Characterization of ech-42, a Trichoderma harzianum endochitinase gene expressed during mycoparasitism.

Abstract

The soil fungus Trichoderma harzianum has been shown to act as a mycoparasite against a range of economically important aerial and soil-borne plant pathogens, being successfully used in the field and greenhouse (1-3). Different factors involved in the antagonistic properties of Trichoderma have been identified, including antibiotics (4-8) and hydrolytic enzymes such as 1,3-3-glucanases, proteases, and chitinases (9).The interaction between Trichoderma and its host is first detectable as chemotropic growth of hyphae of the mycoparasite toward its host (10,11). When the mycoparasite reaches the host, its hyphae often coil around it or are attached to it by forming hook-like structures (12)(13)(14)(15)(16). After these interactions, the mycoparasite penetrates the host mycelium, apparently by partially degrading its cell wall (12,13). Microscopic observations (6,10,13,(17)(18)(19) suggest that Trichoderma spp. produces and excretes mycolytic enzymes, as indicated by localized cell wall lysis at points of interaction. The susceptible host hyphae show rapid vacuolation, collapse, and disintegration (10). Thus, the mycoparasitic process involves (i) chemotropic growth of Trichoderma, (ii) recognition of the host by the mycoparasite and attachment, (iii) excretion of extracellular enzymes, (iv) hyphae penetration, and (v) lysis of the host.Chitin and 1,3-3-glucan are the two major structural components of the cell wall of many plant pathogenic fungi.Therefore, it is expected that the 1,3-3-glucanases, chitinases, and proteases produced extracellularly by Trichoderma (18,(20)(21)(22)(23) play an important role in biocontrol.

Co-expression of two genes, a chitinase (chit42) and proteinase (prb1), implicated in mycoparasitism byTrichoderma hamatum

“…Introduction of multiple copies of the endochitinase chit33 in T. harzianum also resulted in increased biocontrol ability (Dana et al 2001). In contrast, no difference was observed between T. atroviride ech42 overexpression mutants, deletion mutants and wild type (Carsolio et al 1999). Although expression of ech42 and secretion of ECH42 protein during in vitro confrontation with a host pathogen implicated a role for this gene in biocontrol (Carsolio et al 1994), recent diffusion studies have shown ech42 to be involved in pathogen signalling , suggesting a more regulatory role for this enzyme.…”

“…In contrast, no difference was observed between T. atroviride ech42 overexpression mutants, deletion mutants and wild type (Carsolio et al 1999). Although expression of ech42 and secretion of ECH42 protein during in vitro confrontation with a host pathogen implicated a role for this gene in biocontrol (Carsolio et al 1994), recent diffusion studies have shown ech42 to be involved in pathogen signalling , suggesting a more regulatory role for this enzyme. Considerable attention also has focused on the ␤-glucanase genes because a large component of the fungal cell wall is composed of ␤-glucans and, therefore, it is likely that ␤-glucanases play a role in cell-wall degradation during mycoparasitism.…”

“…Direct confrontation assay.-Direct confrontation assays were performed on agar plates covered with cellophane using a modification of the method of Carsolio et al (1994). Fungi were grown at 22 C in the dark on minimal media (MM) agar with 0.2% glycerol.…”

Strain Improvement in Biocontrol Fungi

“…Ail but one transformant expressed higher levels of chitinase activity in comparison to the wild type récipient strain; the maximum level of increase rose 5-fold. Duplicating the copy number of the entire endochitinase gène under its own regulatory séquences is especially suitable for improving the biocontrol capability of Trichoderma as the highly conserved 42-kDa endochitinase-encoding gène, présent in ail mycophagous species of Trichoderma (Fekete et ai, 1996) has been shown to be specifically triggered in mycoparasitic interactions (Carsolio et al, 1994). Triggering occurs when a spécifie "mycoparasitic" protein complex binds to the promoter séquences of the gène and displaces the binding of a catabolite repressor protein (Lorito et al, 1996).…”

Production and partial purification of antifungal chitinase from Bacillus cereus VITSD3

“…The fungal cell wall has a cross-linked complex structure composed of chitin, glucans, and other polymers (AGRIOS, 2005). Enzymes that hydrolyze these components play a significant role in cell wall lysis of the pathogens (ADAMS, 2005;CARSOLIO et al, 1994). The antifungal activity and highly biocompatible quality make the chitin and its derivatives particularly useful for biomedical applications such as wound healing, cartilage tissue engineering, drug delivery and nerve generation (NAGWA et al, 2004).…”