The sequence encoding human HDAC6 (NP_006035.2, UniProtKB - Q9UBN7) was used as a template for cloning all HDAC6 variants used in this report. In general, a nucleotide sequence encoding a given variant was PCR amplified with a set of desired primer pairs and inserted into the pDONR221 plasmid using the BP recombination reaction (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s protocol. The identity of the resulting entry clone was confirmed by Sanger sequencing. Expression plasmids were generated via LR recombination reaction between the entry clone and a required destination vector, which typically introduced a TEV-cleavable tag at the N-terminus of the HDAC6 variant to simplify purification and/or visualization of the resulting fusion. Schematic representations of constructs used in this study are shown in Supplementary Fig. S1 .
Bp recombination reaction
Manufactured by Thermo Fisher Scientific
Sourced in United States
The BP recombination reaction is a molecular cloning technique used for the insertion of DNA sequences into a vector. It enables the rapid and efficient transfer of DNA fragments between different plasmid or virus-based expression systems. The core function of this reaction is to facilitate the directional cloning of DNA fragments by taking advantage of site-specific recombination.
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Lab products found in correlation
Pdonr207, by Thermo Fisher Scientific (2 mentions)
Gateway cloning system, by Thermo Fisher Scientific (2 mentions)
Expand high fidelityplus pcr system, by Roche (2 mentions)
Lsm 700 confocal laser scanning microscope, by Zeiss (2 mentions)
Lr recombination reaction, by Thermo Fisher Scientific (1 mentions)
Pk7gwiwg2 1 vector, by Thermo Fisher Scientific (1 mentions)
Matchmaker gal4 based two hybrid system, by Takara Bio (1 mentions)
Clonexpress entry one step cloning kit, by Vazyme (1 mentions)
13 protocols using bp recombination reaction
1
Cloning and Engineering of HDAC6 Variants
2
Gateway Cloning and Mutagenesis of ATL Constructs
3
Cloning and Tagging CENP Proteins
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Cloning of CENP-M, -N, -S, -T and CENP-X have been described elsewhere [71 (link),97 (link),118 (link)]. Full length coding sequences of CENP-L (IRAUp969E0882D, RZPD Berlin), CENP-W (IRATp970A07105D, imaGenes Berlin), hMis12 (IRAUp969C0611D6, RZPD Berlin), SPC24 (IMAGp958K072621Q, RZPD Berlin), SPC25 (IRAUp969F0887D, RZPD Berlin), Nsl1 (pIC79, Iain Cheeseman), Dsn1 (pIC80, Iain Cheeseman), Nnf1 (pIC81, Iain Cheeseman), CENP-C/H/I [116 (link)] were amplified by PCR (Expand high fidelityPLUS PCR System, Roche, Penzberg, Germany) using primers incorporating flanking attB recombination sites and transferred into vector pDONR221 by BP recombination reaction (Invitrogen, Carlsbad, CA, USA). Genes were then transferred by LR recombination reactions into modified pFP-C and pFP-N (BD Biosciences, Clontech, Palo Alto, CA, USA) based Destination vectors. The fusion constructs have either short (FP-(s)-CENP) or long (FP-(l)-CENP) linkers. In FP-(s)-CENP constructs, the amino acid (aa) linker between the two fused proteins is SGTSLYKKAGFENLYFQGAT , whereas in FP-(l)-CENP constructs the linker is extended to SGTSLYKKAGFGGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSGG .
4
Plasmid Construction for CENP Fusion Proteins
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Plasmids pIC235, pDF180, pDF197, encoding LAP-CENP-R, -S and -T fusions proteins, respectively, were a kind gift of Dan Foltz and Iain Cheeseman. CENP-X was obtained directly by PCR from cDNA prepared from HeLa total RNA. Full-length coding sequences were amplified by PCR (Expand high fidelityPLUS PCR System, Roche, Penzberg, Germany) using primers incorporating flanking attB recombination sites and transferred into vector pDONR221 by BP recombination reaction (Invitrogen, Carlsbad, CA, USA; electronic supplementary material, table S1). Genes were then transferred by LR recombination reactions into modified pFP-C- and pFP-N (BD Biosciences, Clontech, Palo Alto, CA, USA)-based Destination vectors. CLIP-tagged proteins were generated by recombination into a Gateway modified pCLIPm vector (New England Biolabs, Isis Ltd, Bray, Co. Wicklow, Ireland) as described by Prendergast et al. [14 (link)]. All constructs were verified by DNA sequencing (MWG Biotech, Ebersberg, München, Germany).
