Total RNA Extraction and RT-qPCR Analysis in M. truncatula

Total RNA was extracted from c.50 mg M. truncatula leaves using the Nucleospin RNA Plant kit (Macherey‐Nagel) with on‐column DNase treatment. First‐strand cDNA was synthesized from 1 µg of total RNA using the iScript cDNA synthesis kit (Bio‐Rad) or PrimeScript cDNA synthesis kit (Takara‐Bio Inc.). Four‐fold diluted cDNA was used for RT‐qPCR using the 5× HOT FIREPol EvaGreen qPCR mix (Solis BioDyne) or TB Green Premix qPCR mix (Takara‐Bio Inc.) in a QuantStudio 6 Flex Real‐Time PCR system (Applied Biosystems). Three independent biological replicates were processed. MtUbiquitin (UBQ; Medtr3g092130) was used as the internal control due to its invariant expression across infection time points and genotypes (Figure S3). Relative expression was calculated using LinRegPCR v. 2015.1 (Ruijter et al., 2009 (link)). Primers were designed using NCBI Primer‐Blast (Table S2) and product specificity was confirmed by melt curve analysis.