Bimolecular Fluorescence Complementation Assay

For the generation of BiFC constructs, the full length open reading frame (ORF), excluding the stop codon, encoding Trx y2 and FBPase were amplified with iProof™ High-Fidelity DNA Polymerase (Bio-Rad) using oligonucleotides specified in Table S2, which added attB recombination sites at the 5′ and 3′ ends, respectively. The PCR products were cloned in the Gateway vector pDONR207 (Invitrogen) and sequenced. The cloned Trx y2 and FBPase fragments were then transferred to the BiFC vectors [44 (link)] pSPYNE-35S_GW and pSPYCE-35S_GW, respectively, using the LR clonase (Invitrogen). The resulting plasmids, pSPYNE:Trx y2 and pSPYCE:FBPase, were then transformed into the A. tumefaciens strain GV301. For BiFC assays, Agrobacterium strains carrying individual Trx y2, FBPase, Trx x [45 (link)], Trx f1 [25 (link)], glutamine synthetase 2 (GS2) [28 (link)] or 2-Cys Prxs [45 (link)] constructs were mixed at a 1:1 ratio and infiltrated into leaves of 4-week-old Nicotiana benthamiana plants. Leaf sections were analyzed 4 days later by confocal microscopy performed with a Leica SP/2 inverted microscope. Image analysis was performed with the Leica SP/2 software package and the ImageJ bundle provided by the Wright Cell Imaging facility.