The root microbiota is critical for agricultural yield, with growth-promoting bacteria able to solubilise phosphate, produce plant growth hormones, antagonise pathogens and fix N₂. Plants control the microorganisms in their immediate environment and this is at least in part through direct selection, the immune system, and interactions with other microorganisms. Considering the importance of the root microbiota for crop yields it is attractive to artificially regulate this environment to optimise agricultural productivity. Towards this aim we express a synthetic pathway for the production of the rhizopine scyllo-inosamine in plants. We demonstrate the production of this bacterial derived signal in both Medicago truncatula and barley and show its perception by rhizosphere bacteria, containing bioluminescent and fluorescent biosensors. This study lays the groundwork for synthetic signalling networks between plants and bacteria, allowing the targeted regulation of bacterial gene expression in the rhizosphere for delivery of useful functions to plants.

Significance: Inoculation of cereals with diazotrophic (N ₂ -fixing) bacteria offers a sustainable alternative to the application of nitrogen fertilizers in agriculture. While natural diazotrophs have evolved multilayered regulatory mechanisms that couple N ₂ fixation with assimilation of the product NH ₃ and prevent release to plants, genetic modifications can permit excess production and excretion of NH ₃ . However, a lack of stringent host-specificity for root colonization by the bacteria would allow growth promotion of target and nontarget plants species alike. Here, we exploit synthetic transkingdom signaling to establish plant host-specific control of the N ₂ -fixation catalyst nitrogenase in Azorhizobium caulinodans occupying barley roots. This work demonstrates how partner-specific interactions can be established to avoid potential growth promotion of nontarget plants.

Engineering signalling between plants and microbes could be exploited to establish host‐specificity between plant‐growth‐promoting bacteria and target crops in the environment. We previously engineered rhizopine‐signalling circuitry facilitating exclusive signalling between rhizopine‐producing ( RhiP ) plants and model bacterial strains. Here, we conduct an in‐depth analysis of rhizopine‐inducible expression in bacteria. We characterize two rhizopine‐inducible promoters and explore the bacterial host‐range of rhizopine biosensor plasmids. By tuning the expression of rhizopine uptake genes, we also construct a new biosensor plasmid pSIR05 that has minimal impact on host cell growth in vitro and exhibits markedly improved stability of expression in situ on RhiP barley roots compared to the previously described biosensor plasmid pSIR02. We demonstrate that a sub‐population of Azorhizobium caulinodans cells carrying pSIR05 can sense rhizopine and activate gene expression when colonizing RhiP barley roots. However, these bacteria were mildly defective for colonization of RhiP barley roots compared to the wild‐type parent strain. This work provides advancement towards establishing more robust plant‐dependent control of bacterial gene expression and highlights the key challenges remaining to achieve this goal.