Sybr green 2 master mix

RT-qPCR was performed as previously described [59 (link),60 (link)]. Total RNA was prepared using an RNeasy RNA purification system (Qiagen, Hilden, Germany) with DNase I treatment. cDNA was synthesized using an RT kit for RT-qPCR (Qiagen) with a mixture of oligo dT and random primers. The cDNA pool was diluted 5- to 20-fold. The cDNA standard with permissible slopes of PCR efficiencies was prepared by step dilution of the cDNA pool for the relative quantification of mRNA levels. In 20 µL of qPCR mix, 0.25 µM of each primer, 4–10 µL of diluted cDNA, and 10 µL SYBR green 2× Master Mix (Applied Biosystems, Waltham, MA) were added and reacted at 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. Dissociation curves with specific single-peaked PCR and proper amplification slopes for PCR efficiency were confirmed. LinRegPCR software was used for baseline fluorescence correction, and for the calculation of Cq values and PCR efficiency of amplicons [61 (link)]. Primer sequences are listed in Supplementary Materials Table S3.

Overnight cultures (16 to 18 h) of FSL S5-0395 grown in LB broth were subcultured 1:1,000 into LB or N-salts minimal medium (either pH 7 or pH 5.8), and subcultured samples were grown at 37°C with shaking at 200 rpm until cells reached mid-exponential phase (3 h for LB broth, 5 h for N-salts minimal medium). RNA was stabilized with RNA protect (Qiagen) and was collected using the RNeasy kit (Qiagen). DNA was depleted with Ambion DNase I (Life Technologies), and DNA depletion was confirmed with quantitative PCR (qPCR); a cycle threshold (C_T) of 34 for rpoB in DNase-treated RNA samples was used for judging successful DNA depletion. cDNA libraries were prepared with the Superscript reverse transcription kit (Thermo Fisher), according to manufacturer’s instructions. qPCR was performed with SYBR green 2× master mix (Applied Biosystems) in a reaction mixture containing 0.4 μM each primer (see Table S7) and 1 µl of cDNA (approximately 15 ng of cDNA) as the template. Fold expression was calculated by raising the ΔΔC_T to the power of the efficiency calculated for each primer pair (70 (link)). Results are the averages from three independent experiments, each performed in technical duplicates.

qRT-PCR was performed as previously described [40 (link),42 (link)]. Total RNA was prepared with DNase I treatment using RNeasy (Qiagen, Hilden, Germany). cDNA was synthesized using QuantiTect kit (Qiagen), a mixture of oligo dT and random primers, then diluted 5-fold in 10 mM Tris-Cl and 0.1 mM EDTA buffer. A step dilution of the cDNA pool was prepared as a standard for relative expression. cDNA (4–10 µL), 0.25 µM of each primer, and 10 µL SYBR green 2× Master Mix (Applied Biosystems, Waltham, MA, USA) were mixed and filled up to a 20 µL of a reaction mixture. We designed and used primer pairs (Table 1). We validated the primer pairs by melting curve analysis of single amplicons, amplification efficiencies, band size in agarose gel electrophoresis, and DNA sequencing of PCR products. Initial denaturation was performed at 95 °C 10 min, and 40 cycles of PCR were run at 95 °C for 15 sec and at 60 °C for 1 min. Relative mRNA expression levels were obtained as compared with the standard described above.

Total RNA was extracted from mock control and CMV‐inoculated cucumber leaf samples using TRIzol. One microgram of total RNA was treated with DNase (Thermo Fisher Scientific) according to the manufacturer's instructions. First‐strand cDNA synthesis was performed using the qPCRBIO cDNA synthesis kit (PCR Biosystems). Quantitative PCR was performed using 2.5 µl of diluted cDNA, 7.5 µl of SYBR green 2× master mix (Applied Biosystems) and 1 µl of forward and reverse primer (3 µM each), in a final volume of 15 µl of reaction mixture in a StepOne Real‐Time system (Applied Biosystems). The reaction conditions included denaturation at 94 ℃ for 20 s, followed by 40 cycles of 94 ℃ for 3 s and 60 ℃ for 30 s. The host EF‐1α gene was used as an internal control for cDNA normalization. For each replicate, total RNA was extracted from pooled samples of three test plants. Relative expression was quantified using the 2^−ΔΔCt method. Three biological replicate leaf discs (50 mg fresh weight) were taken for analysis and each sample was a pool of three plants.