Regulation of Rhizobial Nodulation Genes by Flavonoid‐Independent NodD Supports Nitrogen‐Fixing Symbioses With Legumes

ABSTRACT Rhizobia and legumes form a symbiotic relationship resulting in the formation of root structures known as nodules, where bacteria fix nitrogen. Legumes release flavonoids that are detected by the rhizobial nodulation (Nod) protein NodD, initiating the transcriptional activation of nod genes and subsequent synthesis of Nod Factors (NFs). NFs then induce various legume responses essential for this symbiosis. Although evidence suggests differential regulation of nodD expression and NF biosynthesis during symbiosis, the necessity of this regulation for the formation of nitrogen‐fixing nodules remains uncertain. Here, we demonstrate that deletion of the Rlv3841 NodD regulatory domain results in a constitutively active protein (NodDFI) capable of activating NF biosynthesis gene expression without the presence of flavonoids. Optimised constitutive expression of nodDFI or nodD3 in nodD null mutants led to wild‐type levels of nodulation and nitrogen fixation in pea and M. truncatula, respectively, indicating that flavonoid‐regulated nodD expression is not essential for supporting symbiosis. These findings illustrate that transcriptional control of flavonoid‐independent NodD regulators can be employed to drive NF biosynthesis, which holds potential for engineering symbiosis between rhizobia and cereals equipped with reconstituted NF receptors. Legumes release flavonoids into the soil, which are detected by the rhizobial NodD protein, initiating the transcription of nod genes for Nod Factors (NF) synthesis. We found that removing the NodD regulatory domain results in a constantly active protein (NodDFI), which independently activates NF biosynthesis genes without flavonoids.