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Appendix S1. Experimental procedure s for cloning
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Construct design pGPTVII-based vectors
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The vector constructs YC3.6-Bar, YC3.6-Kan and YC3.6-Hyg were generated based
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on the plant binary vectors pGPTVII-Bar, pGPTVII-Kan and pGPTVII-Hyg (Walter et al.
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2004) that confer resistance to Basta (bar), kanamycin (nptII) or hygromycin B (hptII),
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respectively. The nos promoter and the polyadenylation site of gene7 from
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Agrobacterium tumefaciens control expression of the corresponding resistance genes.
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The YC3.6 coding DNA was obtained from pNB96-YC3.6 (kindly provided by J.
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Schroeder). A HindIII and blunt-ended EcoRI fragment, containing the dual 35S
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promoter and the following YC3.6 coding region with a XhoI site lying in between, was
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subcloned into HindIII and blunt-ended XhoI sites of pGPTVII vectors. Each resulting
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construct was digested with HindIII and XhoI to release the dual 35S promoter, and
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then ligated with the 636-bp UBQ10 promoter fragment described in the work of Grefen
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et al. (Grefen et al. 2010).
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Construct design pTKan-based vectors
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The vector constructs YC3.6-N, YC3.6-C and D3cpv-C are based on the plant
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binary vector pTKan. pTKan was generated by inserting a 647 bp terminator fragment
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of the rbcS (ribulose bisphosphate carboxylase oxygenase small subunit) from Pisum
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sativum, via PstI/HindIII restriction sites, to the binary vector pJHA212K (Yoo et al.
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2005). In turn, pJHA212K was generated by replacing the 35S-driven resistance gene
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cassette from pPZP212 (Hajdukiewicz et al. 1994) by the mas-driven resistance gene
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cassette of the vector pCGN1547 (McBride and Summerfelt 1990). The pTKan-based
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vectors are characterized by their kanamycin resistance marker (nptII) for selection in
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plants, and their resistance to streptomycin and spectinomycin in bacteria, which is
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mediated by the aadA (aminoglycoside-3-adenyltransferase) gene, located in the
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vector backbone. Expression of the nptII gene is controlled by the 5’- and 3’- regulatory
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regions of the mas (mannopin synthase) gene from A. tumefaciens.
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For pTKan-YC3.6-N a 1226 bp fragment (YC3.6-BamHI.FOR/YC3.6-BglII.REV) and
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a 778 bp fragment (YC3.6-BglII.FOR/YC3.6-XbaISTOP.REV) of the YC3.6 coding
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sequence (CDS) was amplified from YC3.6-Bar with primer pairs indicated in brackets.
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Thereby the stop codon of YC3.6 was removed. After subcloning into the pJET1.2
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vector (Fermentas, www.fermentas.de), both fragments were released using the
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introduced BamHI, BglII and XbaI restriction sites and inserted into the pTKan vector
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backbone using the BamHI and XbaI sites. In a similar way, two fragments of YC3.6
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were amplified from YC3.6-Bar to generate pTKan-YC3.6-C. A 1226 bp fragment was
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amplified using the primer pair YC3.6-XhoI.FOR and YC3.6-BglII.REV, whereas the
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smaller fragment (778 bp) of the YC3.6 CDS was obtained with the primer combination
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YC3.6-BglII.FOR and YC3.6-SpeI.REV. The fragments were subcloned into pJET1.2,
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cut with XhoI, BglII and SpeI respectively, and introduced into pTKan via the XhoI and
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SpeI restriction sites. Finally, a PCR using the primer combination UBQ10prom-KpnI-
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FOR and UBQ10prom-BamHI.REV was performed on YC3.6-Bar. The resulting 661 bp
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fragment of the UBQ10 promoter (Norris et al. 1993) was subcloned into pJET1.2,
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digested with KpnI and BamHI and ligated with the intermediate vector constructs
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pTKan-YC3.6-N and pTKan-YC3.6-C to obtain the basic vector constructs YC3.6-N
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and YC3.6-C. To generate the basic vector D3cpv-C, a 661 bp fragment of the UBQ10
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promoter (Norris et al. 1993) was amplified using the primer pair UBQ10prom-KpnI-
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FOR and UBQ10prom-BamHI.REV and subcloned into pJET1.2. The UBQ10 fragment
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was released using KpnI and BamHI restriction sites and ligated into pTKan to obtain
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pUTKan. Afterwards a 2985 bp fragment of D3cpv was amplified from pcDNA3-D3cpv
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(Palmer et al. 2006) using the primer combination D3cpv-SalI.FOR and D3cpv-
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BcuI.REV. The fragment was subcloned in pJET1.2, released via the introduced SalI
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and BcuI restriction sites and finally fused with the pUTKan vector.
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1
To introduce the N-terminal targeting sequences for nuclear export, nuclear import,
2
and tonoplast localization, oligonucleotides supplied with ATG start codons and
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appropriate restriction sites were designed and inserted into YC3.6-C. For NES-YC3.6
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the primer pair NES-ATG.FOR and NES-ATG.REV has been annealed and inserted
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into YC3.6-C via AatII and SalI restriction sites which resulted in a 62 bp fragment that
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harbours the nuclear export signal of the heat-stable protein kinase inhibitor α (PKIα)
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(Wen et al. 1995). For NLS-YC3.6, the oligonucleotides NLS-ATG.FOR and NLS-
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ATG.REV have been annealed and ligated into YC3.6-C using AatII and SalI restriction
9
sites. The introduced fragment represents the nuclear localization signal that originates
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from simian virus 40 (SV40) large-T protein (Kalderon et al. 1984). To generate TP-
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D3cpv, pUTKan was digested with ApaI and BamHI to generate the 10846 bp
12
backbone fragment of the construct. The vector pcDNA3-D3cpv (Palmer et al. 2006)
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was cut with HindIII and ApaI to obtain a 1962 bp fragment of D3cpv. Annealing of the
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oligonucleotides TP-D3cpv.FOR and TP-D3cpv.REV resulted in a 74 bp tonoplast
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targeting fragment with BamHI/HindIII compatible ends. Finally, all three fragments
16
were fused via their BamHI, HindIII and ApaI restriction sites to obtain TP-D3cpv. To
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generate a plasma membrane localised construct, YC3.6 was fused to the small
18
plasma membrane resident protein Lti6b (Cutler et al. 2000). The CDS of Lti6b was
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amplified from Arabidopsis thaliana Col-0 cDNA using LTI6b-SalI.FOR and LTI6b-
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SpeI.REV. The resulting 188 bp fragment was cloned into pJET1.2, released via the
21
introduced SalI and SpeI restriction sites and finally fused with YC3.6-N to obtain PM-
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YC3.6-Lti6b.
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