Proquest expression vectors

Yeast two hybrid (Y2H) assays were performed using the ProQuest expression vectors (Invitrogen) and Matchmaker Gold Yeast Two-Hybrid System products (TaKaRa). Full length coding sequences were PCR generated from Arabidopsis Col-0 cDNA using the Phusion DNA polymerase system and cloned into the PDONR207 vector using BP Clonase (Invitrogen). The Gateway compatible vectors pGADT7 (prey) and pGBKT7-GW (bait) were used as backbones [56 (link)]. Conserved leucines within the EAR motif of RBE were converted into alanines (CTTGAG CTA AGG CTA to gcTGAG gcAAGG gcA) by site directed mutagenesis. Bait and prey plasmids were obtained by LR reaction (Gateway) and co-transformed into the Y2H Gold yeast strain. SD-Leu-Trp agar plates (DDO) were used to select yeast harboring the bait and prey plasmids. SD-Leu-Trp-His agar plates (QDO) were used to analyze protein interactions. Experiments were repeated three times with similar results. Primers used are listed in S2 Table.

Yeast two-hybrid (Y2H) assays were performed in yeast strain MaV203 using Invitrogen ProQuest expression vectors and product protocols. The open reading frame for each clone was recombined into both pDEST32 [DNA-binding (DB) domain] and pDEST22 [Activation-domain (AD) domain] Gateway-compatible vectors. To assess the effect of IAA3 on TIR1-TIR1 interaction, IAA3 cDNA was cloned into pAG-416-GPD-ccdB containing a GPD constitutive promoter and URA3 selection marker. TIR1-TIR1 or TIR1-IAA3 protein interaction was assessed in media lacking histidine and supplemented with the indicated concentrations of 3-amintotriazole (3-AT).