<i>MtNODULE ROOT1</i> and <i>MtNODULE ROOT2</i> Are Essential for Indeterminate Nodule Identity

Abstract

Symbiotic interactions between legume plants and rhizobia result in the formation of nitrogen-fixing nodules, but the molecular actors and the mechanisms allowing for the maintenance of nodule identity are poorly understood. <i>Medicago truncatula NODULE ROOT1</i> (<i>MtNOOT1</i>), <i>Pisum sativum COCHLEATA1</i> (<i>PsCOCH1</i>), and <i>Lotus japonicus NOOT-BOP-COCH-LIKE1</i> (<i>LjNBCL1</i>) are orthologs of Arabidopsis (<i>Arabidopsis thaliana</i>) <i>AtBLADE-ON-PETIOLE1/2</i> and are members of the <i>NBCL</i> gene family, which has conserved roles in plant development and is essential for indeterminate and determinate nodule identity in legumes. The loss of function of <i>MtNOOT1</i>, <i>PsCOCH1</i>, and <i>LjNBCL1</i> triggers a partial loss of nodule identity characterized by the development of ectopic roots arising from nodule vascular meristems. Here, we report the identification and characterization of a second gene involved in regulating indeterminate nodule identity in <i>M. truncatula</i>, <i>MtNOOT2</i><i>MtNOOT2</i> is the paralog of <i>MtNOOT1</i> and belongs to a second legume-specific <i>NBCL</i> subclade, the <i>NBCL2</i> clade. <i>MtNOOT2</i> expression was induced during early nodule formation, and it was expressed primarily in the nodule central meristem. <i>Mtnoot2</i> mutants did not present any particular symbiotic phenotype; however, the loss of function of both <i>MtNOOT1</i> and <i>MtNOOT2</i> resulted in the complete loss of nodule identity and was accompanied by drastic changes in the expression of symbiotic, defense, and root apical meristem marker genes. <i>Mtnoot1 noot2</i> double mutants developed only nonfixing root-like structures that were no longer able to host symbiotic rhizobia. This study provides original insights into the molecular basis underlying nodule identity in legumes forming indeterminate nodules.