Engineering and Evaluation of Sinorhizobium meliloti Nodulation ( nod ) Gene Reporter Systems in Rhizobia and Non‐Rhizobia

Developing N 2 ‐fixing partnerships between diazotrophs and non‐legumes can enhance soil fertility and reduce dependence on synthetic fertilisers. Unlike legumes, non‐legumes lack the genetic ability to form root nodule symbiosis with rhizobia but can form facultative associations with free‐living diazotrophs. Engineering these microbes by transferring key traits underlying efficient nodule formation and N 2 ‐fixation from well‐characterised rhizobia represents a central aim in synthetic biology to enhance biological nitrogen fixation in non‐legumes. However, the lack of effective tools for identifying compatible and engineerable microbial partners is a key challenge. To address this, we have developed nodulation ( nod ) gene reporters to screen both rhizobia and non‐rhizobia capable of expressing Sinorhizobium meliloti nod genes, which encode bacterial signals initiating nodule formation in legumes. The biosensors include a superfolder GFP reporter controlled by the inducible nod box promoter (P nodA ), plant signal‐dependent activators nodD1 and nodD2 , and a constitutively mScarlet‐I marker, named nodD1 ‐P nodA and nodD2 ‐P nodA . Their functionality was validated across diverse rhizobia and non‐rhizobia using in vitro and in planta induction assays. This reporter system enables high‐throughput identification of novel bacteria capable of recognising and responding to legume signalling molecules that coordinate symbiotic nitrogen fixation.