Ultrastructure, cytochemistry and immunocytochemistry of the interaction between wheat (Triticum aestivum) and leaf rust (Puccinia Recondita f.sp. tritici)
The development of infection structures, derived from urediospores of Puccinia recondita f.sp. tritici in near-isogenic lines of susceptible and resistant wheat, and in non-hosts, viz. maize, oat, sorghum and barley, was examined by fluorescence microscopy and scanning electron microscopy (SEM). The infection structure formation on and in five cereal species follows a similar pattern. In sorghum, fungal development is arrested at the stage of substomatal vesicle formation, while, in maize, most fungal structures collapse during the stage of primary hypha development. On the other hand, in wheat, barley and oat, the fungus forms many branched infection hyphae and haustorial mother cells. There were no significant structural and numerical differences in infection structure development between susceptible and resistant wheat lines. The ultrastructure of intercellular hyphae and D-haustoria of P. recondita f.sp. tritici, and the host response to haustorial invasion, was investigated. The intercellular hyphae share common characteristics with other uredial stage rust fungi. Anastomosis was observed between intercellular hyphae. Two nucleoli were frequently observed in a single nucleus in the haustorium, indicating possible nuclear fusion between the two nuclei in D-haustoria of this fungus. The close association of host organelles, such as the nucleus, Golgi bodies, endoplasmic reticulum , vesicles and mitochondria, with the developing haustorium, was described. The investigation of urediospore formation of P. recondita f.sp. tritici on wheat leaves by SEM and transmission electron microscopy (TEM) showed that one or more protuberances arise sympodially from several different loci on the distal surface of a basal cell, each protuberance developing into a urediospore. At the same site at which one urediospore formed previously, at least one other urediospore initial can form subsequently. A study of the cytochemistry of the interaction between wheat and P. recondita f.sp. tritici, using various enzyme- and lectin-conjugated gold probes, was conducted. This research provided additional information on the nature and composition of the walls of fungal hyphae, the haustorial mother cell, the haustorial neck, the haustorial body and the extrahaustorial matrix. Cellulose, the major component of the host cell wall, was not detect.ed in the extrahaustorial matrix and in the host tubules associated with the invaded haustorium. The composition of walls of the haustorial body of P. recondita f .sp. tritici appears to change as the haustorium matures. The study identified the existence of mannose/glucose, galactose, N-acetylgalactosamine and fucose residues in the extrahaustorial matrix. An antiserum raised against the purified 33 kDa wheat β-1 ,3-glucanase was used to investigate the subcellular localization of the enzyme in P. recondita f.sp. tritici-infected wheat leaves by means of a post-embedding immunogold labelling technique. In the compatible interaction, β-1,3-glucanase was demonstrated to accumulate predominantly in the haustorial wall and extrahaustorial matrix. In the incompatible interaction, strong labelling for β- 1 ,3-glucanase was found in host cell wall appositions, extracellular matrix in the intercellular space, and in electron-dense structures of host origin which only occurred in the incompatible interaction. Using anti-zeatin riboside and anti-isopentenyladenosine antibodies in post-embedding immunocytochemical procedures, cytokinins were localized at the ultrastructural level in P. recondita f .sp. tritici-infected wheat leaves. The sites where cytokinins accumulate were not significantly different between the compatible and incompatible interactions. The cytokinins are mainly present in the fungal cytoplasm of the intercellular hyphal cell, the haustorial mother cell, the haustorial body and extrahaustorial matrix, indicating that cytokinins, primarily of fungal origin, are associated with the nutrient translocation in this host-fungus interaction.