In hot pepper, the sesquiterpene phytoalexin capsidiol is catalyzed by the two final-step enzymes, a sesquiterpene cyclase (EAS) and a hydroxylase (EAH), which are genetically linked and present as head-to-head orientation in the genome. Transcriptomic analysis revealed that a subset of EAS and EAH is highly induced following pathogen infection, suggesting the coregulation of EAS and EAH by a potential bidirectional activity of the promoter (pCaD). A series of the nested deletions of pCaD in both directions verified the bidirectional promoter activity of the pCaD. Promoter deletion analysis revealed that the 226 bp of the adjacent promoter region of EAS and GCC-box in EAH orientation were determined as critical regulatory elements for the induction of each gene. Based on promoter analyses, we generated a set of synthetic promoters to maximize reporter gene expression within the minimal length of the promoter in both directions. We found that the reporter gene expression was remarkably induced upon infection with Phytophthora capsici, Phytophthora infestans, and bacterial pathogen Pseudomonas syringae pv. tomato DC3000 but not with necrotrophic fungi Botrytis cinerea. Our results confirmed the bidirectional activity of the pCaD located between the head-to-head oriented phytoalexin biosynthetic genes in hot pepper. Furthermore, the synthetic promoter modified in pCaD could be a potential tool for pathogen-inducible expression of target genes for developing disease-resistant crops.

Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

在辣椒中，倍半萜烯植物抗坏血酸辣椒素被两个最终步骤的酶催化，即倍半萜烯环化酶（EAS）和羟化酶（EAH），二者之间存在遗传联系并在基因组中以头对头的方向存在。转录组学分析显示，病原体感染后高度诱导了EAS和EAH的一个子集，表明启动子（pCaD）的潜在双向活性可调控EAS和EAH。pCaD在两个方向上的一系列嵌套缺失证实了pCaD的双向启动子活性。启动子缺失分析表明，EAH中EAS和GCC-box的相邻启动子区域为226 bp确定方向是诱导每个基因的关键调控元件。基于启动子分析，我们生成了一组合成的启动子，以在两个方向的启动子的最小长度内最大化报告基因的表达。我们发现，在感染了疫霉疫霉，致病疫霉和细菌性病原体丁香假单胞菌pv后，报告基因的表达被明显诱导。番茄DC3000，但不添加坏死性真菌灰葡萄孢。我们的结果证实了辣椒中以头对头的植物抗毒素生物合成基因之间的pCaD的双向活性。此外，在pCaD中修饰的合成启动子可能是病原体诱导目标基因表达以开发抗病作物的潜在工具。

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