Association of β-1,3-glucanase activity and isoform pattern with systemic resistance to blue mould in tobacco induced by stem injection with Peronospora tabacina or leaf inoculation with tobacco mosaic virus

Abstract

Stem injection of tobacco plants with sporangiospores of Peronospora tabacina or inoculating three to four lower leaves with tobacco mosaic virus (TMV) systemically protected plants against blue mould caused by P. tabacina and systemically increased total β-1,3-glucanase activity. The onset and level of systemic protection was correlated with the elevation of the total enzyme activity in the systemically protected leaves. The total enzyme activity was higher in the protected plants than in the controls at the time of challenge with P. tabacina and during the first 1−4 days after challenge. Direct detection of β-1,3-glucanases after native 15% polyacrylamide gel electrophoresis (PAGE) run with an anodic buffer system revealed the presence of four major isoforms, designated G1, G2, G3 and G4, in the challenged tobacco leaves. The activities of G1 and G2 were higher in leaves of systemically protected plants than in those of the controls during early stages after challenge. G3 was not associated with protection. Its activity in the protected plants did not change before or after challenge. A similar activity was present in the control plants before challenge and at early stage after challenge, but the activity decreased in the control after the appearance of symptoms. The same three isoforms, based on electrophoretic mobilities, were obtained from Ky 14 plants induced by leaf inoculation with TMV or by stem injection with P. tabacina or from Samsun NN plants induced by leaf inoculation with TMV. G4 appeared at the time of symptom expression in control plants, and it appeared to be produced by P. tabacina. Experiments with protoplasts and intercellular fluid indicated that G1, G2 and G3 were predominantly located in intercellular spaces and G3 appeared bound more tightly to cell walls than G1 and G2. These β-1,3-glucanase isoforms were compared on PAGE gels with the four β-1,3-glucanases and 10 PR-proteins previously reported in tobacco. G1 co-migrated with PR-N (2b); G2 co-migrated with PR-O (2c); G3 did not co-migrate with any of the 10 PR-proteins or four β-1,3-glucanases reported previously. Increasing the amount of total protein of crude enzyme extract applied to 15% PAGE gels from 200 μg to 400 μg per lane revealed two more β-1,3-glucanase bands, one co-migrated with PR-2 (2a) and another (designated as G5) that did not co-migrate with any of the 10 PR-proteins or four β-1,3-glucanases previously reported in tobacco. These isoforms were more easily detected on 10% PAGE gels. G5 and PR-2 (2a) were also systemically induced in the TMV-induced plants.