Organized peripheral vascular strand development in nodules is controlled by a bHLH/HLH heterodimer

The Leguminosae family can develop root nodules with symmetrical peripheral vascular-strands (PVSs). Medicago truncatula forms indeterminate nodules with PVSs. The PVSs elongate directly from the root toward the nodule apex, maintaining a symmetrical organization and facilitating the formation of the cylindrical nodule structure. By combining genetic, biochemical, and genomic tools, we have shown that two basic Helix-Loop-Helix groups of transcription factors, MtbHLH1 (renamed Nodule Vascular bundle Development 1 (NVD1)) and NVD2, control the development of symmetrical PVSs in M. truncatula. In nvd1 nodules, PVSs drift toward the infection zone, generating aberrantly shaped nodules. NVD1 activates its expression along with NVD2, a transcriptional regulator. NVD1 functions downstream of auxin signaling. Transcriptome sequencing of nvd1 and nvd2 nodules, combined with visualization of auxin and cytokinin (CK) signal outputs, revealed disrupted auxin and CK signaling in nvd nodules. Furthermore, ectopic expression of the auxin biosynthetic enzyme (MtYUCCA8) under pMtNVD1 and pMtNVD2 resulted in defective PVSs. Mutant nvd2 nodules display asymmetric PVSs. NVD2 regulates the transcriptional activity of NVD1 by forming heterodimers with it. The formation of symmetrical PVSs depends on the balanced presence of NVD1 and NVD2. Our findings highlight the pivotal role of the NVD1-NVD2 interaction in shaping the development of symmetrical PVSs.