Validation of RNA-Seq Gene Expression by qRT-PCR

Samples as above were used for the qPCR analyses which were run on a QUANTSTUDIO 3 Real-Time System (Thermo Fisher Scientific, Waltham, MA, United States) using SYBR green I with ROX as an internal loading standard. The reaction mixture was 10 μL, comprising 5 μL of 2X Maxima^TM SYBR Green qPCR Master Mix (Maxima SYBR Green/ROX qPCR Master Mix 2X, Thermo Fisher Scientific, United States), 0.5 μL of 1:10 diluted cDNA and 100 nM primers (Integrated DNA Technologies, Coralville, IA, United States). Furthermore, non-templates were run as a negative control using only total RNA without reverse transcription to monitor for genomic DNA contamination and the same was done by using water with water. Primers were designed using Primer 3.0 software (Rozen and Skaletsky, 2000) as reported in Supplementary Table 1. The thermal conditions for all genes were: 10 min at 95°C, 40 cycles 15 s at 57°C, and 20 s at 72°C. Fluorescence was read following each annealing and extension phase. All runs were followed by a melting curve analysis from 55 to 95°C. The linear range of template concentration to threshold cycle value (Ct value) was determined by preparing a dilution series, using cDNA from three independent RNA extractions analyzed in three technical replicates. Primer efficiencies for all primer pairs were calculated using the standard curve method. All amplification plots were analyzed with the QUANTSTUDIO 3 software to obtain Ct values (Pfaffl, 2001).

The following groups of genes were analyzed: Calcium-related genes: calcium-binding EF hand family protein (Solyc10g006700), calmodulin (Solyc04g058160), calcium-binding phospholipase D (Solyc01g091910). Oxidative stress-related genes: ubiquinol oxidase (Solyc08g075550), polyphenol oxidase F, chloroplastic, PPO (Solyc08g074630), peroxidase (Solyc03g025380), catalase (Solyc01g100630). Proton pump-related genes: V-type proton ATPase subunit a (Solyc11g072530), proton pump interactor 1 (Solyc08g068850), proton pump interactor 1 (Solyc05g008780). Defense-related genes: 4-coumarate-CoA ligase-like protein (Solyc06g035960), β-1,3-glucanase (Solyc01g060020), chymotrypsin inhibitor-2 (Solyc09g084450), Kunitz-type protease inhibitor (Solyc03g098780), multidrug resistance protein ABC transporter family (Solyc05g014500), pathogenesis-related protein 1a (Solyc01g106620), pathogenesis-related protein P2 (Solyc01g097240), pathogenesis-related protein-1 (Solyc01g106610), polygalacturonase (Solyc12g096730), sesquiterpene synthase (Solyc07g052130), trypsin inhibitor-like protein precursor (Solyc11g022590), wound induced protein (Solyc07g054780), wound/stress protein lipoxygenase, LH2 PLAT domain-containing protein (Solyc03g096550), wound-induced proteinase inhibitor 1 (Solyc09g084470). Heat shock proteins (Hsps) and chaperones: heat shock protein Hsp90 (Solyc07g047790), heat shock protein (Solyc03g117630), heat shock protein 22 Mitochondrial (Solyc08g078700), heat shock protein 70 (Solyc03g082920), heat shock transcription factor 1 (Solyc02g079180), hsc1 heat shock protein 70 kDa (Solyc06g076020), hsc70.3 er21 ethylene-responsive heat shock protein cognate 70 (Solyc04g011440), hsp40 Chaperone protein (Solyc11g071830), hsp90 heat shock protein 90 (Solyc06g036290), NEF Heat shock protein 4 (Solyc07g043560), SHsfA7 Heat stress transcription factor A3 (Solyc09g065660). DNA binding: DNA primase/helicase (Solyc02g022830), DNA-directed RNA polymerase (Solyc02g083350). Receptor-like genes: receptor-like serine/threonine-protein kinase (Solyc03g025130), serine/threonine-protein kinase (Solyc03g112950), TIR-NBS-LRR resistance protein Toll-Interleukin receptor (Solyc00g294230), TIR-NBS-LRR disease resistance-like protein (Solyc07g052790), CC-NBS-LRR, resistance protein (Solyc10g047320). Phytohormone-related genes: ACC oxidase (Solyc09g008560), auxin-induced SAUR-like protein (Solyc01g111000), ethylene-responsive nuclear protein (Solyc02g070040), ethylene-responsive transcription factor 4 (Solyc12g009240), ethylene-responsive TF1 pathogenesis-related transcriptional factor (Solyc03g093550). Photosynthesis: chloroplastic RuBisCO small subunit 3B (Solyc02g085950), RuBisCO activase 1 (Solyc09g011080).

Four different reference genes TC194780a, actin 1 (ACT1); {"type":"entrez-nucleotide","attrs":{"text":"X14449","term_id":"19272","term_text":"X14449"}}X14449, elongation factor 1α (EF1); {"type":"entrez-nucleotide","attrs":{"text":"DQ205342","term_id":"77963734","term_text":"DQ205342"}}DQ205342, β-tubulin (TUB), and TC193502a, ubiquitin (UBI) (see Supplementary Table 1 for primers), according to Løvdal and Lillo (2009). The best of the four genes was selected using the NormFinder software (Andersen et al., 2004). The relative expression mRNA levels of each gene were calibrated and normalized with the level of the most stable reference genes, EF1 and UBI [in agreement with (Løvdal and Lillo, 2009)]. For each treatment, three biological replicates and three technical replicates were analyzed.