Immunogold localization of an extracellular beta-1,3-glucanase of the ergot fungus Claviceps purpurea during infection of rye

Immunogold labelling and electron microscopy were used to investigate beta- 1,3-glucanase secretion by Claviceps purpurea during ergot disease of rye i n situ. Molecular cytology allowed us to explore the hypothesis that this e nzyme might degrade host phloem callose to maintain flow of assimilates for fungal nutrition and therefore beta-1,3-glucanase could play a role in pat hogenicity. An extracellular endo-beta-1,3-glucanase was purified from axen ic culture and an antibody was raised. Enzyme activity staining and immunob lotting showed that the antibody was monospecific for beta-1,3-glucanase pr esent in fungal protein populations. Mycelia printings of plate-grown cultu res displayed spot-like and streaky immune-signals suggesting a secretion o f beta-1,3-glucanase at hyphal tips and young hyphae. The enzyme was immune gold localized predominantly in cell walls of mycelia from axenic culture. In Western blots of honeydew fractions, one beta-1,3-glucanase was immunor eactive. In inoculated plants, immunogold labelling was found in all infect ion stages and limited to the host-pathogen interface. Gold labelling was d etected over fungal protoplasts in vacuoles and in tubular-vesicular comple xes and multivesicular bodies, which fused with the fungal plasma membrane, indicating that they are part of the secretion pathway. The labelling of t he fungal secretory organelles and lack of labelling in any host area apart from the interface verified the fungal origin of beta-1,3-glucanase immune -detected in infected ovaries, Antigenic sites were located in external, su bcuticular, penetrating and intercellular hyphae, indicating that the enzym e was secreted throughout the colonization process in planta. Intense label ling regularly associated with fungal cell,walls extended into adjacent hos t walls, indicating a migration of the fungal beta-1,3-glucanase into the h ost apoplast. The gold labelling over host periplasmic spaces showed that t he enzyme reached the deposition sites of callose, pointing to an enzymatic suppression of putative plant defense reactions. The host phloem was colon ized inter- and intracellularly. Hyphae penetrated into the pectic middle l amella of sieve plates and intense immune-labelling for beta-1,3-glucanase in this area supports a phloem unblocking hypothesis.