Isolation and partial characterization of a complementary protein from the mycoparasite Piptocephalis virginiana that specifically binds to two glycoproteins at the host cell surface

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Immunofluorescence microscopy was used to detect in the mycoparasite Piptocephalis virginiana the presence of a complementary glycoprotein that binds specifically to the host cell surface glycoproteins b and c, reported earlier from our laboratory. Germinated spores of P. virginiana treated with cell wall extract of the host Mortierella pusilla, primary antibody prepared against cell wall glycoproteins b and c, and fluorescein isothiocyanate (FITC) – goat anti-rabbit IgG conjugate showed fluorescence. Immunobinding analysis identified from the mycoparasite a protein of 100 kDa that binds with the host glycoproteins b and c, separately as well as collectively. Its purification was achieved by (i) 60% ammonium sulfate precipitation, (ii) heat treatment, (iii) Sephadex G-100 gel filtration, and (iv) preparative polyacrylamide gel electrophoresis (PAGE). The purity was ascertained by sodium dodecyl sulphate (SDS) – PAGE and Western blot analysis. Positive reaction to periodic acid – Schiff s reagent revealed its glycoprotein nature, and mannose was identified as a major sugar component. The specificity of the polyclonal antibody raised against electrophoretically purified complementary protein in rabbit was confirmed by dot immunobinding and Western blot analyses. Immunofluorescence microscopy revealed surface localization of the protein on the germ tubes of P. virginiana. Fluorescence was also observed at the surface of the germinated spores and hyphae of the host M. pusilla, after treatment with complementary protein from P. virginiana, primary antibody prepared against the complementary protein, and FITC – goat anti-rabbit IgG conjugate.Key words: biotrophic mycoparasite, cell surface agglutinin, glycoprotein immunobinding, immunofluorescence, mucoraceous host.