qRT-PCR Analysis of Gene Expression

RNA was extracted according to the method described by Guo et al (2012) [45 (link)]. cDNA was synthesized using Prime Script Kit (Takara, Dalian, China) following the manufacturer’s instructions. The cDNA concentration used for qRT-PCR was 50 ng/µL. The iCycleriQ™ Multicolor PCR Detection System (Bio-Rad, Hercules, CA, USA) machine was used for qRT-PCR and the procedure was designed following Zhang et al. [18 (link)]. Briefly, the qRT-PCR cycling conditions were as follows: pre-denaturation at 95 ℃ for 1 min, followed by 40 cycles of denaturation at 95 ℃ for 10 s, annealing at 56 °C for 30 s, and extension at 72 °C for 30 s. The fluorescent signal was measured at the end of each cycle, and melting curve analysis was performed by heating the PCR product from 56 to 95 °C in order to verify the specificities of the primers. The actin mRNA (CaActin2, accession No. AY572427) from pepper and the actin-97 mRNA (Nbactin-97, accession No. LOC109206422) from N. benthamiana were used as references, respectively [46 (link),47 ]. All the primer specificities used for the qRT-PCR were assessed using NCBI Primer BLAST (Supplementary Table S3). Three independent biological replicates were carried out. The relative expression of genes was calculated using the 2^−△△Ct method described by Schmittgen and Livak (2008) [48 (link)].