Transfer cells mediate nitrate uptake to control root nodule symbiosis.

Root nodule symbiosis enables nitrogen fixation in legumes and, therefore, improves crop production for sustainable agriculture. Environmental nitrate levels affect nodulation and nitrogen fixation, but the mechanisms by which legume plants modulate nitrate uptake to regulate nodule symbiosis remain unclear. Here, we identify a member of the Medicago truncatula nitrate peptide family (NPF), NPF7.6, which is expressed specifically in the nodule vasculature. NPF7.6 localizes to the plasma membrane of nodule transfer cells (NTCs), where it functions as a high-affinity nitrate transporter. Transfer cells show characteristic wall ingrowths that enhance the capacity for membrane transport at the apoplasmic-symplasmic interface between the vasculature and surrounding tissues. Importantly, knockout of NPF7.6 using CRISPR-Cas9 resulted in developmental defects of the nodule vasculature, with excessive expansion of NTC plasma membranes. npf7.6 nodules showed severely compromised nitrate responsiveness caused by an attenuated ability to transport nitrate. Moreover, npf7.6 nodules exhibited disturbed nitric oxide homeostasis and a notable decrease in nitrogenase activity. Our findings indicate that NPF7.6 has been co-opted into a regulatory role in nodulation, functioning in nitrate uptake through NTCs to fine-tune nodule symbiosis in response to fluctuating environmental nitrate status. These observations will inform efforts to optimize nitrogen fixation in legume crops.