Regulation via repression: Regulator of symbiosome differentiation (RSD) acts as zone-specific repressor to mediate nodule development

Medicago truncatula forms indeterminate nodules having successive development zones. Indeterminate nodules maintain an oxygen gradient to uncouple symbiosome differentiation at invasion zone and nitrogen fixation at fixation zone. The oxygen gradient maxima to minima from apex to base of nodule length depends on leghemoglobins ( LgHbs ) expression which is responsible for oxygen buffering as LgHbs create micro-oxic environment. In this study, we demonstrate the mode of action of Regulator of Symbiosome Differentiation (RSD), a transcriptional repressor. RSD interacts with Nodule Inception (NIN) and NIN-like protein 2 (NLP2). Studies confirm NIN-NLP2 activate LgHbs expression. RSD expression and protein localization remains confined within invasion and interzone.  We identified this zone-specific expression of RSD is crucial as RSD represses LgHb expression at invasion zone. Manipulation of the stoichiometry of RSD, NIN, and NLP2 in planta disrupts LgHb expression and impairs functional nodule development. To unravel, the dynamics of the invasion zone-specific regulation, we performed translatome and transcriptome profiling using the RSD promoter, to identify putative targets of RSD. Collectively, our findings suggest that RSD-mediated repression of NIN and NLP2-targets are crucial for symbiosome differentiation. RSD recruitment in the nitrogen-fixing clade also recommends its evolution during nodulation and conserved functionality in other legume species as well. Thus RSD, as a repressor from its invasion zone optimizes the genetic network crucial for functional nodule development.