Search

In tobacco leaves, pathogenesis-related (PR) 1 proteins are abundantly induced by hypersensitive reaction to the infection with tobacco mosaic virus (TMV) and by treatment with salicylic acid, and are secreted into the intercellular spaces. To study the distribution of PR 1 proteins outside of the cells, the immunogold technique was used with anti-PR 1 antibody. When sections of salicylate-treated tobacco leaf were reacted with antibody against PR 1a and then with protein A-gold complex, most of the gold label was localized in the intercellular spaces in the region between cells, and a little label was found in the cytoplasmic matrix and in small electron-dense granules inside the cells. When salicylate-treated leaves were incubated with polygalacturonase and/or cellulase to liberate protoplasts or single cells from the tissue, most of PR 1 proteins were solubilized far before complete liberation of single cells, suggesting their localization in free spaces or a region susceptable to maceration, such as the secondary cell wall.

The localization of pathogenesis-related (PR) proteins induced in tobacco leaves by treatment with potassium salicylate or a hypersensitive response to tobacco mosaic virus (TMV) infection was studied using immunochemical methods. Total PR protein levels increased with time after these treatments. The proportion of PR proteins in the intercellular spaces to the total content in the leaf discs rapidly rose in the later stage of the treatment to about 75% on the 9th day after salicylate treatment and to more than 80% on the 6th to 9th day after TMV inoculation. After 5 days of salicylate treatment, the amounts of PR proteins in the peeled leaf epidermis were two fold those in the mesophyll tissue. Only five percent or less of the total PR proteins in the epidermal and mesophyll tissues of salicylate-treated leaves were detected in the isolated epidermal and mesophyll protoplasts. The sugar content in highly purified PR la, lb and lc was less than one mole of monosaccharide per mole of each protein. These results show that the PR proteins are non-glycoproteins secreted into the intercellular spaces.

Pathogenesis-related proteins (PR proteins), that are induced in tobacco leaves in hypersensitive response to infection with tobacco mosaic virus (TMV) or by treatment with chemicals, were found to be also inducible in a dedifferentiated system, tobacco suspension culture. Quantitative determination of these proteins using anti PR 1a IgG showed that their increase started at about the end of cell growth period and that their production could be enhanced by the addition of potassium salicylate, Eosin Yellowish and plant hormones (GA3, IAA and 2,4-D). The production depended on the concentration of the chemical inducer and the cell line used. In BY-2 cell line, PR proteins amounted to 12 μg at day 5 and then increased exponentially with time, reaching 280 μg or 70 μg per g fr wt of cells at day 9 with or without the addition of 25 μm potassium salicylate. More than 90% of the induced PR proteins was found in the medium and less than 10% in the cells at day 9. Peroxidase activity in the medium was constant throughout the experiment although total activity in the flask increased with cell growth, indicating that PR proteins are actively secreted into the medium.

Salicylic acid (SA) has been proposed to be an endogenous signal for systemic acquired resistance to infection by pathogens in plants. In general, most SA is found in an inactive form as SA β-glucoside (SAG). SAG seems to be a storage form of SA from which bioactive SA can be generated. Recent reports indicate that β-glucosidase might be involved in regulating the signaling activity of phytohormones. Therefore, it seems likely that SA β-glucosidase, the enzyme that hydrolyzes SAG to yield free SA, might also play an important role by regulating the level of free SA. Since hydrolysis of SAG seems to occur in intercellular spaces, we attempted to isolate SA β-glucosidase activity from the intercellular spaces of SA-treated tobacco leaves, where we found considerable amounts of the enzymatic activity. Furthermore, increased levels of SA and SA β-glucosidase activity were found in the leaves after treatment with exogenous SA. The role of SA β-glucosidase in plant defense systems is discussed.

Constitutive expression of a type of stress-inducible proteins including pathogenesisrelated (PR) 1 protein and ubiquitin-related protein in an interspecific hybrid of Nicotiana glutinosa × Nicotiana debneyi was noted. In the two parental species and in tobacco, these proteins are not expressed in healthy plants but they are induced by stresses such as the formation of local lesions after viral infection and treatment of salicylic acid. A second type of stress-inducible genes, such as the genes for basic β-1,3-glucanase and putative proteinase inhibitor were regulated normally, and were not expressed constitutively in the hybrid. In the transgenic hybrid, into which a chimeric gene consisting of 5′ upstream of tobacco PRla gene and β-glucuronidase (GUS) gene was introduced, very high GUS activity was expressed constitutively even at healthy state. An abnormal response by this hybrid to plant hormones was also noted. A possible mechanism for the unregulated expression of the stress-inducible genes in the interspecific hybrid is discussed.

Pathogenesis-related (PR) proteins, which are induced in response to pathogenics and chemicals, were synthesized as larger precursors with in vitro translation systems, and these precursors were processed by dog pancreatic microsome membrane to become of mature size. Two-dimensional gel electrophoresis showed that at least six products were synthesized in vitro after immunoprecipitation with an antibody against PR1a (most acidic PR protein). These findings suggest the presence of at least six different kinds of precursors of the PR1 group (PR1), and also suggest the presence of a microheterogenous multi-gene family which expresses PR1 proteins in tobacco leaves. A comparison of the induction kinetics of mRNAs for PR1 proteins and the syntheses of these proteins in vivo and in vitro showed the similarity of these processes, suggesting that the synthesis of PR1 proteins is regulated by a transcriptional step and occurs without delay by induction of the production on of their mRNAs. A comparison between in vitro and in vivo pulse labeling systems prepared from the same leaves as to the incorporation efficiency of [35S]methionine into PR1 proteins showed that the in vivo incorporation ratio was at least 50 times higher than the in vitro one. However, for the incorporation of a small subunit of ribulose bisphosphate carboxylase, as a control protein, the ratio was two to three times higher in the in vivo case. These findings indicate that mRNAs for PR1 proteins are preferably translated to other normal proteins in chemically treated cells.

Stresses induce the synthesis of a large variety of proteins in plants. A set of novel soluble proteins, called pathogenesis-related (PR) proteins, are induced as a result of a hypersensitive reaction of host plants when plants are infected by incompatible pathogens. These proteins form one class of stress-induced proteins. Since the occurrence of PR proteins is coordinated with systemic acquired resistance of host plants to attack by pathogens, a role for these proteins in plant defense mechanisms has been postulated. The occurrence, nature and regulated expression of genes for PR proteins are described.