'Pathogenesis-related' P1(p14) Protein. Vacuolar and Apoplastic Localization in Leaf Tissue from Tomato Plants Infected with Citrus Exocortis Viroid; in vitro Synthesis and Processing

Abstract

SUMMARYTomato 'pathogenesis-related' Pl(pl4) protein was synthesized in vitro, mRNAs were isolated from leaves showing characteristic symptoms of viroid infection, followed by chromatography on oligo(dT)-cellulose and the poly(A) + fraction was translated with a rabbit reticulocyte lysate system. No significant differences were found between the levels of [3SS]methionine incorporation directed by mRNA preparations from healthy and viroid-infected leaves. Only mRNA from infected leaves incorporated label into a protein that could be immunoprecipitated with rabbit IgG specific for tomato P 1 (p 14) protein. Analysis by SDS-PAGE of the immunoprecipitated protein from the in vitro translation system revealed that P 1 (p 14) was translated as a precursor protein of 2K to 3K larger than the Pl(pl4) that accumulated in vivo. This protein was converted to the mature form when translation was carried out in the presence of canine pancreatic microsomal membranes. By using Protein A-gold immunocytochemistry we have detected Pl(pl4) concentrated in dense inclusion bodies within the vacuole as well as in association with electron-dense material present in the intracellular spaces. The presence of the additional polypeptide sequence in the newly synthesized protein indicates that Pl(pl4) undergoes post-translational processing. The additional sequence is probably the signal peptide that directs its transport through the endoplasmic reticulum into the vacuolar compartment of tomato leaf cells and/or the intercellular space.

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

“…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).…”

“…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.…”

“…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.…”

Purification and Serological Characterization of Three Basic 15-Kilodalton Pathogenesis-Related Proteins from Tomato

“…electron-dense material in the intercellular spaces and with vacuolar 'inclusion bodies' (29,30). Hence, it was concluded that P14 could be involved in cell degeneration during senescence or pathogenic attack.…”