum in the presence of low concentrations (0.1%, w/v) of glucose and high concentrations (38 mM) of ammonium sulfate, or in the presence of high concentrations (1%, w/v) of glucose and low concentrations (0.38 mM) of ammonium sulfate also stimulated chit33-mRNA accumulation, although to a lower degree than induction by N-acetylglucosamine. Transfer of T. harzianum cultures to either 40 °C or 4 °C initiated a very rapid expression of chit33 in the absence of an inducer, yet only at very low levels (5%) of the induced control. Confrontation experiments, using the gfp gene as a reporter and R. solani as a host, showed that chit33 is expressed only during but not before the stage of overgrowth on R. solani. These data show that Chit33 is an enzyme involved in mycoparasitism; and its formation is controlled by induction, by either carbon or nitrogen starvation and, to a low degree, also under conditions of temperature stress.