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Regulation of sesquiterpene cyclase gene expression : Characterization of an elicitor- and pathogen-inducible promoter

The promoter for a tobacco (Nicotiana tabacum) sesquiterpene cyclase gene, a key regulatory step in sesquiterpene phytoalexin biosynthesis, has been analyzed. The EAS4 promoter was fused to the [beta]-glucuronidase (GUS) reporter gene, and the temporal and spatial expression patterns of GUS activity were examined in stably transformed plants and in transient expression assays using electroporated protoplasts of tobacco. No GUS activity was observed in any tissues under normal growth conditions. A low level of GUS activity was detected in wounded leaf, root, and stem tissues, whereas a much higher level was observed when these tissues were challenged with elicitors or microbial pathogens. The GUS expression pattern directed by the EAS4 promoter was identical to the induction patterns observed for the endogenous sesquiterpene cyclase genes. Neither exogenous salicylic acid nor methyl jasmonate induced GUS expression; and H2O2 induced GUS expression to only a limited extent. Although the EAS4 promoter contains cis-sequences resembling previously identified transcriptional control motifs, other cis-sequences important for quantitative and qualitative gene expression were identified by deletion and gain-of-function analyses. The EAS4 promoter differs from previously described pathogen-/elicitor-inducible promoters because it only supports inducible gene expression and directs unique spatial expression patterns.

Sesquiterpene cyclase is not a determining factor for elicitor- and pathogen-induced capsidiol accumulation in tobacco

Abstract. The induction of sesquiterpene cyclase, a key phytoalexin biosynthetic enzyme, and the accumulation of phytoalexins in relation to the induction of a hypersensitive response (HR) and cell necrosis in tobacco (Nicotiana tabacum L.) were investigated. When tobacco leaves were inoculated with virulent or avirulent isolates of Ralstonia solanacearum, steady-state levels of mRNA complementary to cDNA of the sensitivity-related (sts) gene str319 were dramatically induced. This cDNA clone is greater than 90% homologous with a gene coding for 5-epi-aristolochene synthase (EAS), previously described as a branch-point enzyme regulating the synthesis of capsidiol, the major sesquiterpenoid phytoalexin found in tobacco. Accumulation of EAS transcripts in leaves after inoculation with virulent and avirulent strains of R. solanacearum, or after treatment with necrotizing or non-necrotizing elicitins was rapid but transient, and restricted to the site of infiltration. Two highly similar sesquiterpene cyclase activities, 5-epi-aristolochene synthase and a vetispiradiene synthase-like activity, were found in extracts of elicitin-challenged and R. solanacearum-inoculated tobacco. Under all conditions tested, the induction of cyclase activity was closely correlated with induction of the cyclase mRNA level. In contrast, high levels of capsidiol were found only after treatment with the necrosis-inducing elicitin cryptogein, or after infiltration with HR-inducing bacterial strains. Low levels of capsidiol did accumulate after application of capsicein, an elicitin that induces little or no necrosis on tobacco, or after infection with a virulent bacterium. Hence, capsidiol accumulation, not 5-epi-aristolochene synthase gene expression or total sesquiterpene cyclase enzyme activity, appears to be a good marker for the HR of tobacco.