The full-length CDS fragments of WRKY63 were PCR-amplified and cloned into the pCR8/GW/TOPO vector (Invitrogen). Point mutation constructs were constructed by specific primer sets then cloned into the pCR8/GW/TOPO vector (Invitrogen). WRKY63 was recombined into the PK7WGF2 (Invitrogen) to generate the GFP:WRKY63 plasmid. To construct WRKY63pro::WRKY63:GFP, approximately 2-kb promoter with the genome sequence of WRKY63 was cloned into the pCR8/GW/TOPO vector (Invitrogen), then recombined into the pMDC107 binary vector. The transgenic plants were generated by using the floral dip method.
Pcr8 gw topo vector
Manufactured by Thermo Fisher Scientific
Sourced in United States, Japan
The PCR8/GW/TOPO vector is a plasmid designed for direct cloning of Taq polymerase-amplified PCR products. It contains a TOPO cloning site, which allows for rapid and efficient insertion of PCR products. The vector also includes Gateway recombination sites, enabling easy transfer of the insert into other expression vectors.
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Lab products found in correlation
Gateway lr clonase 2 enzyme mix, by Thermo Fisher Scientific (7 mentions)
Gateway technology, by Thermo Fisher Scientific (4 mentions)
Lr clonase 2, by Thermo Fisher Scientific (4 mentions)
Gateway system, by Thermo Fisher Scientific (4 mentions)
Pgem t easy vector, by Promega (3 mentions)
Phusion polymerase, by Thermo Fisher Scientific (3 mentions)
Biolistic pds 1000 he, by Bio-Rad (3 mentions)
Blasticidin, by Thermo Fisher Scientific (3 mentions)
Lr clonase, by Thermo Fisher Scientific (3 mentions)
Pcmv 2xmyc dest, by Thermo Fisher Scientific (2 mentions)
P3xflag hd dest, by Thermo Fisher Scientific (2 mentions)
Homo sapiens vps54 cdna, by OriGene (2 mentions)
Gateway recombination technology, by Thermo Fisher Scientific (2 mentions)
Pegfp n1, by Takara Bio (2 mentions)
Pet 53 dest vector, by Merck Group (2 mentions)
Primestar max dna polymerase, by Takara Bio (2 mentions)
Quikchange site directed mutagenesis kit, by Agilent Technologies (2 mentions)
Gateway cloning technology, by Thermo Fisher Scientific (2 mentions)
Gateway lr clonase enzyme mix, by Thermo Fisher Scientific (2 mentions)
Tcs sp5 confocal spectral microscope imaging system, by Leica (2 mentions)
85 protocols using pcr8 gw topo vector
1
WRKY63 Gene Cloning and Transgenic Expression
2
Cloning and Characterization of COQ8 Proteins
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Sequences of all oligonucleotides used in this study can be found in Supplementary Table S1. yCOQ8 was amplified from yeast genomic DNA, cloned into pCR8/GW/TOPO vector (Thermo Fisher, Waltham, MA, USA) and then transferred to the centromeric (CEN) pCM189 yeast expression vector adapted to the Gateway cloning system (Thermo Fisher, Waltham, MA, USA). The coding sequence of human COQ8B was amplified from human cDNA and cloned into pCR8/GW/TOPO vector (Thermo Fisher, Waltham, MA, USA). We used a reverse primer that included the sequence encoding either the V5 or the HA tag. COQ8BV5 was then subcloned into the pCM189 yeast expression vector, whereas COQ8HA was cloned into the pCDNA5 using the Gateway cloning system.
The yCOQ8‐COQ8B and yCOQ3‐COQ8B hybrid genes were constructed by sequential PCR as described (Nguyen et al.,2014 ) and cloned in pCM189. Both constructs encode the V5 epitope on the 3′terminus.
All mutagenesis reactions were performed on fragments cloned in pCR8/GW/TOPO vector using the QuikChange Lightning site‐directed mutagenesis kit (Agilent, Santa Clara, CA, USA). The correctness of all constructs was confirmed by direct sequencing.
