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      Developmental, hormonal, and pathogenesis-related regulation of the tobacco class I beta-1,3-glucanase B promoter.

      Plant Molecular Biology

      Cytokinins, pharmacology, Down-Regulation, Ethylenes, Gene Expression Regulation, Enzymologic, drug effects, Genes, Reporter, Glucan Endo-1,3-beta-D-Glucosidase, chemistry, genetics, Indoleacetic Acids, Plant Growth Regulators, Plants, Genetically Modified, Plants, Toxic, Promoter Regions, Genetic, Recombinant Fusion Proteins, biosynthesis, Tobacco, enzymology, growth & development, microbiology, Tobacco Mosaic Virus, physiology

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          Abstract

          The class I beta-1,3-glucanases are antifungal vacuolar proteins implicated in plant defense that show developmental, hormonal, and pathogenesis-related regulation. The tobacco enzymes are encoded by a small gene family with members derived from ancestors related to the present-day species Nicotiana sylvestris and N. tomentosiformis. We studied the expression in transgenic tobacco plants of a chimeric beta-glucuronidase (GUS) reporter gene fused to 1.6 kb of upstream sequence of the tobacco class I beta-1,3-glucanase B (GLB) gene, which is of N. tomentosiformis origin. Expression of the GUS reporter gene and the accumulation of class I beta-1,3-glucanase and its mRNA showed very similar patterns of regulation. In young seedlings the reporter gene was expressed in the roots. In mature tobacco plants it was preferentially expressed in lower leaves and roots and was induced in leaves by ethylene treatment and by infection with tobacco mosaic virus (TMV). Furthermore, it was down-regulated in cultured leaf discs by combinations of the hormones auxin and cytokinin. Histological studies of GUS activity showed that the GLB promoter shows highly localized expression in roots of seedlings. It is also expressed in a ring of cells around necrotic lesions induced by TMV infection, but not in cells immediately adjacent to the lesions or in the lesions themselves. The results of deletion analyses suggest that multiple positive and negative elements in the GLB promoter regulate its activity. The region from -1452 to -1193 containing two copies of the heptanucleotide AGCCGCC, which is highly conserved in plant-stress and defense-related genes, is necessary for high level expression in leaves. Additional regions important for organ-specific and regulated expression were: -568 to -402 for ethylene induction of leaves; -402 to -211 for expression in lower leaves and cultured leaf discs and for TMV induction of leaves; and -211 to -60 for expression in roots.

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          Most cited references 23

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          Biochemical Mechanisms of Disease Resistance

           A Bell (1981)
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            The effect of T-DNA copy number, position and methylation on reporter gene expression in tobacco transformants.

            Inter-transformant variability in the expression of introduced genes was studied in the R1 and R2 generations of 10 tobacco transformants, produced by Agrobacterium-mediated transformation. In replicated and physiologically equivalent material, tranformants showed considerable variability in the expression of the reporter gene uidA as shown by transcript levels and beta-glucuronidase (GUS) activity. However, homozygous R2 material could be investigated for seven of the transformants and among these, and in one line in which two inserts could segregate independently, this inter-transformant variability was reduced to simple bimodal expression. The two levels of expression for GUS activity in leaves were high or low (approximately 2.5 or 0.3 nmol cm-2 min-1 respectively), with no continuous variation. Transformants in the high group had single T-DNA insertions, while those in the low group had multiple T-DNA insertions, at the same or different loci. Within each group, although T-DNA was apparently integrated at different sites in the plant genome, there was no evidence of position effects. GUS activity levels of the transformants were very similar in the field and in environmentally controlled conditions under high or low light. Plants with multiple insertions and low expression also tended to have increased methylation of the integrated T-DNA.
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              Tobacco genes encoding acidic and basic isoforms of pathogenesis-related proteins display different expression patterns.

              The induction by cytokinin stress and ethylene of nine different tobacco mosaic virus-inducible mRNA classes (termed A-I) encoding pathogenesis-related (PR) proteins was studied. The induced mRNA levels were compared to basal levels in healthy tobacco plants grown in tissue culture and in a greenhouse. Cytokinin stress and ethylene were found to induce different subsets of the mRNAs, indicating that ethylene is not the primary inducing signal in cytokinin-stressed shoots. mRNAs F, H and G encoding the basic hydrolytic enzymes chitinase, beta-1,3-glucanase and a basic equivalent of PR-1, respectively, were found to be expressed at high levels in roots of healthy plants. mRNAs D, I and B encoding the acidic equivalents of the proteins proved to be present at low levels in healthy plants. These results indicate that genes encoding basic and acidic isoforms of pathogenesis-related proteins are differentially regulated.
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                8018877

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