6. Materials and Methods
6.6 Plasmid construction
Cloning success was monitored by endonuclease digestion using at least two independent restriction enzymes, and following PCR amplification steps, sequences were checked by external sequencing at Eurofins Scientific (Brussels, Belgium). If not indicated otherwise, cloning was performed in E. coli strains Dh5α or Top10. All primer sequences used for cloning purposes are listed in Tab. S4.
Most constructs were created by use of the GatewayTM technology (Invitrogen, Thermo Fisher Scientific, Waltham, USA). To this end, a PCR product flanked by attB-sites was amplified by PfuX7 Polymerase (Nørholm, 2010), and purified from an agarose gel using the GeneJET Gel Extraction Kit (Cat.-No. K0832; Thermo Fisher Scientific). Plasmids were purified using the GeneJET Plasmid Miniprep Kit (Cat.-No. K0502; Thermo Fisher Scientific), and DNA concentrations were measured with a plate-reader photospectrometer (Infinite F200 PRO; Tecan, Männedorf, Switzerland). BP and LR reactions were performed with Gateway™ BP Clonase™ II Enzyme mix (Cat.-No. 11789020; Thermo Fisher Scientific) and Gateway™ LR Clonase™ II Enzyme mix (Cat.-No. 11791100; Thermo Fisher Scientific) according to the manufacturer’s instructions, but with half total reaction volumes and additional reductions of the respective clonase enzyme mixes. The composition of the BP and LR reaction mixtures is presented in Tab. 2.
Tab. 2| Compositions of BP and LR reaction mixtures. TE buffer: 10mM Tris-HCl, pH 8.0; 1mM EDTA.
BP reaction LR reaction
attB-PCR product (7.5-75 ng) 0.5-3.5 µl Entry Clone (25-75 ng) 0.5-3.5 µl pDONR vector (150 ng/µl) 0.5 µl Destination Vector (150 ng/µl) 0.5 µl
TE buffer to 4 µl TE buffer to 4 µl
BP clonase II enzyme mix 0.5 µl LR clonase II enzyme mix 0.25 µl
Following overnight incubation at room temperature (22°C), 0.5 µl of Proteinase K (provided with the clonase enzyme kits) was added to the reactions, the mixture was incubated for 0.5-3 hours at 37°C, cooled down on ice, and subsequently transformed into E. coli cloning strains.
6.6.1 Destination vectors
All promoter constructs were cloned to yield Destination vectors containing the GatewayTM cassette (GW) behind the promoter sequence by replacing the AscI/XhoI fragment of pAM-PAT-ProCDKA;1-GW (Addgene plasmid #79750; Nowack et al., 2006). Ligation was performed using T4 DNA Ligase (Cat.-No. EL0011; Thermo Fisher Scientific) according to the manufacturer’s instructions. The promoter sequences of PRT1, ATL9, and EIN2 were amplified from Col-0 genomic DNA (gDNA) using primers proPRT1_fw/proPRT1_rev, proATL9_fw/proATL9_rev, and proEIN2_fw/proEIN2_rev, respectively. The final vector pAM-PAT-proPRT1ΔCTCT-GW (lacking CTCT in the 3’ region of the promoter due to amplification problems of a repetitive sequence) is listed on TAIR with the stock number CD3-2396.
To construct pAM-PAT-ProUBQ10 GW (Addgene #79751), a plasmid that contained the UBQ10 promoter (generated and kindly provided by Stefan Pusch1 (Pusch et al., 2011)) was used as a template to amplify the UBQ10 promoter sequence using the primer pair
1 German Cancer Research Center, 69120 Heidelberg, Germany
(proUBQ10_fw/proUBQ10_rev). The PCR product was subsequently cloned into the AscI/XhoI sites of pAM-PAT-ProCDKA;1-GW (Addgene plasmid #79750; Dissmeyer and Schnittger (2011)), yielding pAM-PAT-ProUBQ10 GW (Addgene #79751). All destination vectors were cloned using E. coli DB3.1.
