Heterologous Expression of CrSS Protein

The coding sequence of CrSS was amplified with primers oxSS-F and oxSS-R (Supplemental Table S1), which introduce an AgeI restriction site in both extremities. The PCR product was digested and ligated in an AgeI/XmaI-digested pEAQ-HT plasmid in order to generate the pEAQ-HT:CrSS-6HIS construct. The plasmid was electroporated in A. tumefaciens GV3101, and cells were prepared for infiltrations as described in the section on VIGS methodology. Agrobacteria harboring either an EV (pEAQ-HT) or the pEAQ-HT:CrSS-6HIS (oxCrSS) vector were mixed with Agrobacteria harboring a GFP-expressing vector (pEAQ-HT:GFP-6HIS) in a final OD₆₀₀ = 0.25 each. Catharanthus roseus plants (cv Little Bright Eyes) were vacuum infiltrated and leaf areas exhibiting GFP fluorescence were collected at 6 d post infiltration for further analyses. Proteins were extracted as described previously (Carqueijeiro et al., 2020 ) and resolved on 8% SDS–PAGE, followed by Coomassie Brilliant Blue staining or western blotting. Western blot analysis was performed with an anti-HIS tag primary antibody (#ab213204, Abcam) and a horseradish peroxidase-conjugated secondary antibody (#1706515, Bio-Rad) following standard protocols. The alkaloid content of agroinfiltrated leaves was determined using UPLC-MS analysis as described previously (Dugé de Bernonville et al., 2017 (link)).