Pentr d topo cloning kit

Entry vectors for Gateway cloning were generated using the pENTR/D-TOPO Cloning Kit (Invitrogen) or via BP reaction of PCR products and pDONR221 vectors. Vectors for expression in N. benthamiana were generated by LR reaction of entry vectors (TOPO or pDONR221) and desired destination vectors (pAB^{30 (link)} or pBiFC-2in1^{31 (link)}). For expression of full-length ETR1, an inducible BiFC vector, pBiFC-ind-CN, was cloned by combining features of pAB and pBiFC-2in1. For subcellular localization studies an inducible expression system was used^{23 (link)}. BiFC^{31 (link)} and roGFP2^{26 (link)} studies were performed using d35-driven constitutive expression of the gene of interest. Constructs for heterologous expression of full-length and deletion mutants in E. coli were integrated by Gibson assembly^{32 (link)} into pETEV16b.

The AeSSPs sequences were amplified by PCR from A. euteiches gDNA with specific primers (Additional file 6: ST3b). The CACC cloning site was added to each forward primer. PCR products were purified using the PCR Clean-Up Kit (Promega, Madison, WI, USA) and introduced in the pENTRY-D-TOPO vector (pENTR/D-TOPO Cloning Kit, Invitrogen). Positive clones were introduced in a pK7FWG2 vector (Invitrogen) or pAMpAT/YFP vector. After sequencing, positive clones were introduced in A. tumefaciens GV3101 and A. rhizogenes ARqua1. For the KDEL fusion, the GFP construct was amplified from the obtained pK7FWG2 vector by PCR using primers that introduced a C-terminal SEKDEL sequence (Additional file 6: ST3b). Cloning was performed as previously reported using a pK2GW7 vector (Invitrogen). For leaf infiltration, A. tumefaciens GV3101-transformed strains were syringe-infiltrated as described in [61 (link)]. For M. truncatula roots transformation, A. rhizogenes ARqua1 strains were used and confocal imaging was performed at 28 dpi, as described in [29 (link)].

The dKdm2 cDNA was subcloned to the pENTR vector using the pENTR/D-TOPO Cloning kit (Invitrogen, K240020). The pTWG vector (from the DGRC #1076¹) was used as the destination vector and the attL x attR reaction was mediated by the LR Clonase II enzyme mix (Invitrogen, 11791-020), resulting in the ‘pUASt-dKdm2⁺-eGFP’ transgenic vector. After injecting the vector into w¹¹¹⁸ embryos, the UAS-dKdm2⁺-eGFP transgenic lines were established by standard fly genetics.

The hygromycin resistance cassette from pSELECT100 [19 (link)] and a gateway cloning site with luciferase reporter [49 (link)] were transferred to the high-copy pGEM backbone to form pNOC-Dlux. The LDSP 3′ UTR and terminator was amplified by PCR on Nannochloropsis genomic DNA, using primers given in Additional file 14: Table S4. The PCR product was blunt cloned with Zero Blunt^® PCR Cloning Kit (Invitrogen, ThermoFisher Scientific), sequenced and transferred to the SacI and AflII sites in the pNoc-Dlux plasmid. The elongation factor (EF) promoter was amplified by PCR from Nannochloropsis genomic DNA (primers given in Additional file 14: Table S4) and inserted in the pENTR gateway entry vector by using pENTR™/D-TOPO^® Cloning Kit (Invitrogen, ThermoFisher Scientific), sequenced, and transferred to pNoc-Dlux-LDSP terminator by a LR clonase reaction (Invitrogen). The luciferase reporter was removed by digestion with AscI and SacI and replaced with venus fluorescent protein (Additional file 9: Figure S8A) or green fluorescent protein (Additional file 9: Figure S8B) genes, amplified by PCR with the primers given in Additional file 14: Table S4, blunt cloned as described above, sequenced and inserted into the HpaI and MluI sites.

The full-length cDNAs of pfdn2, mgr, and pfdn5 were amplified from cDNA clones obtained from Drosophila Genomics Resources Center (DGRC) and sub-cloned into Gateway^® pENTR^TM vector (pENTR™⁄D-TOPO^® Cloning Kit, Invitrogen). Myc or Flag tags were added into the N-terminus of the gene sequences by LR recombination reactions (Gateway^® LR Clonase^® II Enzyme mix, Invitrogen) using pAMW or pAFW destination vectors respectively. The oligos that were used to amplify various DNA fragments were listed in Table 1.

A full length cDNA clone of SlABCB4 was obtained from the National Bioresource Project (NBRP)-Tomato (http://tomato.nbrp.jp/indexEn.html) with clone ID number, LEFL2031I14. The KOD, plus DNA polymerase (Toyobo, Osaka, Japan), and pENTR D-TOPO Cloning Kit (Invitrogen, Carlsbad, CA, USA) were used. A full length cDNA of SlABCB4 was cloned into the pENTR D-TOPO entry vector (Invitrogen, Carlsbad, CA, USA) using the In-Fusion cloning system (Takara Bio Inc., Kusatsu, Japan), following the method described by Park et al. [30 (link)]. In brief, amplification of the entry clone and linearization of the entry vector was done by using the in-fusion primers, which were generated using the In-Fusion cloning online tools (Takara Bio Inc., Kusatsu, Japan) (Supplementary Table S1). The Cauliflower mosaic virus 35S promoter driven expression constructs with no tag, C- and N-terminal GFP tags; pGWB2-SlABCB4, pGWB5-SlABCB4-GFP and pGWB6-GFP-SlABCB4 [31 (link)], respectively, were generated using the Gateway LR reaction (Invitrogen).