5
Cloning and expression of TMK1 and AHA proteins
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Full-length and truncated variants TMK1, AHA1 and AHA2 were amplified by PCR from Col-0 cDNA and cloned into a protoplast transient expression vector (HBT vectors obtained from L. Shan and P. He) or the plant binary vectors pGWB641 and pGWB644. Stable transgenic lines were generated using standard Agrobacterium tumefaciens-mediated transformation in the tmk1-1 tmk4-1 mutant or Col-0 (ref. 43 (link)). The full-length cDNAs of TMK1 and AHA1 were amplified by PCR, and then cloned into the pDONR221-P1P4 and pDONR221-P3P2 vectors using the BP recombination reaction (Invitrogen), respectively. pDONR221-P1P4-TMK1 was recombined with pDONR221-P3P2-AHA1 into pFRETgc-2in1-NC to generate pFRET-mEGFP-AHA1+TMK1-mCherry44 (link). pDONR221-P3P2-AHA1 was recombined with pENTRL1-pLac-LacZalpha-L4 (Invitrogen) into pFRETgc-2in1-NC to generate pFRET-mEGFP-AHA1. pDONR221-P1P4-TMK1was recombined with pENTRL3-pLac-Tet-L2 (Invitrogen) into pFRETgc-2in1-CC to generate pFRET-MK1-mCherry. The AHA2 C-terminal region was cloned into pDest-565, and expressed in Escherichia coli (Rosetta, BL21) (a list of the primers is provided in Supplementary Table 1 ).
6
Agrobacterium-Mediated Transient Expression of THI20
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The primer pair was designed based on the THI20 ORF (Trans-THI20, Table 2 ). The partial BP adaptors were used for targeted fragment amplification. The whole sequence was amplified with BP site primers, followed by inserting into the pDONR207 vector by a BP recombination reaction (Invitrogen, Carlsbad, CA, USA). The targeted fragment was then inserted into the pK7GWIWG2(I) vector to obtain pK7GW12WG2-THI20 by an LR recombination reaction (Invitrogen, Carlsbad, CA, USA). The resulting plasmid was confirmed by sequencing, followed by transforming into Agrobacterium tumefaciens strain LBA4404 by electroporation.
Seeds of N. benthamiana were grown on MS agar. After 30 days, sterile leaves were immersed in a solution of A. tumefaciens strain LBA4404 with the recombinant plasmid and cultured on MS agar plates subsequently. After 3 days, the leaves were transferred to MS agar plates with 100 mg/L kanamycin.
Seeds of N. benthamiana were grown on MS agar. After 30 days, sterile leaves were immersed in a solution of A. tumefaciens strain LBA4404 with the recombinant plasmid and cultured on MS agar plates subsequently. After 3 days, the leaves were transferred to MS agar plates with 100 mg/L kanamycin.
7
Characterizing Ammonium Transporter 1;3
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The fragment of AtAMT1;3 cDNA carrying a C-terminal EGFP coding sequence was amplified by PCR from plamised pCAMBIA1300-AMT1;3K75R,K233R-EGFP, and then, the entry clone pDON-AMT1;3-EGFP was created by performing a BP recombination reaction (Invitrogen). We performed an LR recombination reaction to mobilize the fragment AMT1;3-EGFP into the pDR vector (according to the Gateway Technology manuals). The pDR-AMT1;3K75R,K233R-EGFP and the pDR-AMT1;3 (a gift from the laboratory of Wolf Frommer (Carnegie Institution for Science, Stanford, CA)) vectors were transformed into the triple MEP deletion yeast strain 31019b (mep1D mep2D mep3D ura3) by the lithium acetate method. Transformants were selected on minimal medium lacking uracil. Growth complementation assays were performed on solid yeast nitrogen base (YNB) medium (without an N source; BD Biosciences, BD233520) supplemented with 2% (wt/vol) galactose.
