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

We investigated the regulation of chit33 expression in Trichoderma harzianu m CECT 2413. This gene encodes the Chit33 endochitinase, which is a major c omponent of the fungus' chitinolytic enzyme system and is important for bio control. 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 f luorescent protein were used. Northern analysis and data obtained with the reporter systems were compatible, thus showing that the 1.4-kb fragment bea rs all necessary information for the regulation of chit33 gene expression, chit33 is weakly expressed during growth on chitin and Rhizoctonia solani c ell walls. The addition of N-acetylglucosamine transiently induced chit33 e xpression in resting cells of the fungus. The addition of either glucose or glycerol prevented induction of chit33 gene expression by chitin or cell w alls. 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 concent rations (0.38 mM) of ammonium sulfate also stimulated chit33-mRNA accumulat ion, although to a lower degree than induction by N-acetylglucosamine. Tran sfer of T. harzianum cultures to either 40 degreesC or 4 degreesC initiated a very rapid expression of chir33 in the absence of an inducer, yet only a t very low levels (5%) of the induced control. Confrontation experiments, u sing the afp 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. sola ni. These data show that Chit33 is an enzyme involved in mycoparasitism; an d its formation is controlled by induction, by either carbon or nitrogen st arvation and, to a low degree, also under conditions of temperature stress.