Molecular genetics of nodule number regulation: Cloning, characterization and functional studies of the root determined nodulator1 (RDN1) gene in Medicago truncatula

Nitrogen is critical to life. However, the majority of nitrogen on earth (in the atmosphere) is inert and unavailable to nearly all organisms. Metabolically diverse prokaryotes are the only organisms capable of fixing atmospheric nitrogen; rhizobia set up a symbiosis with legume plants allowing the plants to benefit from this ability. Since nodulation and the subsequent nitrogen fixation processes are energy intensive, the host plant must balance hosting of the rhizobia by limiting the number of nodules it forms through a mechanism called Autoregulation of Nodulation (AON). My study of mutants in the model legume Medicago truncatula defective in AON allowed identification of loss-of-function alleles of the ROOT DETERMINED NODULATION1 (RDN1) gene (Medtr5g08952). I identified RDN1 by genetic mapping, transcript profiling, and rescue of the mutant phenotype. RDN1 is predicted to encode a 357-amino acid protein and is a member of an uncharacterized, highly conserved gene family unique to green plants. The promoter drives expression in the vascular cylinder and subcellular localization places RDN1 in the secretory pathway, consistent with a role for RDN1 in intracellular and long distance signaling in plants. I used grafted plants to show that RDN1 regulatory function occurs in the root before the shoot-derived suppression signal regulated by SUNN, another AON gene. Using a combination of gene expression assays, analysis of sunn/RDN1 double mutants and shoot-to-root reciprocal grafting I showed SUNN and RDN1 act in the same signaling pathway. RDN genes from poplar, rice and Arabidopsis can rescue the MtRDN1 mutant suggesting RDN1 protein function is retained in non-legumes. I report multiple root defects in Arabidopsis and Medicago mutants with defects in RDN genes. Together these findings help establish RDN as a family of proteins with previously uncharacterized regulatory functions involved not only AON but also root growth and lateral root development in land plants. Building on RDN's AON role, I also developed a split root inoculation system to understand the timing of autoregulation of nodulation in M. truncatula and discovered evidence for a previously unknown secondary AON signal.