Rhizopine biosensors for plant-dependent control of bacterial gene expression

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 (<i>RhiP</i>) 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 <i>RhiP</i> barley roots compared to the previously described biosensor plasmid pSIR02. We demonstrate that a sub-population of <i>Azorhizobium caulinodans</i> cells carrying pSIR05 can sense rhizopine and activate gene expression when colonizing <i>RhiP</i> barley roots. However, these bacteria were mildly defective for colonization of <i>RhiP</i> 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.