8
Cloning and Transformation of Arabidopsis
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The coding sequences (CDS) from PvTDIFL3MR3 and PvTDIFL3MR2 were amplified using PCR with gene specific primers (Supplementary Table 2 ). The CDS of PvTDIFL1 was artificially synthesized (Sunny Technology, Shanghai, China). The products were cloned into a Gateway™ entry vector pDONR201 using the BP recombination reaction (Invitrogen). The clones were analyzed using DNA sequencing. The correct fragments were subsequently recombined into the destination vector, pK2GW7 (Karimi et al., 2002 (link)). Transformation of A. thaliana was performed using the Agrobacterium tumefaciens-mediated floral dip method (Clough and Bent, 1998 (link)).
9
Overexpression and RNAi of OsAHL1 in Rice
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The native promoter [an upstream fragment (1,753 bp) starting from the base next to the start codon of OsAHL1] was amplified from genomic DNA of upland rice cv. IRAT109 and placed to pCAMBIA1300-EGFP to control GFP expression. Truncated and full-length cDNA of OsAHL1, without the termination codon, were placed respectively on pCAMBIA1300-EGFP for fusion expression under the control of the CaMV 35S promoter.
The full-length cDNA of OsAHL1 was amplified from IRAT109 by RT-PCR and inserted into pCAMBIA1300 under the control of the CaMV 35S promoter for overexpression (I17). To make a dsRNAi construct of OsAHL1 (RNAi) a 189 bp fragment of OsAHL1 (nucleotides 1143–1331) was generated by PCR and was cloned into pCB2004B2 through attB/attP (BP) recombination cloning41 . The attB1 and attB2 are the 2 sequences for the BP recombination reaction (Invitrogen). All of the constructs were transformed into the japonica rice cv. Zhonghua 11 by the Agrobacterium-mediated (stain EHA105) transformation method42 (link).
The full-length cDNA of OsAHL1 was amplified from IRAT109 by RT-PCR and inserted into pCAMBIA1300 under the control of the CaMV 35S promoter for overexpression (I17). To make a dsRNAi construct of OsAHL1 (RNAi) a 189 bp fragment of OsAHL1 (nucleotides 1143–1331) was generated by PCR and was cloned into pCB2004B2 through attB/attP (BP) recombination cloning41 . The attB1 and attB2 are the 2 sequences for the BP recombination reaction (Invitrogen). All of the constructs were transformed into the japonica rice cv. Zhonghua 11 by the Agrobacterium-mediated (stain EHA105) transformation method42 (link).
10
Bimolecular Fluorescence Complementation Assay
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Full length of BZR1 or BZR15S were amplified by PCR and cloned into the pDONR221-P1P4 vector, PIF4 or ARF6 were cloned into the pDONR221-P3P2 vector using the BP recombination reaction (Invitrogen), respectively. The 2in1 LR reaction were performed with destination vector pBiFCt-2in1-CC and different pDONR221 vectors33 (link). Agrobacterial suspensions containing p35S:PIF4-nYFP-p35S:RFP-p35S:BZR1-cYFP, p35S:PIF4-nYFP-p35S:RFP-p35S:BZR15S-cYFP, p35S:ARF6-nYFP-p35S:RFP-p35S:BZR1-cYFP, or p35S:ARF6-nYFP-p35S:RFP-p35S:BZR15S-cYFP constructs were injected into the lower epidermis of tobacco leaves. The transfected plants were kept in the greenhouse for at least 36 h at 22 ℃, and then treated with mock solution or 1 mM H2O2 for 30 min. Fluorescent signals were visualized by using the LSM-700 laser scanning confocal microscope (Zeiss) and the signal intensities of YFP and RFP were determined by ImageJ software.
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