| Summary: | The engineering of nitrogen fixing bacteria in the soil provides an opportunity to reduce the dependency of agriculture on inorganic fertiliser produced by industry, which leads to ground water pollution and the release of potent greenhouse gases. However, natural diazotrophs lack plant host specificity for root colonisation and employ multilayered regulatory mechanisms that couple the rate of nitrogen fixation with the assimilation of fixed nitrogen, preventing effective release of ammonia to the environment. Native regulation can be overcome by using a synthetic signal from the desired plant host to responsive root colonising bacteria for plant specific control of nitrogen fixation. A trans-kingdom signal was previously developed using bacteria rhizopine molecules allowing specific induction of bacterial gene expression in association with target plant species. Use of this signal to induce bacterial gene expression was tested in the model cereal-associative rhizobium A. <em>caulinodans</em>. By engineering bacterial genetic circuitry for rhizopine perception, we were able to improve the sensitivity of rhizopine induced gene expression by 103-fold. The rhizopine system was used to demonstrate tight transcriptional control of the NifA master transcriptional regulator of <em>nif</em> genes for nitrogen fixation <em>in vitro</em>, thereby developing a rhizopine responsive diazotrophic strain which can be assayed for plant growth promotion on rhizopine producing barley lines. To test the feasibility of controlled synthetic symbiosis in a nodule environment, rhizopine control of nitrogen fixation was assayed in the model symbiotic rhizobium <em>S. meliloti</em>. Rhizopine signalling was also amplified via two inducible relay signals, allowing plant dependent control of nitrogen fixation in the cereal associative gammaproteobacterium <em>E. radicincitans</em>. This work demonstrates a step towards establishing effective control of nitrogen fixation for plant growth promotion and how the engineering of stringent partner-specific symbiosis could be established in the field for agriculturally relevant cereals.
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