Gene expression analysis
Harvested nodules and roots were frozen in liquid nitrogen and ground. Total RNA extraction was performed using the RNAzol ® RT reagent following the manufacturer’s recommendations (Sigma-Aldrich, USA). After the DNAse treatment with RQ1 DNAse (Promega, USA), 1 µg or total RNA was used for the cDNA synthesis (GoScript ™ Reverse Transcription System, Promega). Quantitative real-time PCRs were performed in the Agilent AriaMx System using the GoTaq® qPCR Master Mix (Promega, USA). Two reference genes (MtC27 and a38) were used to normalize the transcript level. The complete list of primers is reported in Table S1. Data were quantified using the Aria Software and analyzed with RqPCRBase, an R package working on R computing environment for analysis of quantitative real-time PCR data (Rancurel et al., 2019).