Immunolocalization of 1,3-β-Glucanases Secreted byGaeumannomyces graminisvar.triticiin Infected Wheat Roots

Abstract

The distribution of extracellular 1,3-β-glucanase secreted by Gaeumannomyces graminis var. tritici (Ggt) was investigated in situ in inoculated wheat roots by immunogold labelling and transmission electron microscopy. Antiserum was prepared by subcutaneously injecting rabbits with purified 1,3-β-glucanase secreted by the pathogenic fungus. A specific antibody of 1,3-β-glucanase, anti-GluGgt, was purified and characterized. Double immunodiffusion tests revealed that the antiserum was specific for 1,3-β-glucanase of Ggt, but not for 1,3-β-glucanase from wheat plants. Native polyacrylamide gel electrophoresis of the purified and crude enzyme extract and immunoblotting showed that the antibody was monospecific for 1,3-β-glucanase in fungal extracellular protein populations. After incubation of ultrathin sections of pathogen-infected wheat roots with anti-1,3-β-glucanase antibody and the secondary antibody, deposition of gold particles occurred over hyphal cells and the host tissue. Hyphal cell walls and septa as well as membranous structures showed regular labelling with gold particles, while few gold particles were detected over the cytoplasm and other organelles such as mitochondria and vacuoles. In host tissues, cell walls in contact with the hyphae usually exhibited a few gold particles, whereas host cytoplasm and cell walls distant from the hyphae were free of labelling. Furthermore, over lignitubers in the infected host cells labelling with gold particles was detected. No gold particles were found over sections of non-inoculated wheat roots. The results indicate that 1,3-β-glucanase secreted by Ggt may be involved in pathogenesis of the take-all fungus through degradation of callose in postinfectionally formed cell wall appositions, such as lignitubers.