The yCOQ8‐COQ8B and yCOQ3‐COQ8B hybrid genes were constructed by sequential PCR as described (Nguyen et al.,
All mutagenesis reactions were performed on fragments cloned in pCR8/GW/TOPO vector using the QuikChange Lightning site‐directed mutagenesis kit (Agilent, Santa Clara, CA, USA). The correctness of all constructs was confirmed by direct sequencing.
3
AMAT Gene Cloning and Sequencing
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AMAT DNA sequences were PCR-amplified from genomic DNA using the outF/outR primer pair (Table S21 ). AMAT cDNAs (ORF) were obtained by RT-PCR amplification of berry skin RNA samples using the c5F/c7R primer pair (Table S21 ). PCR products were cloned into pCR®8/GW/TOPO® vector (Thermo Fisher Scientific). The whole insert was sequenced with vector primers and gene-specific primers.
4
LRRK2 Mutant Constructs Generation
5
Cloning and Expression of VPS52 Constructs
6
Constructs for LRRK2, JIP4, and CORO1C
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Constructs for 3xFLAG-LRRK2 and mNeonGreen-JIP4 have been described previously (Beilina et al., 2014 (link), 2020 (link); Bonet-Ponce et al., 2020 (link)). JIP4, LRRK2, and CORO1C cDNAs were amplified with PCR and cloned into the pCR8/GW/TOPO vector (Thermo Fisher Scientific). Each was then subcloned into pDEST vectors using Gateway technology (Thermo Fisher Scientific). LRRK2 was subcloned into the pDEST-HaloTag and pDEST-mScarlet vectors; JIP4 was subcloned into pDEST-HaloTag and pDEST-SNAPtag, and CORO1C was subcloned into pDEST-HaloTag.
LAMP1-RFP, LAMP1-mNeonGreen, mCherry-SEC61B, mCherry-Climp63, and TMEM192-3xHA plasmids were purchased from Addgene (Addgene#1817, #98882, #90994, #136293, and #102930) (Sherer et al., 2003 (link); Shibata et al., 2008 (link); Nixon-Abell et al., 2016 (link); Abu-Remaileh et al., 2017 (link); Chertkova et al., 2017 ).
mNeonGreen-SEC61B and HaloTag-SEC61B were cloned by using the mCherry-SEC61B plasmid obtained from Addgene and replacing the tags using IN-FUSION. The LAMP1-HaloTag, ARL8B-mCherry, and VPS13C-HaloTag plasmids were gifts from Juan Bonifcacino (National Institutes of Health) and Pietro De Camilli (Yale University), respectively. All expression constructs used in this study are summarized in Supplemental Table 1.
LAMP1-RFP, LAMP1-mNeonGreen, mCherry-SEC61B, mCherry-Climp63, and TMEM192-3xHA plasmids were purchased from Addgene (Addgene#1817, #98882, #90994, #136293, and #102930) (Sherer et al., 2003 (link); Shibata et al., 2008 (link); Nixon-Abell et al., 2016 (link); Abu-Remaileh et al., 2017 (link); Chertkova et al., 2017 ).
mNeonGreen-SEC61B and HaloTag-SEC61B were cloned by using the mCherry-SEC61B plasmid obtained from Addgene and replacing the tags using IN-FUSION. The LAMP1-HaloTag, ARL8B-mCherry, and VPS13C-HaloTag plasmids were gifts from Juan Bonifcacino (National Institutes of Health) and Pietro De Camilli (Yale University), respectively. All expression constructs used in this study are summarized in Supplemental Table 1.
7
Cloning and Expression of VPS52 Constructs
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Constructs for 2x-myc- and 3x-flag- tagged LRRK2 full length and domains, GAK, BAG5, RAB29 and GUS have been described previously (Beilina et al., 2014 (link); Taymans et al., 2011 (link)). VPS52 full length and domain constructs were PCR amplified with the following primers: VPS52 full length: F_flVPS52 and R_flVPS52; ΔC-VPS52: F_ΔcVPS52 and R_ΔcVPS52; ΔN -VPS52: F_ΔnVPS52 and R_-ΔnVPS52; ΔC/ΔN-VPS52(Sac2): F_SacVPS52 and R_SacVPS52 and cloned into pCR8/GW/TOPO vector (Thermo Scientific). Full-length VPS52 and domains were transferred into the pCMV-2xmyc-DEST, pDEST53-DEST, pAcGFP-DEST and p3xflag-HD-DEST vectors using Gateway recombination technology (Thermo Scientific). The generation of a plasmid encoding Syndetin-GFP was previously described. A plasmid encoding VPS54-GFP was generated by using Homo sapiens VPS54 cDNA (Origene, MD) subcloned into pEGFP-N1 (Clontech) vector by Gibson assembly using F-VPS54 and R-VPS54; F_vector and R_vector primers.