6.6.2 PRT1 expression vectors
For expression of PRT1-3HA from p35S, a Gateway® Entry Clone containing the CDS of PRT1 plus a 17 bp Kozak sequence (GCTTAGCCGCCATGGGA) upstream of ATG was provided by C. Naumann, and recombined with pAM-PAT-p35S::GW-3HA (Bernoux et al., 2008). Primers used by C. Naumann to amplify the BP product were PRT1_kozak_ss and PRT1new_as.
3HA-PRT1 was constructed by fusion PCR. To this end, the 3HA-tag was amplified using primers Fw_HA and Rev_HA-PRT1 from plasmid pENTR-K2-GUS (obtained from F. Faden), and PRT1 was amplified from plasmid pAM-PAT-p35S::PRT1-3HA using Fw_PRT1 and Rev_PRT1+stop.
Subsequently, fusion PCR of both PCR products was conducted using primers Fw_attB1-HA and Rev_attB2-PRT1. The fusion product was used in a BP reaction with pDONR 201 (Invitrogen) to yield pEN-3HA-PRT1, and the BP product was used in LR reactions with pAM-PAT destination vectors bearing the requested promoter sequences (i.e., pAM-PAT-ProUBQ10 GW, pAM-PAT-proPRT1ΔCTCT -GW, and pAM-PAT-proEIN2-GW). pEN-3HA-PRT1 was furthermore subjected to site-directed mutagenesis to obtain pEN-3HA-PRT1V28A, pEN-3HA-PRT1C29A, and pEN-3HA-PRT1C195A (see below, chapter 6.7.5).
PRT1 was also cloned as the genomic locus sequence including the 5’ UTR followed by a C-terminal quadruple-Gly linker and a 3HA-tag. For this purpose, the genPRT1 sequence was amplified from gDNA with primers attB1-genPRT1_fw and HA-genPRT1_rev, and the 3HA-tag was obtained from pEN-3HA-PRT1 using primers genPRT1-HAT_fw and attB2-HAT_rev. Due to amplification problems which were already encountered during amplification of the PRT1 promoter, the sequence of genPRT1 in the final vector contained a deletion of “CT” within a repetitive sequence of the 5’ UTR.
Fusion-PCR was performed with attB1-genPRT1_fw and attB2-HAT_rev. The PCR product was recombined into pDONR 201 and subsequently subjected to LR reaction with pAM-PAT-proEIN2-GW.
To create GFP-PRT1 expression vectors, mGFP5 was amplified including a C-terminal Ser-Ala linker from pEarleyGate 103 (Earley et al., 2006) using primers attB1_mGFP5_fw and mGFP5_rev, and PRT1 was amplified from pEN-3HA-PRT1 (WT, C29A, or C195A) with primers GFP-PRT1_fw and Rev_PRT1+stop. Subsequent fusion-PCR employed primers attB1_mGFP5_fw and Rev_attB2-PRT1, and these products were used in BP reactions with pDONR 201. LR reactions were performed with pAM-PAT-proUBQ10 GW or pAM-PAT-proPRT1ΔCTCT-GW Destination Vectors.
For BiFC assays, a plasmid for expression of C-terminally YFPN-tagged PRT1 (pUBC-YFPN-PRT1) was kindly provided by C. Naumann. Alternatively, pEN-3HA-PRT1 and pEN-3HA-PRT1V28A were recombined with pUBN-YFPN-Dest (Grefen et al., 2010), to obtain WT and V28A variants of pUBN-YFPN-3HA-PRT1.
For expression of PRT1 with N-terminal 8xHisMBP tag in E. coli, an Expression Clone carrying the PRT1 CDS with a 5’ recognition sequence of the Tobacco Etch Virus (TEV) protease in pVP16 (Clontech Laboratories, Mountain View, USA) was provided by C. Naumann (Naumann et al., 2016). For the
production of the mutated variants of PRT1, an Entry Clone containing the PRT1 CDS with the TEV recognition sequence at the 5’ end (pEN-TEV-PRT1) was received from C. Naumann (cloning of this Entry Clone is described in Mot et al. (2018)), and was subjected to site-directed mutagenesis (see below). The modified Entry Clones were recombined with pVP16. For N-terminal GST-tagged variants, the same Entry Clones were recombined with pDEST™15 Vector (Invitrogen). For expression of PRT1 variants from pVP16, E. coli Bl21 (DE3) was used as the expression strain, and pDEST™15 constructs were expressed in E. coli Rosetta™ 2(DE3)pLysS (a kind gift of M. Trujillo).
