2.6. RNA extraction and cDNA synthesis

Infiltrated wild-type N. benthamiana leaf material was harvested 5-days post-infiltration (dpi) for transient experiments, or 5-dpi from GVA infiltration for stable experiments (7-dpi from TRV mixture infiltration), and three infiltrated leaves per plant were pooled together and subsequently frozen in liquid nitrogen. Total RNA was extracted from 100mg aliquots using the Spectrum™ Plant Total RNA Kit (Sigma, USA) following the manufacturer’s instructions. First-strand complementary DNA (cDNA) synthesis was performed using 1µg of DNase-treated total RNA with Random Primers (Promega, USA) and Maxima Reverse Transcriptase (Thermo Fisher Scientific, USA), following the manufacturer’s instructions. cDNA synthesis was confirmed with an end-point PCR by amplifying the housekeeping gene actin.