Immunogold Localization of the Citrus Exocortis Viroid-Induced Pathogenesis-Related Proteinase P69 in Tomato Leaves

Abstract

Citrus exocortis viroid induces in tomato plants (Lycopersicon esculentum) synthesis and accumulation of a pathogenesis-related protein (P69) previously reported to be a proteinase (Vera P, Conejero V [1988] Plant Physiol 87: 58-63). By immunogold/ transmission electron microscopy, we have studied the distnbution of this protein in thin sections of parenchymatous leaf tissue. The enzyme was present intra-and extracellularly. The intracellular location was limited to the vacuole and was always associated with engulfed cell material. When extracellularly located, the enzyme was associated with a dispersed, electron-dense material in the intercellular spaces. This latter location was confirmed after analysis of intercellular washing fluids obtained by vacuum infiltration of leaves. These observations provide new data for the understanding of viroid pathogenesis and the biological role of the pathogenesis-related proteinase P69. stress responses. These enzymes may participate in biochemical defenses against pathogens.Here we present results of an immunological approach to determine the cellular site of accumulation of proteinase P69 in tomato leaf tissues infected with CEV. We have used a monospecific antiserum against P69 proteinase (25) to localize this protein by immunogold staining. We have found P69 in the vacuole and intercellular spaces of leaf parenchyma cells.

cDNA Cloning of Viroid-Induced Tomato Pathogenesis-Related Protein P23 (Characterization as a Vacuolar Antifungal Factor)

“…Figure 2A shows that P23 is present in the vacuole of leaf mesophyll cells in association with dense inclusion bodies. Unlike other tomato PR proteins (Vera et al, , 1989a(Vera et al, , 1989b, P23 was not detected in the leaf intercellular spaces. No significant labeling was found in ultrathin sections of CEVd-infected tomato leaves incubated with preimmune serum (Fig.…”

“…Additionally, tobacco cell cultures subjected to osmotic stress also contain inclusion bodies in which osmotin preferentially accumulates (Singh et al, 1987). Unlike the tomato PR proteins Pl(pl4) and P69, which are present in both vacuolar inclusion bodies and intercellular spaces (Vera et al, , 1989a(Vera et al, , 1989b, P23 was not detected in the apoplast. The specific vacuolar compartmentation of P23 is otherwise coincident with what is general for most basic PR proteins described to date (Bol et al, 1990).…”

“…Further characterization presented in this work has revealed that tomato PR P23 is a vacuolar protein that seems to be selectively localized in association with inclusion bodies. The nature of these vacuolar structures remains obscure, but we have found them to be present in cells from viroid-infected tissues and absent from healthy ones (Vera et al, 1989a(Vera et al, , 1989b. Additionally, tobacco cell cultures subjected to osmotic stress also contain inclusion bodies in which osmotin preferentially accumulates (Singh et al, 1987).…”

“…Tissue sample preparation for EM was performed as previously described (Vera et al, , 1989a(Vera et al, , 1989b. Ultrathin sections were placed on 200-mesh carbon-coated nickel grids and processed for protein A-colloidal gold immunostaining using the specific P23 antisera diluted 1:200.…”

A novel extracellular matrix protein from tomato associated with lignified secondary cell walls.

“…To determine more precisely the association of the tomato TLRP with the xylem elements, we examined ultrathin sections of resin-embedded leaf sections by immunoelectron microscopy as described previously for other proteins from tomato plants (Vera et al, 1989a(Vera et al, , 1989b. After the samples had been processed with antiTLRP serum and protein A-gold particles, the antigens were shown to be specifically localized in the cell walls of tracheary elements ( Figures 6A, 68, 6D, and 6F).…”