6.6.3 EIN2 expression vectors
To express a UFT construct of EIN2646-1294 with a C-terminal Myc-YFPC tag, a plasmid containing the sequence of a synthetic codon-optimized Ub (Faden et al., 2016) with Lys to Arg exchanges at positions K29 and K48 fused to the CDS of EIN2646-1294 (pEN-UbK29,48R-EIN2646-1294) was kindly provided by C. Naumann, and used as a PCR template. Primers attB1-Ub and EIN2-myc_rev were used for amplification. The Myc-tag was amplified from vector pE-SPYNE-GW (Schütze et al., 2009) using primers EIN2_myc_fw and attB2_Myc_noS, and both PCR fragments were fused by PCR using primers Ub_fw and Myc_rev. Subsequently, an adapter PCR primed by attB1_Ub and attB2_Myc_noS was conducted, and the product was subjected to BP reaction with pDONR 201, to yield pEN-UbK29,48R -EIN2646-1294-Myc. Phe-646 (TTC) of EIN2646-1294 was subsequently changed to Gly (GGT) by site-directed mutagenesis (see below), and both Entry Clones (containing EIN2646-1294 or Gly-EIN2647-1294) were recombined with pUBC-YFPC-Dest (Grefen et al., 2010).
To create the BiFC construct pUBC-YFPC-3HA-UbK29,48R-EIN2C-term-3HA, an Entry Clone was first constructed by PCR fusion of 3HA (amplified from a plasmid containing the K2 construct (Faden et al., 2016) using primers Kozak-HAT_fw/HAT_rev_1), and UbK29,48R-EIN2646-1294 (amplified from pEN-UbK29,48R-EIN2646-1294 using primers Ubiq_fw/EIN2-KpnI-AscI_rev; primer EIN2-KpnI-AscI_rev leads to the 3’ addition of sequential KpnI (FD0524, Thermo Fisher Scientific) and AscI restriction sites). Fusion PCR was performed using primers Kozak-HAT_fw/EIN2-KpnI-AscI_rev. The fusion PCR product was subjected to an adapter PCR using attB1_Kozak/attB2_EIN2, and recombined into pDONR 201 to yield pEN-3HA-UbK29,48R-EIN2646-1294-KpnI-AscI. This Entry Clone was used in mutagenesis PCR to create pEN-3HA-UbK29,48R-Gly-EIN2647-1294-AscI. Both constructs were subsequently ligated with KpnI-3HA-AscI (including a [Gly-Ala]2 linker in front of the 3HA sequence) which was amplified from K2 using primers GAGA-HAT_fw/HAT_rev2. Ligation was performed using T4 DNA Ligase (Cat.-No.
EL0011; Thermo Fisher Scientific) according to the manufacturer’s instructions, and resulted in Phe- and Gly variants of pEN-3HA-UbK29,48R-EIN2C-term-3HA. These Entry Clones were recombined with pUBC-YFPC-Dest to create the final constructs. In an alternative BiFC assay, the above described plasmid variants of pUBC-YFPC-UbK29,48R-EIN2C-term-Myc were used.
