An antifungal exo-alpha-1,3-glucanase (AGN13.1) from the biocontrol fungus Trichoderma harzianum

Abstract

Trichoderma harzianum secretes alpha-1,3-glucanases when it is grown on polysaccharides, fungal cell walls, or autoclaved mycelium as a carbon source (simulated antagonistic conditions). We have purified and characterized one of these enzymes, named AGN13.1. The enzyme was monomeric and slightly basic. AGN13.1 was an exo-type alpha-1,3-glucanase and showed lytic and antifungal activity against fungal plant pathogens. Northern and Western analyses indicated that AGN13.1 is induced by conditions that simulated antagonism. We propose that AGN13.1 contributes to the antagonistic response of T. harzianum.

FIG. 1

Purification of AGN13.1 from T. harzianum. Samples containing α-1,3-glucanase from each purification step were analyzed by SDS-PAGE. Lanes: std., molecular mass standards; 1, ammonium sulfate precipitation from a culture filtrate from T. harzianum grown on 0.5% A. niger cell walls (10 μg of protein); 2, adsorption and digestion of S-glucan (5 μg of protein); 3, gel filtration pool (5 μg of protein).

FIG. 2

HPLC profiles of the reaction products of AGN13.1 acting on S-glucan. S-glucan (4 mg/ml) was incubated for 14 h at 37°C in the absence of enzyme (A) or with 2 μg of the purified enzyme (B). In the abbreviation “G_n,” n represents the degree of polymerization.

FIG. 3

Antifungal properties of AGN13.1. (A) Inhibition of spore germination of P. aurantiogriseum by AGN13.1 at different concentrations. (B) Inhibition of hyphal growth of A. niger by AGN13.1 at different concentrations: solid circles, 0 μg/ml; open circles, 90 μg/ml; open squares, 180 μg/ml; open triangles, 270 μg/ml. In panels A and B, error bars indicate standard deviations. The experiment was performed three times with similar results. (C) Photographs of A. niger under the light microscope growing in the absence (left panel) or presence (right panel) of 270 μg of AGN13.1 per ml. Magnification, 100-fold. (D) Detail of the photographs shown in panel C at 400-fold magnification. As indicated above, the left panel corresponds to the control without AGN13.1, and the right panel corresponds to 270 μg of AGN13.1 per ml.

FIG. 4

Expression pattern of agn13.1. (A) Northern blot analysis. Total RNA was extracted from cultures grown for 9 h on 2% glucose (lane 1), 0.1% glucose (lane 2), 1.5% chitin (lane 3), 0.5% P. syringae cell walls (lane 4), 0.5% A. niger cell walls (lane 5), and with nitrogen starvation (lane 6). Total RNA (50 μg) was then subjected to electrophoresis on an agarose gel under denaturing conditions, transferred to a nylon membrane, and hybridized against a 1.9-kb probe of the coding sequence of agn13.1 (upper panel) or 18S rDNA (bottom panel). (B) Western blot analysis. Filtrates were obtained from cultures grown for 48 h on 2% glucose (lane 1), 0.1% glucose (lane 2), 1.5% chitin (lane 3), 0.5% P. syringae cell walls (lane 4), 0.5% P. aurantiogriseum cell walls (lane 5), 0.5% A. niger cell walls (lane 6), and with nitrogen starvation (lane 7). Samples (25 μg) were subjected to SDS-PAGE on a 12% polyacrylamide gel, and protein was transferred to a nitrocellulose membrane and probed with specific rabbit polyclonal AGN13.1 antibodies (dilution 1:1,000). AGN13.1 was visualized with peroxidase-conjugated goat anti-rabbit secondary antibodies (dilution of 1:1,000).