8
Generating PAS2 Transgenic Lines
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The pas2-1 mutants are EMS alleles in Col-0 background. To generate the PPAS2::GFP-PAS2 (GFP-PAS2) transgenic line, a DNA fragment containing 1.5-kb PAS2 promoter and the full-length cDNA clone of PAS2 were amplified using primers in Table S1 online and cloned into the PCR8/GW/TOPO vector (Thermo Fisher Scientific, Waltham, MA, USA). After sequencing, both fragments were cloned to the pH7WGF2 vector [55 (link)]. The verified construct was introduced into pas2-1 using Agrobacterium tumefaciens-mediated transformation [56 (link)]. A GFP-PAS2-expressing Arabidopsis thaliana transgenic line was crossed with one expressing mCherry-SYP32, -Got1p, -VTI12, -RabA1g or -RabG3f [46 (link)] to generate dual labeled transgenic lines, respectively. The point mutations were generated directed on the GFP-PAS2 construct using a QuikChange® Site-Directed Mutagenesis Kit (Agilent, Santa Clara, CA, USA). The mutated constructs were transformed into pas2-1 heterozygous background. The T1 generation was screened for pas2-1 homozygous plants. A GFP-CESA3-expressing Arabidopsis thaliana transgenic line [3 (link)] was crossed with pas2-1 heterozygous plants. F2 seedlings homozygous for pas2-1 by PCR genotyping were used for confocal imaging and other analysis.
9
Molecular Cloning of Soybean Genes
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All CDSs were amplified by PCR using PrimeSTAR MAX DNA Polymerase (TAKARA) with primer sets and cDNA (Aspergillus-fermented soybean at DAS 5) as the template. Primer sequences were as follows: GmIOMT1 (Glyma.05G147000.1): 5′-CACCATGTCGGGTGATTTAGCATACAAG-3′ and 5′-TCACAGACGTCTACACAGGG-3′; OMT2 (Glyma.07G048900.1): 5′-ATGGCTCCATCATTGGAAACC-3′ and 5′-TTACTTATAAAATTCCATAACCCAG-3′; and F6H (Glyma.18G080400.1): 5′-ATGGATCTTCAACTTCTCTACTTC-3′ and 5′-CTAATTATGAACAGTTTTGGGAATG-3′. Amplified fragments were introduced into pENTR-D-TOPO vector (Thermo Fisher Scientific) for GmIOMT1 or pCR8/GW/TOPO vector (Thermo Fisher Scientific) for OMT2 and F6H. CDSs of OMT were introduced into the pET-53-DEST vector (Merck Millipore) using a Gateway LR Clonase II Enzyme Mix (Thermo Fisher Scientific). For GmIOMT1 and F6H expression in hairy root, the genes were introduced into pASG-GW and pAKG-GW vectors (Supplementary Method ) using a Gateway LR Clonase II Enzyme Mix.
10
Generation of Transgenic Hybrid Aspens
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For AtGC1::GUS and CaMV35S::AHA2 constructs, AtGC1 and AHA2 were cloned into the pCR8/GW/TOPO vector (Thermo Fisher Scientific, Waltham, MA, United States) and transferred to the pGWB433 and pGWB402 vectors via the Gateway LR reaction (Nakagawa et al., 2007 (link)). Construction of AtGC1::AHA2 was described previously (Wang et al., 2014 (link)). The binary vectors (pGWB433-AtGC1::GUS, pGWB402-CaMV35S::AHA2, and pPZP211-AtGC1::AHA2) were transformed into Agrobacterium tumefaciens strain GV3101 (pMP90). Transgenic hybrid aspens were generated using the vectors, essentially as described by Eriksson et al. (2000) (link).
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