Engineering and evaluation of Sinorhizobium meliloti nodulation ( nod ) gene reporter systems in rhizobia and non-rhizobia

Developing N□-fixing partnerships between diazotrophic microbes and non-legumes can enhance soil fertility and reduce dependence on synthetic fertilizers. 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 root nodule symbiosis in non-legumes remains a central aim in synthetic biology to enhance biological nitrogen fixation in cereals. Such a symbiosis will require specific symbiotic signaling that mimics the rhizobium-legume interaction. However, lack of effective tools for identifying compatible and engineerable microbial partners is a key challenge. To address this, we have developed inducible nodulation ( nod ) gene reporters to screen both rhizobial and non-rhizobial strains capable of expressing Sinorhizobium meliloti nod genes, which encode bacterial signals initiating nodule formation in legumes. The reporters 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 in various S. meliloti backgrounds using in vitro induction and two in planta induction approaches. These advancements facilitated the identification of both rhizobia and non-rhizobia capable of expressing S. meliloti nod genes, thereby supporting the development of synthetic N□-fixing symbioses in cereals.