Subcellular localization by immunocytochemistry of the extracellular protease produced by Nectria galligena Bres. in infected apple tissue

Abstract

Existing evidence implicates an extracellular protease of Nectria galligena Bres. in the initiation of the accumulation of antifungal amounts of benzoic acid in immature apple fruit. It is the purpose of the present work to examine further this relationship between protease and the host cells by immunocytochemical techniques. Thin sections of infected apple tissue, fixed, and embedded in Lowicryl K4M at −40°C were reacted with monospecific antibodies raised in rabbits against the purified protease. Using a secondary tetramethylrhodamine isothiocyanate conjugated antibody, specific fluorescent localization of intercellular and intracellular fungal hyphae and host cell cytoplasm was demonstrated, thus establishing the presence of the protease in infected tissue. In a transmission electron microscope study, this was confirmed, and the localization and distribution of the enzyme further resolved by protein A-gold immunocytochemistry. Specific binding of colloidal gold was evident over, (1) intercellular and intracellular fungal hyphae, (2) electron-dense deposits located within and around hyphae and adjacent to partially degraded apple cell walls, and (3) peripheral vacuolar cytoplasm of intact host cells. These observations suggest that protease synthesized in hyphae of N. galligena is secreted into an extracellular matrix whence it may diffuse through host cell walls and detached, but entire, plasmalemmae observed in infected tissues. The protease is, therefore, able to reach sites in the host where it may elicit benzoic acid or where it may release an endogenous elicitor for that purpose. This is essential supporting evidence for the hypothesis that the accumulation of benzoic acid is initiated by fungal protease in vivo.