Quantifying Gene Expression in Tomato

cDNA synthesis and real-time PCR were performed as described^{14 (link),58 (link),59 (link)}. Briefly, cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad). Real-time PCR primers for pin1, pr1a1, bHLH3, bHLH6, PL, and PE were designed with Beacon Designer software (Premier Biosoft International) avoiding template secondary structure (Table 2). Primer efficiencies (Table 2) were calculated using serial dilutions of MoneyMaker genomic DNA and CFX Manager 3.0 software (Bio-Rad). Reference transcripts and primers were chosen based on published works: act41 and ubi3^{60 (link)}. Stable expression between treatments was validated using the Best Keeper program and three independent RNA samples from each treatment per biological replicate. Real-time PCR experiments utilized the CFX96 Real-Time System, and data were analyzed with the CFX Manager 3.0 software (Bio-Rad). The mean Cq of each target transcript was normalized by the mean Cq of each reference gene using the formula: 2^{(−(Cq target-Cq reference))}. As previously described^{60 (link)}, we determined the relative expression ratio (RER) of the target gene by dividing the normalized target RNA by a calibrator consisting of the average of the normalized values of the control samples (expression after water treatment in most experiments; expression after infection with wildtype Xhg in the AvrHah1 target experiments (Table 1)).