For Ub-EIN2C-term-YFP constructs, the CDS of EIN2646-1294 was amplified from pEN-UbK29,48R-EIN2646-1294 -Myc using primers ss_UBQ_EIN2 and YFP-EIN2_rev. The CDS of a synthetic codon-optimized Ub (without any amino acid exchanges) was amplified from a plasmid containing the K2 construct (Faden et al., 2016) using primers attB1_Ub and as_UBQ_EIN2. YFP was obtained from pEarleyGate 101 (Earley et al., 2006) by applying primers EIN2-YFP_fw and attB2-YFP. All three PCR products were combined in a reaction for fusion PCR using primers attB1-Ub and attB2-YFP. The product was recombined into pDONR 201, and subjected to Phe646 (TTC) to Gly (GGT) mutation to yield both
pEN-Ub-EIN2646-1294-YFP and Ub-Gly-EIN2647-1294-YFP (or: EIN2C-term-YFP and Gly-EIN2C-term-YFP). To construct pEN-Met-EIN2647-1294-YFP (or: pEN-Met-EIN2C-term-YFP), the sequence of EIN2647-1294-YFP was amplified from pEN-Ub-EIN2646-1294-YFP using primers attB1-Met-EIN2 and attB2-YFP, and subjected to a BP reaction with pDONR 201. All three Entry Clones were recombined with pAM-PAT-ProUBQ10 GW.
pEN-Ub-EIN2646-1294-YFP and Ub-Gly-EIN2647-1294-YFP were furthermore introduced into pAM-PAT-proEIN2-GW.
6.6.4 Further expression vectors
The PRT7 expression vector was created by Taq DNA polymerase-mediated amplification of the PRT7 CDS with primers PRT7_CDS_fw and PRT7-attB2 from complementary DNA (cDNA) of Col-0.
Subsequently, an adapter PCR was conducted using primers adapter_TEV and PRT7-attB2 to add the TEV protease recognition sequence, as well as the attB1 site to the 5’ end of the amplicon. This product was introduced into pDONR 201 by BP reaction, and into pVP16 for E. coli Bl21 (DE3) expression with N-terminal HisMBP-tag by LR reaction.
An Entry clone containing the CDS of ATE1 was created by C. Naumann using primers ATE1_pos2_ss and ATE1_as for amplification from cDNA. For in-vitro studies within this work, this Entry Clone was used in a LR reaction with pDEST™17 Vector (Invitrogen) for N-terminal fusion with a hexa-His tag.
The construct was expressed from E. coli BL21-CodonPlus® (DE3)-RIL.
PCO1 and PCO4 with N-terminal hexa-His tag were provided as purified enzymes by M. White and E.
Flashman (White et al., 2017).
For UBRPRT6 expression, an Entry Clone containing the CDS of PRT61-193 with N-terminal TEV recognition sequence and C-terminal simple HA-tag was provided by P. Reichman. This was included in a LR reaction with pDEST™15 for N-terminal GST fusion. The construct was transformed into E. coli BL21-CodonPlus® (DE3)-RIL. In contrast, purified recombinant GST-PRT6119-189 was provided by H.
Zhang and F. Theodoulou.
To create pEN-YFP, the sequence of YFP was amplified from pEN-Ub-EIN2646-1294-YFP using primers attB1-YFP and attB2-YFP, and the product was recombined into pDONR 201. LR into pAM-PAT-ProUBQ10 GW yielded pAM-PAT-pAM-PAT-ProUBQ10::YFP.
A plasmid containing mCherry was co-transformed as a control during BiFC experiments. The respective plasmid was kindly provided by G. Furlan and M. Trujillo as a vector containing mCherry for cytoplasmic and nuclear localization (Furlan et al., 2017). The plasmid for expression of YFP with a plastidial localization signal (Fig. S19) was provided by C. Naumann.
To create the GUS reporter constructs, an Entry Clone containing the GUS CDS was provided by N.
Dissmeyer (IPB, Halle Saale), and originally obtained from Bekir Ülker1. This Entry Clone was recombined with Destination vectors pAM-PAT-proPRT1ΔCTCT-GW and pAM-PAT-proATL9 (see above).
The vector for expression of GUS under control of the promoter of IQD4 was kindly provided by J.
Quegwer and K. Bürstenbinder and was previously published in Bürstenbinder et al. (2017).
1 Institute of Cellular and Molecular Botany, University of Bonn, 53115 Bonn, Germany
For expressing eGFP from the PRT1ΔCTCT promoter, pEN-L1-F-L2 (Karimi et al., 2007) was recombined with pAM-PAT-proPRT1ΔCTCT-GW.