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

AbstractOnly the Leguminosae family can develop root nodules with peripheral vasculature, an adaptation that grants them an advantage in optimizing nitrogen fixation efficiency.Medicago truncatuladevelops indeterminate nodules that possess peripheral vascular-strands encircling the central infection zone. How vascular-strands shifted from the nodule central part to the periphery remains unresolved. Here we show, MtbHLH1 (renamed as Nodule Vascular bundle Development 1) is required for the proper organization of vascular strands. Innvd1nodules, vascular strands pass through the infection zone.NVD2, an HLH transcription factor that lacks a DNA-binding domain, is activated by NVD1. Mutantnvd2nodules display a similar partially central vasculature.NVD2is expressed along the nodule vascular bundle and NVD2:GFP fusion protein localizes to the nodule vascular endodermis. The formation of the peripheral vasculature is dependent on the proper stoichiometry of NVD1 and NVD2 heterodimers, as NVD2 controls NVD1-mediated transcriptional activation by sequestering NVD1. Transcription ofNVD1is activated by auxin and Auxin Responsive transcription Factor (MtARF5). Transcriptome sequencing ofnvd1andnvd2nodules and visualization ofin situauxin and cytokinin signal outputs indicated aberrant auxin/cytokinin balance in these nodules. Our findings showed that the NVD1-NVD2 heterodimer plays a key role in the formation of an orderly peripheral vascular bundle around Medicago nodules.