Plant materials and growth conditions

The Miyakojima MG-20 ecotype of L. japonicus⁵⁶ was used as WT in this study. The Gifu B-129 ecotype of L. japonicus⁵⁷ was used as a crossing partner for map-based cloning of NRSYM1. The nrsym1-1 and nrsym1-2 mutants were isolated from the M₂ generation of WT that had been mutagenized with 0.4% EMS. M₂ seeds were collected from ~10,000 M₁ plants provided from LegumeBase. A description of the har1-7 mutants was published previously⁹. Plants were grown with or without Mesorhizobium loti MAFF 303099 in autoclaved vermiculite with Broughton and Dilworth (B&D) solution⁵⁸ that does not contain a nitrogen source. The plants were grown under a 16 h light/8 h dark cycle at 24 °C in a growth cabinet. For measurement of relative nodule size, plants were grown with or without M. loti on a 1% agar plate containing B&D medium under the same light conditions. For the nitrate response assay, different concentrations of KNO₃ (0–50 mM) were supplemented into B&D solution. The solution was exchanged every 7 days after inoculation with newly prepared B&D solution containing different concentrations of KNO₃ to maintain the original KNO₃ concentrations.