MICROWAVE FIXATION OF RUST-INFECTED WHEAT LEAVES - PRESERVATION OF FINE-STRUCTURE AND DETECTION OF CELL-SURFACE ANTIGENS, LECTIN-BINDING, AND SUGAR-BINDING SITES

Wheat leaves infected with stem rust (Puccinia graminis tritici) were infiltrated with fixative, subjected to microwave irradiation, and sliced with a vibratome. The slices were probed with antibodies, lectins, or neoglycoproteins, and processed for electron microscopy. In tissue irradiated for 10 sec to 40-degrees-C, 45-degrees-C, or 50-degrees-C, the quality of structural preservation was indistinguishable from that in control tissue subjected to conventional fixation (3 h in fixative at room temperature). The best preservation of fungal antigenic cell surface material was achieved with 10 sec of microwave-induced heating to 45-degrees-C in the presence of fixative, followed by 10 min in fixative at room temperature. Under these conditions, twice as many antigenic sites were detected on the fungal surface than in non-irradiated ("power-off" control, or conventionally-fixed) tissue, The microwave fixation protocol with heating to 45-degrees-C was used in experiments to probe infected tissue with lectins or neoglycoproteins. Most of these probes had been labelled with biotin, and this label was detected with goat anti-biotin IgG and rabbit anti-goat IgG/gold. The gold markers were localized mainly at some distance outside the outer wall layer of hyphal cells, indirectly confirming the presence of unstained extramural material that had been detected in earlier work in freeze-substituted specimens. Of seven lectins, all with demonstrated ability to bind to cross sections of intercellular hyphal walls, only concanavalin A and wheat germ lectin bound to the fungal surface. Of four neoglycoproteins, alpha-D-glucosyl- and alpha-D-mannosyl-BSA bound to this surface, but only the binding of the glucosyl conjugate was inhibitable with hapten. We concluded that the surface composition of these cells is less complex than previously suggested from studies using post-embedding cytochemistry.