The function of vacuolar β-1,3-glucanase investigated by antisense transformation. Susceptibility of transgenic Nicotiana sylvestris plants to Cercospora nicotianae infection

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Abstract
Vacuolar class I β-1,3-glucanases (EC 3.2.1.39) are believed to be important in the induced defense reaction of plants to fungal infection. We used antisense transformation to test this hypothesis and to identify other possible physiological functions of this enzyme. Nicotiana sylvestris plants were transformed with antisense constructions containing the region from position 27 to 608 of the coding sequence of the basic, vacuolar β-1,3-glucanase gene GLA of tobacco regulated by cauliflower mosaic virus 35S RNA expression signals. Plants homozygous for this transgene showed a marked, ca. 20-fold reduction in the constitutive expression of class I β-1,3-glucanase antigen in their leaves. RNA blot analysis indicated that the antisense plants expressed low levels of the sense transcript of the host β-1,3-glucanase gene and the antisense transcript of the transgene. Immune blot analysis of plant extracts indicated that only expression of the N. sylvestris homologue of class I tobacco β-1,3-glucanase and not the acidic, class II isoforms of the enzyme was blocked in the antisense plants. Class I isoforms of β-1,3-glucanase and chitinase were coordinately induced in leaves of untransformed and empty-vector-transformed N. sylvestris plants treated with ethylene or infected with the fungal leaf pathogen Cercospora nicotianae. In antisense plants, chitinase but not β-1,3-glucanase was induced under these conditions indicating that antisense transformation effectively blocks constitutive as well as induced expression of class I β-1,3-glucanase. Under greenhouse conditions, antisense plants developed normally and were fertile. The plants did not exhibit increased susceptibility to C. nicotianae infection. These results suggest that expression of the β-1,3-glucanase isoform blocked by antisense transformation is not necessary for ‘house-keeping’ functions of N. sylvestris nor defense against the fungal pathogen tested.