Expression of PR1 Genes in Transgenic Plants

Contigs predicted to encode a basic form of pathogenesis-related protein 1-like in O. europaea var. sylvestris were searched in the NCBI databases. Those showing homology with regions of PR1-encoding genes from other species were selected for gene expression analysis in leaf tissue from control and transgenic plants by qRT-PCR (Supplementary Table 1).

Primer sequences for the endogenous control genes and the predicted O. europaea var. sylvestris PR1 genes are shown in Supplementary Table 2. Primer pairs were chosen to generate fragments between 70 and 140 bp and designed using Primer 3 software¹. Primer specificity was tested by performing conventional PCR and confirmed by the presence of a single melting curve during qRT-PCR. Serial dilutions (1:10, 1:20, 1:50, and 1:200) were made from a pool of cDNA from each treatment and time point, and calibration curves were performed for each gene. For qRT-PCR, the reaction mixture consisted of the first-strand cDNA template, primers (500 nmol final concentration) and SYBR Green Master Mix (SsoAdvanced Universal SYBR Green Supermix, Bio-Rad) in a total volume of 20 μl. The PCR conditions were as follows: 30 s at 95°C, followed by 40 cycles of 15 s at 95°C and 30 s at 60°C, 3 min at 72°C, and 1 min at 95°C. The reactions were performed using an iQ5 real-time PCR detection system (Bio-Rad). The ubiquitin gene was used as an endogenous control for normalization. Relative quantification of the expression levels for the target was analyzed using comparative Ct methods. An arbitrary value of 1 was given to the control, non-transformed line. All reactions were carried out in triplicate.