A roGFP2-based bacterial bioreporter for redox sensing of plant surfaces.

The redox environment of the phytobiome, i.e. the plant-microbial interface, can strongly influence the outcome of the interaction between microbial pathogens, commensals and their host. We describe a noninvasive method using a bacterial bioreporter that responds to ROS and redox-active chemicals to compare microenvironments perceived by microbes during their initial encounter of the plant surface. A reduction-oxidation sensitive variant of green fluorescent protein (roGFP2), responsive to changes in intracellular levels of reduced and oxidized glutathione, was expressed under the constitutive and promoters in the epiphytic bacterium, 299R (299R/roGFP2). Analyses of 299R/roGFP2 cells by ratiometric fluorometry showed concentration-dependent responses to several redox active chemicals, including hydrogen peroxide (HO), dithiothreitol (DTT), and menadione. Changes in intracellular redox were detected within five minutes of addition of the chemical to 299R/roGFP2 cells, with approximate detection limits of 25 μM and 6 μM for oxidation by HO and menadione, respectively, and 10 μM for reduction by DTT. Caffeic acid, chlorogenic acid and ascorbic acid mitigated the HO-induced oxidation of the roGFP2 bioreporter. Aqueous washes of peach and rose flower petals from young blossoms created a lower redox state in the roGFP2 bioreporter than washes from fully mature blossoms. The bioreporter also detected differences in surface washes from peach fruit at different stages of maturity and between wounded and nonwounded sites. The 299R/roGFP2 reporter rapidly assesses differences in redox microenvironments and provides a noninvasive tool that may complement traditional redox-sensitive chromophores and chemical analyses of cell extracts.