Purification and characterization of an extracellular alpha-glucosidase protein from Trichoderma viride which degrades a phytotoxin associated with sheath blight disease in rice

Aims: To purify and characterize an extracellular alpha -glucosidase from T richoderma viride capable of inactivating a host-specific phytotoxin, desig nated RS toxin, produced by the rice sheath blight pathogen, Rhizoctonia so lani Kuhn. Methods and Results: The host-specific RS toxin was purified from both cult ure filtrates (culture filtrate toxin, CFTox) and R. solani-inoculated rice sheaths (sheath blight toxin, SBTox). Sodium dodecyl sulphate-polyacrylami de gel electrophoresis analyses of extracellular proteins, purified from a biocontrol fungus T. viride (TvMNT7) grown on SBTox and CFTox separately, w ere carried out. The antifungal activity of the purified high molecular wei ght protein (110 kDa) was studied against RS toxin as well as on the sclero tial germination and mycelial growth of R. solani. Enzyme assay and Western blot analysis with the antirabbit TvMNT7 110-kDa protein indicated that th e protein was an alpha -glucosidase. The 110-kDa protein was highly specifi c to RS toxin and its Michaelis-Menten constant value was 0.40 mmol l(-1) w hen p-nitrophenyl alpha -D-glucopyranoside was used as the substrate. The i soelectric point of the protein was 5.2. N-terminal sequencing of the alpha -glucosidase protein showed that its amino acid sequence showed no homolog y with other known alpha -glucosidases. Conclusions: This appears to be the first report of the purification and ch aracterization of an alpha -glucosidase capable of inactivating a host-spec ific toxin of fungal origin. The alpha -glucosidase is specific to RS toxin and is different from the known alpha -glucosidases. Significance and Impact of the Study: As RS toxin could be inactivated by t he microbial alpha -glucosidase enzyme, isolation of the gene that codes fo r the enzyme from T. viride and transfer of the gene to rice plants would l ead to enhanced resistance against sheath blight pathogen by inactivation o f RS toxin.