Characterization of accumulation of tobacco PR-5 proteins by IEF-immunoblot analysis.

Accumulation of group 5 pathogenesis-related (PR) proteins in tobacco was characterized by immunological analysis combined with isoelectric focusing. This method clearly distinguished 3 subclasses of PR-5 proteins; basic (osmotin), neutral (osmotin-like protein: OLP) and acidic (PR-S). A high accumulation of PR-5 proteins was detected only in root tissues in which neutral osmotin-like protein was mainly accumulated, in addition to a small amount of osmotin. Immunohistochemical analysis revealed that OLP accumulated in the cortex of the root. Cultured tobacco cells accumulated large amounts of neutral PR-5 proteins (OLP) in cells and the acidic form (PR-S) in the medium. Adaptation to salt stress was associated with a higher accumulation of basic PR-5 (osmotin) and less neutral PR-5. In an analysis of the effect of biotic and abiotic stimuli on the synthesis of PR-5 proteins in leaf tissues, ethylene was found to induce a high accumulation of basic and neutral PR-5 proteins. Tobacco mosaic virus (TMV) infection induced accumulation of all major PR-5 proteins, but induction of OLP was less than that of the other isoforms. Systemic induction of PR-5 proteins in upper non-infected leaves was not observed. Salicylate induced only a small accumulation of PR-S. These results indicate that each PR-5 protein has an independent regulatory pathway for its gene expression.