The glycosylation status and the role of carbohydrate moieties in the heterogeneity of cucumber anionic virus-inducible peroxidase.

Three forms of anionic peroxidase (PRX) from hypersensitively reacting cucumber cotyledons were purified to homogeneity and different methods were used to analyze the nature of their carbohydrate chains. Immunoblot analysis with betaF1 antiserum showed that all three forms are highly glycosylated and contain asparagine N-linked glycans commonly found in other plant glycoproteins. Mobility shift analysis showed that chemical deglycosylation converted PRXs 1, 2 and 3 to the same-sized (35 K) products. Enzymatic deglycosylation with alpha-mannosidase converted PRX1 and PRX2 to immunoreactive products migrating in mobility shift polyacrylamide gels at the positions of PRX2 and PRX3, respectively. PRX3 treated with alpha-mannosidase yielded a product with Mr similar to that obtained with the chemical deglycosylation. Cleavage of the PRXs 1, 2 and 3 by formic acid at the Asp-Pro site resulted in peptide maps and the putative glycopeptide(s) were recognized using betaF1 antiserum. Only one glycopeptide was observed for each of the forms. Lectin-affinity blot analysis using biotin-conjugated lectins suggested that virus-inducible PRX contains complex-type N-glycosyl carbohydrate chain(s). These results indicate that heterogeneity of cucumber virus-inducible PRX is not caused mainly by differences in the terminal alpha-linked mannose residues.

Differential sensitivity of purified isoforms of cucumber anionic virus-inducible peroxidase to exogenous proteases.

Three different molecular forms, isoforms, of the major virus-inducible anionic peroxidase (PRX) of cucumber (Cucumis sativus L.) were purified to homogeneity from crude extracts of hypersensitively reacting cotyledons and subjected to proteolysis with five exogenous endoproteinases. The PRX isoforms were fully resistant to degradation by trypsin and chymotrypsin even though at a prolonged incubation. Partial proteolysis with pepsin yielded peptides which were similar in size and serological properties. When papain was used, the peptides released from PRX1 isoform differed both in size and number but not serologically from the peptides released from isoforms PRX2 and PRX3 confirming similar primary structure of polypeptide chains. PRX3 was the only substrate giving a peptide map after incubation with protease K. Under experimental conditions used in this work, PRXI and PRX2 were degraded completely with protease K. These results indicate that PRX1, PRX2, and PRX3 contain similar antigenic determinants and indicate very similar but not identical primary structures. Several practical implications of the present study are also mentioned.

Induction and distribution of amylolytic activity in Cucumis sativus L. in response to virus infection.

Inoculation of cucumber (Cucumis sativus L. cv. Laura) cotyledons with tobacco necrosis virus (TNV) causes both qualitative and quantitative changes in the total and fractionated protein extracts as well as in amylolytic activity. Using a specific test it was demonstrated that the virus infection strongly enhances a major band (Rf 0.0645) of amylolytic activity, predominantly located in apoplast space. The accumulation of this extracellular amylolytic activity is regulated by a time-dependent manner and is correlated with the development of necrotic lesions. The amylolytic activity may be related to degradation of starch shown to be accumulated in the immediate vicinity of necrotic lesions associated with the hypersensitive response (HR). The possible biological function of the identified amylolytic activity in the term of ,,pathosmosis" is also discussed.

Immunological identification of a basic homologue to acidic virus-inducible cucumber peroxidase.

Eight basic, fast-moving pathogenesis-related peroxidase PR-PRX isoenzymes were found to accumulate in cotyledons of cucumber (Cucumis sativus L., cv.Laura) reacting hypersensitively to tobacco necrosis virus (TNV). These isozymes were provisionally designated a, b, c, d, e, f, and g in the order of decreasing electrophoretic mobility in native cathodic polyacrylamide gels. Besides the differential mode of compartmentalization, some of these isoenzymes serologically cross-reacted with the antiserum prepared against the most prominent and best characterized cucumber PR-protein, an anionic PR-PRX. The immunoblot analysis confirmed that, by analogy with the tobacco PR-proteins 1, acidic and basic PR-PRXs also belong to a family of differently charged isomers.

Intra- and extracellular isoforms of PR-3 class chitinase in virus-infected cucumber plants.

Cucumber (Cucumis sativus L. cv. Laura) contains three different isoforms of chitinase (EC 3.2.1.14), thought to be involved in the defense against a pathogen. Using a highly specific rabbit antiserum raised against a predominant, extracellularly localized, virus-inducible acidic chitinase (p28), two additional enzyme isoforms with differential mode of compartmentalization were identified. Immunoblot analysis of the fractionated plant extracts separated by denaturing and native (anodic and cathodic) polyacrylamide gel electrophoresis (PAGE) revealed that the chitinase p25 (M(r) 25.5 K), is a basic, extracellular isoform and the chitinase p24 (M(r) 24.6 K) is an acidic, probably intracellular isoform of the enzyme.

Serological relationships between tobacco necrosis virus-inducible cucumber peroxidase and corresponding peroxidase isoenzymes in other Cucurbitaceae.

Pathogenesis-related peroxidases (PRXs), found in leaves of different cucurbits infected with tobacco necrosis virus (TNV), were compared serologically using a highly specific rabbit antiserum raised against PRX purified from TNV-infected cotyledons of Cucumis sativus L., cv. Laura. After native polyacrylamide gel electrophoresis (PAGE) of intercellular washing fluid extracts from a large number of species of this family, immunoblot analysis revealed the presence of cross-reacting protein bands in all species tested. Despite the serological relatedness, the analysis also revealed that the antiserum recognized only fast-moving PRX isoenzymes localized in the apoplast space.

Identification and partial characterization of beta-1,3-glucanase from virus-infected cucumber cotyledons.

One of the five "pathogenesis-related" (PR) proteins known to accumulate in Cucumis sativus L. cv. Laura and to react hypersensitively to tobacco necrosis virus (TNV) was shown to to have beta-1,3-glucanase activity. The TNV-induced acidic beta-1,3-glucanase activity increased 6-fold after infection and had extracellular localization and estimated M(r) of 25,700. The beta-1,3-glucanase activity was investigated with a new method of activity staining using a conjugated substrate in overlay gels. By using the antiserum against tobacco beta-1,3-glucanase purified to homogeneity, a close serological relationship was demonstrated between cucumber and tobacco beta-1,3-glucanases on immunoblots.

A virus-inducible cucumber anionic peroxidase has a serological counterpart in different plant species.

A highly specific rabbit antiserum raised against anionic peroxidase (PRX) purified from cucumber (Cucumis sativus L., cv. Laura) cotyledons infected with tobacco necrosis virus (TNV) was used to further investigate the antigenic relatedness of peroxidases in the selected plant species. Immunoblot analysis using native polyacrylamide electrophoresis (PAGE) revealed that the antiserum recognized only fast-moving anionic PRX isoenzymes and that cross- reacting protein bands were present in five plant species belonging exclusively to the Solanaceae family. There was no serological cross-reaction indicated in virus-infected plant species from three other dicotyledonous families (Amaranthaceae, Chenopodiaceae and Leguminosae). Marked qualitative, as well as quantitative differences in the degree of serological relatedness were apparent.