The overexpression of an endo-β-1,3-glucanase, engl1, in Trichoderma virens

Abstract

Many strains of Trichoderma spp. have been shown to be effective biocontrol agents against foliar and soilborne plant pathogens. The production and secretion of hydrolytic enzymes appear to play an important role in these antagonistic interactions. We overexpressed a gene (engl1) that encodes an endo-β-1,3-glucanase under the control of a constitutive promoter, gpd, and its own inducible promoter in T. virens. Transformants were selected following four separate transformation events and designated as: GLN series, which contained extra copies of the native gene and the GOE, GA, and GA100 series, which were constitutively overexpressing transformants. Many of the transformant strains secreted significantly (P < 0.01) more glucanase than the wild-type strain, Tv29-8, when grown under non-inducing conditions (mannitol as a carbon source). Culture filtrate from overexpressing transformants liberated from 2-30 times more glucose from laminarin than Tv29-8. The presence of multiple copies of the ORF of engl1 in overexpressing transformants was confirmed by Southern blotting analysis using the ORF of engl1 and the promoter of gpd. When crude protein extracts were analyzed by SDS-PAGE and stained with Coomassie blue R-250, a band of ~80 kDa was visualized with the overexpressing transformants, but not with Tv29-8. Glucanase activity gels revealed the presence of as many as five active β-1,3-glucanases in all strains when grown under non-inducing conditions. While active protein was found in the crude protein extracts, the extracts at the concentrations tested did not inhibit the germination of Phytophthora parasitica var. nicotianae encysted zoospores. Three of the nine transformant strains were found to grow (in vitro) significantly greater than Tv29-8 and were able to colonize cotton roots to a greater extent than Tv29-8. An ID-DI curve was created for a cotton-Pythium ultimum pathosystem and an LD₅₀ of 0.3 g of P. ultimum infested rice particles per 1.8 l of Metro-mix potting medium was established. Cotton seeds coated with a wheat bran-peat moss base containing strains of T. virens were planted into potting mix infested with 0.2 or 0.3 g of P. ultimum inoculum. The overexpression transformant strains were no more effective than Tv29-8 in protecting cotton seeds against infection by the pathogen.