A recently evolved <i>Nodulin</i> recruited by <scp>NIN</scp> for root hair rhizobial infection in <i>Medicago truncatula</i>

Summary In root nodule symbiosis, rhizobia enter into legume nodules either by intracellular infection via root hairs or by intercellular infection such as crack entry. The formation of the infection thread (IT) is key to root hair infection, but the underlying mechanisms are unclear. Although transcriptome studies have revealed gene regulatory networks of some central regulators such as NODULE INCEPTION (NIN), the specific roles of many downstream genes in IT development and their integration mechanism into the symbiotic pathway remain unknown. Through genetics, cell biological approaches and evolutionary analysis, we characterized the role and evolutionary origin of Medicago truncatula Nodulin 6 ( N6 ) in rhizobial infection. MtN6 is specifically induced by rhizobia in root hairs. Mtn6 mutant shows rhizobial infection defects with abnormal ITs in root hairs and nodules. MtN6 encodes a cell wall‐localized protein which is secreted into infection chamber and ITs. NIN regulates rhizobia‐induced MtN6 expression by directly binding its promoter. N6 is only present in legumes adopting root hair rhizobial infection mode and is likely to have originated from Nodulin/glutamine synthase‐like protein by gene duplication. We conclude that N6 is a newly formed gene recruited by NIN for root hair rhizobial infection during the evolution of root nodule symbiosis.