Abstract:
Presence of surface glycoprotein in Piptocephalis virginiana that
recognizes the host glycoproteins band c, reported earlier from our
laboratory, was detected by immunofluorescence microscopy.
Germinated spores of P. virginiana treated with Mortierella pusilla cell
wall protein extract, primary antibodies prepared against glycoproteins
band c and FITC-goat anti-rabbit IgG conjugate showed fluorescence.
This indicated that on the surfaces of the biotrophic mycoparasite P.
virginiana , there might be a complementary molecule which recognizes
the glycoproteins band c from M. pusilla. Immunobinding analysis
identified a glycoprotein of Mr 100 kDa from the mycoparasite which
binds with the host glycoproteins band c, separately as well as
collectively. Purification of this glycoprotein was achieved by (i) 60%
ammonium sulfate precipitation, (ii) followed by heat treatment, and
(iii) Sephadex G-IOO gel filtration. The glycoprotein was isolated by
preparative polyacrylamide gel electrophoresis by cutting and elution.
The purity of the protein ·was ascertained by SDS-PAGE and Western
blot analysis. Positive reaction to periodic acid-Schiff reagent revealed
the glycoprotein nature of this 100 kDa protein. Mannose was identified
as a major sugar component of this glycoprotein by using a BoehringerMannheim
Glycan Differentiation Kit.
Electrophoretically purified glycoprotein was used to raIse
polyclonal antibody in rabbit. The specificity of the antibody was
determined by dot-immunobinding test and western-blot analysis. Immunofluorescence mIcroscopy revealed surface localization of the
protein on the germ tube of Piptocephalis virginiana. Fluorescence was
also observed at the surfaceJ 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.
