Transcriptomic insights into the mode of action of carvacrol against copper-resistant Xanthomonas euvesicatoria pv. perforans reveals impairment of copper resistance pathway alongside widespread metabolic shifts

Background Bacterial spot of tomato and pepper is caused by four genetic groups in the genus Xanthomonas. Intensive copper-based management against the cosmopolitan Xanthomonas euvesicatoria pv. perforans ( Xep ) has led to the development of resistance in many bacterial populations. Carvacrol has been shown to control copper resistant Xep as well as improve the efficacy of copper-based bactericides against Xep . To understand the mode of action of carvacrol against copper resistant Xep , RNA-seq analysis of copper resistant Xep exposed to carvacrol treatment was conducted. Results We treated copper resistant Xep strain GEV485 with carvacrol at an MIC of 32 mg/L and sequenced the RNA at time points 1 hr and 4 hr after treatment, including untreated controls. Reads from RNA sequencing were mapped onto the complete genome of copper resistant Xep LH3 for downstream bioinformatic analyses. Significant differentially expressed genes (DEGs) were identified and mapped onto the complete annotated Xep LH3 pathway. Our result shows that carvacrol exerts global regulation on the Xep genome. We identified rapid downregulation of genes involved in oxidative phosphorylation and other bacterial energy generating pathways, which inexplicably have impacts on ribosome biogenesis, DNA synthesis and repair, lipopolysaccharide and fatty acid metabolism, motility, efflux, secretion systems, quorum sensing and biofilm formation, alongside regulation of other pathways. Interestingly, there was a downregulation of the multicopper oxidase gene copA as well as dynamic regulation of other copper associated and efflux genes. Conclusion We identified regulated genes in pathways in Xep that provide novel insights on the mode of action of carvacrol against copper resistant Xep . Our results point to a rapid collapse of the proton motive force (PMF) driving energy balance accompanied by impairment of critical functions in Xep . The identification of carvacrol-induced downregulation of copper resistance genes in the copLAB operon shows that the mode of action of carvacrol is different from the mode of action of copper bactericides which are known to upregulate copper resistance genes. The results in this study provide an improved foundation for understanding the mode of action of carvacrol against copper resistant Xep , potentially aiding efforts in formulation of the molecule for improved management of bacterial spot of tomato and pepper.