Tripure isolation reagent

To analyze nitrate-induced gene expression, M. truncatula wild-type (R108), cra2, nin-2, or nlp1 seedlings were grown on BNM or FP plates supplemented with 50 nM aminoethoxyvinylglycine (Sigma-Aldrich) for 3 days, then transferred to plates with BNM or FP containing 10 mM KCl or KNO₃ for another 3 days. To analyze rhizobium-induced gene expression, R108, nin-2, or nlp1 seedlings were grown on BNM or FP agar plates for 3 days, then inoculated with Sm1021 (OD₆₀₀ ≈ 0.05) and grown for another 3 days. Total RNA was extracted from M. truncatula roots using the TRIpure isolation reagent (Aidlab, Beijing, China) according to the manufacturer's instructions. RNA was quantified using a NanoDrop 2000 instrument (Thermo Fisher, Waltham, MA, USA) and then reverse transcribed into cDNA using the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen, Beijing, China). Gene transcript levels were analyzed by real-time PCR using TB Green Premix Ex Taq (Takara, Dalian, China) with the StepOnePlus PCR system (Thermo Fisher, Waltham, MA, USA). Relative transcript levels were normalized against that of the reference gene (MtUBQ10). Statistical significance based on three biological replicates was calculated using the 2^−ΔΔCt method. The primers used for qRT-PCR are listed in Supplemental Table 1.