2.3. Plant RNA extraction, cDNA synthesis and gene expression

Total RNA was isolated from tobacco leaves harvested at 0, 6 and 24 hr post inoculation (hpi) with Pta using the RNeasy® Plus Mini Kit (Qiagen, USA), according to the manufacturer's instructions. Quantitative reverse‐transcription PCR (qRT‐PCR) assays were performed as described previously (Song, Sim, Kim, & Ryu, 2016). The expression of candidate defense priming genes was analysed using the following primer pairs: NbPR1a‐F/R (5′‐AATATCCCACTCTTGCCG‐3′ and 5′‐CCTGGAGGATCATAGTTG‐3′, respectively), NbSABP‐F/R (5′‐ACCATCAGACCAAGATGT‐3′ and 5′‐TGGCTAAGAGTGGAAGGT‐3′, respectively), NbHIN1‐F/R (5′‐AATATCCCACTCTTGCCG‐3′ and 5′‐CCTGGAGGATCATAGTTG‐3′, respectively), and NbPR2‐F/R (5′‐ACCATCAGACCAAGATGT‐3′ and 5′‐TGGCTAAGAGTGGAAGGT‐3′, respectively). PCR was performed using the following thermocycler parameters: 95°C for 10 min, followed by 44 cycles at 95°C for 30 s, 60°C for 30 s and 72°C for 42 s. Transcript levels of each tobacco gene were calibrated and normalized using the housekeeping gene NbACT (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"U60489","term_id":"1498347","term_text":"U60489"}}U60489), and relative quantification of specific mRNA levels was performed using the comparative 2–Δ(ΔCt) method (Livak & Schmittgen, 2001).