Generation of promoters enabling high-level constitutive gene expression in both plants and Escherichia coli

Abstract Functional validation of genetic components in plants often requires cloning them separately into both plant and bacterial expression vectors, a process that is both time-consuming and laborious. This study aimed to simplify this workflow by developing plant-bacteria dual-host promoter systems that drive high-level constitutive expression in both environments. To achieve this, two variants of the chloramphenicol acetyltransferase promoter (PCAT), a bacterial σ factor-dependent promoter, were integrated into the cauliflower mosaic virus 35S promoter (P35S), and their performance was evaluated using a hygromycin phosphotransferase (HPT)-GFP fusion reporter. One of these variants, PCAT1, conferred hygromycin resistance to Escherichia coli (DH5α and BL21 (DE3)) and maintained high-level expression comparable to the original P35S in onion epidermal cells. A hybrid P35S enhancer-PNOS system also conferred hygromycin resistance to E. coli, but its activity in inducing GFP signals in onion cells remained lower than that of P35S. Due to its compact size (89 bp) and efficiency, PCAT1 can serve as a module for converting standard plant vectors into dual-host systems, accelerating gene characterization and the development of new gene-based tools. Competing Interest Statement The authors have declared no competing interest. Abbreviations - CDS - Coding sequence - GFP - Green fluorescent protein - HPT - hygromycin phosphotransferase - Hyg - hygromycin - HygR - hygromycin-resistant - LB - Luria-Bertani - ND - neutral density - P35S - cauliflower mosaic virus 35S promoter - PCAT - chloramphenicol acetyltransferase promoter - PCR - polymerase chain reaction - PEG - polyethylene glycol - PNOS - nopaline synthase promoter - SD - Standard deviation - Spec - spectinomycin - SpecR - spectinomycin-resistant - TNOS - nopaline synthase terminator - uORF - upstream open reading frame