Gene Expression Analyses

Total RNA extraction from M. truncatula roots was performed using the innuPREP RNA kit (Analytik Jena AG, Germany) and quantified using a DeNovix Ds-11+Spectrophotometer (DeNovix Inc., United States). cDNA synthesis was performed using the reverse transcriptase SuperScriptII (Invitrogen by Thermo Fisher Scientific, Germany) as described before (Kuhn et al., 2010). Quantitative real-time expression analyses were carried out using the MESA Green 231qPCR Master Mix Plus (Eurogentec, Germany) in a CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories GmbH, Germany). A total of 1 μl cDNA (1:5) was used per well as template with the following PCR protocol: 5 min 95°C, 15 s 95°C, 20 s 56°C, 30 s 72°C (40 cycles). Plant transcript levels and transcript levels of the translation elongation factor 1-alpha of R. irregularis (RiTEF1α, {"type":"entrez-nucleotide","attrs":{"text":"DQ282611","term_id":"82792159","term_text":"DQ282611"}}DQ282611) were normalized to the translation elongation factor 1-alpha of M. truncatula (MtTEF1α, Medtr6g021800) while fungal transcripts were normalized to RiTEF1α. Transcript levels of genes were determined in three technical replicates in each independent biological replicate. Numbers of biological replicates are indicated in the corresponding figure legends. All primers are listed in Supplementary Table S2.