'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

SUMMARY Tomato ‘pathogenesis-related’ P1(p14) 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 [35S]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 P1(p14) protein. Analysis by SDS-PAGE of the immunoprecipitated protein from the in vitro translation system revealed that P1 (p14) was translated as a precursor protein of 2K to 3K larger than the P1(p14) 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 P1(p14) 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 P1(p14) 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.