A bean epicuticular glycoprotein is present in the extracellular matrices around infection structures of the anthracnose fungus, Colletotrichum lindemuthianum.

Monoclonal antibodies were raised to Colletotrichum lindemuthianum (Sacc. & Magn.) Briosi & Cav. infection structures which had been isolated from leaves of Phaseolus vulgaris L. by isopycnic centrifugation and immunomagnetic separation. One of the antibodies, UB29, recognized a carbohydrate epitope in a 200 kDa glycoprotein present on the surface of conidia, germ-tubes and appressoria when they developed on host tissue. Intracellular hyphae were not labelled. Immunogold labelling showed that the antigen was confined to the extracellular matrices around such structures. No such antigen was detected by immunofluorescence or Western blotting when the fungus developed in vitro. UB29 recognized a glycoprotein of identical Mr located in the epicuticular wax layer of uninoculated bean hypocotyls. The results suggest that a host epicuticular glycoprotein is present in the extracellular matrices of fungal structures during growth on the plant surface.

Immunomagnetic isolation of viable intracellular hyphae of Colletotrichum lindemuthianum (Sacc. & Magn.) Briosi & Cav. from infected bean leaves using a monoclonal antibody.

A method is described for isolating intracellular hyphae (IH, i.e. infection vesicles and primary hyphae) of Colletotrichum lindemuthianum (Sacc. & Magn.) Briosi & Cav. from infected leaves-of bean (Phaseolus vulgaris L.). 1H were recovered from homogenates of infected leaves after filtration through a 45 µm nylon mesh and isopyenic centrifugation on Percoll. 1H were then affinity-purified by immunomagnetic separation using Dynabeads coated with monoclonal antibody UB25, specific for IH surface glycoproteins. The method yielded 7 × 104 IH g-1 f. wt leaf tissue, with 27% purity and 62% viability, as judged by staining with fluorescein diacetate. The Viability of isolated IH was confirmed by their ability to grow in nutrient medium and by the normal ultrastructure of their cytoplasm. The host plasma membrane and matrix layer which surround IH in planta were absent from isolated IH. Staining with lectins. Calcofluor and aniline blue showed that the walls of IH contain N-acetylgalactosamine. α-linked mannose residues and ß-linked polysaccharides, including-chitin and ß-1,3-glucans. Potential uses of the isolated IH are discussed.

Identification of glycoproteins specific to biotrophic intracellular hyphae formed in the Colletotrichum lindemuthianum-bean interaction.

Monoclonal antibodies (MAbs) specific for intracellular for hyphae (IH, i.e. infection vesicles and primary hyphae). Appressoria/germ tubes and conidia of Colletotrichum lindemuthianum (Sace, & Magn.) Briosi & Cav. isolated from infected leaves of Phaseolus vulgoria L. were obtained using a co-immunization procedure. One of the MAhs; UB25, bound specifically to IH in immunofluorescence immunogold and Western blot assays: it showed no affinity for conidia, conidial germ tubes, appressoria or appressorial germ tubes growing in vitro, of for any plant components. Immunogold labeling of infected tissue prepared by high pressure freezing, freeze-substitution and low temperature embedding showed that the UB25 antigen was present in the interfacial matri surrounding IH and in the fungal wall. The antigen was confined to infection vesicles and primary hyphae in contact with host protoplast and could not be detected in primary hyphae growing in intercellular spaces. UB25 recognizes a protein epitope present in a set of N-linked glycoproteins. These glycoproteins are expressed at an early stage of intracellular development, suggesting a possible role in biotrophy or recognition.