NODULES WITH ACTIVATED DEFENSE 1 is required for maintenance of rhizobial endosymbiosis in Medicago truncatula.

The symbiotic interaction between legume plants and rhizobia results in the formation of root nodules, in which symbiotic plant cells host and harbor thousands of nitrogen-fixing rhizobia. Here, a Medicago truncatula nodules with activated defense 1 (nad1) mutant was identified using reverse genetics methods. The mutant phenotype was characterized using cell and molecular biology approaches. An RNA-sequencing technique was used to analyze the transcriptomic reprogramming of nad1 mutant nodules. In the nad1 mutant plants, rhizobial infection and propagation in infection threads are normal, whereas rhizobia and their symbiotic plant cells become necrotic immediately after rhizobia are released from infection threads into symbiotic cells of nodules. Defense-associated responses were detected in nad1 nodules. NAD1 is specifically present in root nodule symbiosis plants with the exception of Morus notabilis, and the transcript is highly induced in nodules. NAD1 encodes a small uncharacterized protein with two predicted transmembrane helices and is localized at the endoplasmic reticulum. Our data demonstrate a positive role for NAD1 in the maintenance of rhizobial endosymbiosis during nodulation.