GoKawiilExperimental model and subjects
Plant materials and growth conditions
Arabidopsis thaliana Col-0 was used as the wild-type ecotype. The genotypes RAP2.12 1 –28 –FLuc, RAP2.12–GFP and Δ13RAP2.12–GFP were as previously described2,3. Seeds were sown in a 3:1 soil/vermiculite mixture, stratified at 4 °C in the dark for 3 days, then germinated at 22 °C/20 °C with a 16 h:8 h light/dark photoperiod and 100 μmol photons m−2 s−1 intensity. For in vitro propagation, seeds were sterilized using 70% ethanol for 1 min and incubated in 10% sodium hypochlorite (NaClO) for 10 min, followed by 6 washes in 1 ml sterile distilled water. For growth in liquid medium, 100 μl of seed suspension, corresponding to 20–40 seeds, was inoculated in 1 ml of sterile half-strength MS medium (basal salt mixture 2.15 g l−1, pH 5.7) supplemented with 1% sucrose in each well of 6-well plates. For growth in solid media, seeds were incubated in the dark at 4 °C for 2 days and subsequently on half-strength MS medium59, supplemented with 1% (w/v) sucrose and 0.8% (w/v) agar, and grown at 22 °C with a 16:8 day/night photoperiod at 100 μmol photons m−2 s −1 intensity.
Yeast strains and culture
A haploid parental strain BY4742 (Matα; His3-Δ1; Leu2-Δ0; Lys2-Δ0; Ura3-Δ0; Euroscarf #Y10000) was cotransformed following the LiAc/SS carrier DNA/PEG method60 with PCO4–pAG415GPD and different versions of the DLOR–pAG413GPD (C-, D- or R-DLOR). All plasmids used for yeast expression were produced in previous work61. Before transformation, cells were grown at 30 °C on YPDA (20 g l−1 peptone, 10 g l−1 yeast extract, 20 g l−1 glucose (Duchefa) and 20 mg l1 adenine hemisulfate (Sigma-Aldrich), supplied with 20 g l−1 agar (Duchefa) when necessary). Transformants were selected on SD medium containing 6.7 g l−1 yeast nitrogen base (DIFCO), 1.37 g l−1 yeast dropout medium (Sigma-Aldrich) and 20 g l−1 glucose, plus supplements (0.16 M uracil, 0.8 M histidine–HCl, 0.8 M leucine and 0.32 M tryptophan (Sigma-Aldrich) when complete), with 20 g l−1 agar when solid.
Bacterial strains
Bacterial strain Escherichia coli BL21 (DE3) was used for expression of recombinant PCOs. Bacteria were cultured in 2YT medium at 37 °C until the optical density at 600 nm (OD 600 ) reached 0.6. Protein expression was induced by addition of 0.8 mM isopropyl-β-d-thiogalactoside (Sigma-Aldrich) at 18 °C for 16 h with shaking at 170 rpm in an incubator (Eppendorf).
Method details
DNA construct generation
For generation of the 35S:RAP2.3–nLuc construct, an 806-nucleotide synthetic string containing an Arabidopsis codon-optimized nLuc sequence including the RAP2.2 intron (Supplementary Table 7) was synthesized in the pMK-RQ backbone by GeneArt (Thermo Fisher Scientific). The destination vector pK7GWnL2 was generated through ligation between pK7GW2 (ref. 62) and GWnLuc-intron after restriction using XbaI and MluI (Thermo Fisher Scientific). The Arabidopsis RAP2.3 CDS was amplified from Col-0 complementary DNA without stop codon (RAP2.3Δstop), with overlapping AttB sites introduced by PCR using GoTaq DNA polymerase (Promega). Entry clone vector was then generated as a BP reaction between the RAP2.3 CDS PCR product and pENTR/D-TOPO (Life Technologies). The resulting entry vector was recombined into the generated pK7GWnL2 destination vector using LR clonase mix II (Thermo Fisher Scientific). Primers used for RAP2.3Δstop cloning and screening are listed in Supplementary Table 8. For generation of the 35S:RAP2.3–GFP construct, the entry vector containing RAP2.3 CDS was recombined with a pK7GW2F62 destination vector using LR clonase mix II (Thermo Fisher Scientific).
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