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

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.

Transcriptional Down-Regulation by Abscisic Acid of Pathogenesis-Related β-1,3-Glucanase Genes in Tobacco Cell Cultures1

“…The results shown in Table I confirm the basic pattern of regulation of ␤GLU I and CHN I by auxin and cytokinin reported previously (Shinshi et al, 1987;Vö geli-Lange et al, 1994b): the activity of both ␤GLU and CHN was high in auxin-and cytokinin-free medium, reduced in medium containing auxin or cytokinin alone, and markedly reduced in medium containing both hormones. The important point is that ABA effectively inhibited the induction of ␤GLU I but did not affect induction of CHN I in the presence of different combinations of auxin and cytokinin.…”

“…High levels of ethylene-dependent transcription of ␤GLU I genes requires a cis-acting, ethylene-responsive element that binds ethylene-responsive binding proteins in nuclear extracts (Ohme-Takagi and Shinshi, 1995). Different regions of the Glb promoter are important for high-level, ethylenedependent expression and for the down-regulation of expression by auxin and cytokinins (Vö geli-Lange et al, 1994b). We found that inhibition of ␤GLU I expression by ABA was not appreciably affected by combinations of auxin and cytokinin that down-regulate expression of ␤GLU I and CHN I.…”

“…GUS activity was measured by a modification of the fluorometric method of Jefferson et al (1987) described by Vö geli- Lange et al (1994b) and is expressed as picomoles of 4-methylumbelliferol produced per minute. Protein concentration was measured by the Bradford (1976) method with bovine ␥-globulin as the standard.…”

“…The 1.6-kb 5Ј sequence of Glb used in the construct had been shown earlier to contain the elements needed for organ-specific expression, induction by ethylene and TMV infection, and down-regulation in cultured tissues by auxin and cytokinin (Vö geli-Lange et al, 1994b). A 35S-GUS transformant carrying the GUS-reporter gene and driven by a 528-bp cauliflower mosaic virus 35S RNA promoter was used as a positive control (Hart et al, 1993).…”

Constitutive expression of an inducible β-1,3-glucanase in alfalfa reduces disease severity caused by the oomycete pathogenPhytophthora megasperma f. spmedicaginis, but does not reduce disease severity of chitin-containing fungi

“…The hydrolytic enzymes chitinase and [3-1,3-glucanase are induced in plants by pathogen attack or treatment with biotic or abiotic elicitors, and are also regulated by developmental cues such as changes in hormone levels Zhu et al, 1993;V6geli-Lange et al, *To whom correspondence should be addressed.…”