Molecular characterization of pathogen-inducible bi-directional promoter from hot pepper (Capsicum annuum)

In hot pepper, the sesquiterpene phytoalexin capsidiol is catalyzed by the two final step enzymes, a sesquiterpene cyclase (EAS) and a hydroxylase (EAH) that 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 co-regulation of EAS and EAH by a potential bi-directional activity of the promoter (pCaD). A series of the nested deletions of pCaD in both directions verified the bi-directional promoter activity of the pCaD. Promoter deletion analysis revealed that the 226 base pair 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 the infection with Phytophthora capsici, Phytophthora infestans, and a bacterial pathogen, Pseudomonas syringae pv. tomato DC3000, but not with a necrotrophic fungi Botrytis cinerea. Our results confirmed the bi-directional activity of the pCaD located between the head-to-head oriented phytoalexin biosynthetic genes in the hot pepper. Furthermore, the synthetic promoter modified in the pCaD could be a potential tool for pathogen-inducible expression of target genes for developing disease-resistant crops.