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

Abstract

Monoclonal antibodies (MAbs) specific for intracellular hyphae (IH, i.e. infection vesicles and primary hyphae), appressoria/germ tubes and conidia of Colletotrichum lindemuthianum (Sacc. & Magn.) Briosi & Cav. isolated from infected leaves of Phaseolus vulgaris L. were obtained using a co-immunization procedure. One of the MAbs, UB25, bound specifically to IH in immunofluorescence, immunogold and Western blot assays: it showed no affinity for conidia, conodial germ tubes, appressoria or appressorial germ tubes growing in vitro, or for any plant components. Immunogold labelling of infected tissue prepared by high pressure freezing, freeze-substitution and low temperature embedding showed that the UB25 antigen was present in the interfacial matrix surrounding IH and in the fungal wall. The antigen was confined to infection vesicles and primary hyphae in contact with host protoplasts 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.