Upcoming Webinar: Advancing Sustainable Agriculture with Droplet Microfluidics

Engineering biological nitrogen (N2) fixation in non-leguminous crops is a major goal for sustainable agriculture. Unlike rhizobia colonizing in legume root nodules, associative and free-living diazotrophs in cereals do not possess the symbiotic signalling machinery required to initiate nodulation (Nod Factor or NF production). Thus, engineering an orthogonal symbiotic signalling in cereal-colonising microbes through either NFdependent or independent cascades will be a critical contribution to synthetic root nodule symbiosis in cereals. However, a critical bottleneck of these approaches is lack of tools to identify suitable symbionts for synthetic cereal root nodules.

To tackle this problem in the context of engineering orthologous NF-based signalling, we have developed and validated Sinorhizobium meliloti nodulation (nod) gene reporters with two aims. First, to provide insight into the activation of symbiotic signalling and regulatory mechanisms in the native strain S. meliloti, and second, to rapidly identify rhizobial and non-rhizobial strains capable of expressing S. meliloti nodulation genes in response to plant-derived symbiotic signals. The functionality of the biosensors was validated across diverse model rhizobia and non-rhizobia using in plant induction assays. The reporters were then integrated with a unique plasmid-ID system and introduced into corn-associated N2-fixing isolates to identify engineerable candidates for future transfer of S. meliloti nodule formation capacity.

In parallel, we integrated the nod reporter with a droplet-based microfluidic platform to perform high-throughput screening of a Tn5-mutagenized S. meliloti library, enabling the high-throughput isolation of mutants exhibiting elevated nod gene expression. Together, these approaches establish a scalable platform for identifying and engineering cereal-associated microbes with enhanced symbiotic potential.