Lr clonase enzyme mix

Unless otherwise specified, all cloning experiments were performed by Gateway recombination. Briefly, 150 ng of the destination vector and donor vector containing the gene of interest were co-incubated with 1X LR Clonase Enzyme Mix (Invitrogen #11789–020) overnight at room temperature. The reaction mixture was then transformed into 50 μl of Stbl3 bacteria, spread on LB plates with corresponding antibiotics. MAVS and BILF1 mutants are generated either by standard restriction-ligation procedures or site-directed mutagenesis according to manufacturer’s protocol (NEB # E0554). Oligos used for mutagenesis are listed in Table S6.

Coding regions of GALT1, GALT3, and GALT4 were obtained from the RIKEN Bioresource Center, while the coding region of GALT6 was obtained from The French Plant Genomic Resource Center (http://cnrgv.toulouse.inra.fr/). N-terminal 6x-His tag fusion gene constructs were amplified using Q5 high fidelity DNA Taq polymerase (New England Biolabs, Ipswich, MA, USA), initially cloned into the pENTR/D-TOPO vector (Life technologies, Grand Island, NY, USA), and eventually cloned into the destination vector pMDC32 gateway vector using LR clonase enzyme mix (Life Technologies Grand Island, NY, USA). Primers used for amplification are listed in Additional file 1: Table S6. Gene constructions were transformed into Agrobacterium strain GV3101 by the freeze thaw method, and the transformants were grown overnight in Luria-Bertani (LB) medium. Bacterial cells were harvested by centrifugation and suspended in a buffer containing 10 mM MES, 10 mM MgCl₂, and 50 μM acetosyringone (OD₆₀₀ = 0.2). Leaves from 6-week-old WT N. tabacum cv. Petit Havana were used for Agrobacterium-mediated transient expression. Four days after infiltration, leaves were harvested, and microsomes were prepared according to the method described by Liang et al. [45 (link)] with minor modifications.

Full length GALT3, GALT4, and GALT6 devoid of stop codons was cloned into the pENTR/D-TOPO vector (Life technologies, Grand Island, NY, USA) and sequenced. The resulting plasmids were cloned in the destination vector pEarlyGate 101 by a gateway cloning strategy, using LR clonase enzyme mix (Life Technologies Grand Island, NY, USA) to generate the YFP N-terminal fusion constructs. These gene constructions were transformed into Agrobacterium strain GV3101 and infiltrated into tobacco leaves as described in the above section except that the bacterial concentration was lower (OD₆₀₀ = 0.05). The GALT3-YFP, GALT4-YFP, and GALT6-YFP constructions were co-expressed with either the ER marker HDEL-GFP or the Golgi marker sialic acid transferase (ST)-GFP to ascribe subcellular localization. Transformed plants were incubated under normal growth conditions and imaged 2 days post-infiltration using an upright Zeiss LSM 510 META laser scanning microscope (Jena, Germany), with a 40 × oil immersion lens and an argon laser. For imaging the expression of YFP constructs, the excitation line was 514 nm, and emission data were collected at 535–590 nm; whereas for GFP constructs, the excitation line was 458 nm, and the emission data were collected at 505–530 nm.

A 578 bp NtHop/Sti1 fragment (Nucleotides +1150 to +1728 relative to the ATG codon) was amplified from tobacco leaf cDNA (Oligonucleotide primers: NtHOP-RNAi-for: 5′-CACCTTAACTTGGACAATTCCT-3′; NtHOP-RNAi-rev: 5′-AGAGCAGCAAGAGTATTTCAATC-3′). The resulting amplicon was inserted into the pENTR-D/TOPO^® vector (Thermo Fisher Scientific) according to the manufacturer’s instructions. The fragment was subsequently recombined into the destination vector pK7GWIWG2(II) (Karimi et al., 2002 (link)) using LR-Clonase^® Enzyme Mix (Thermo Fisher Scientific). The resulting plasmid was transformed into Agrobacterium tumefaciens strain C58C1 harboring pGV2260 (Deblaere et al., 1985 (link)). The transformation of tobacco was carried out following Horsch et al. (1985) (link).

The sequence of human METTL3 cDNA was amplified from pENTR-METTL3 (Vigene Bioscience, CH8066235). The PCR products were first cloned into pENTR1A no ccDB (Adegene, #17398) by Gibson Assembly Mix (NEB, #E2611) according to the manufacturer’s protocol. The cloned products were then recombined into the pLEX_307 (Addgene, #41392) using the LR Clonase Enzyme Mix (Thermo Fisher, 11791020). The METTL3 mutant plasmid was constructed by Mut Express II Fast Mutagenesis Kit (Vazyme, C214-01) according to the manufacturer’s protocol. All sgRNA was cloned into the px330 vector (Addgene, #42230), sgRNAs, and primers used for the construction are shown in Supplementary Tables.
1 × 10⁶ hESCs were infected with the lentiviruses. 0.25 μg/ml puromycin (Thermo Fisher, A1113803) was added 7 ~ 10 days after the infection.

The 35Sp::GFP-NtHop/Sti1 construct was generated using the Gateway^® Cloning system (Thermo Fisher Scientific, Waltham, MA, United States): The coding sequence of NtHop/Sti1 was amplified from Nicotiana tabacum cv. Samsun NN cDNA by PCR (Oligonucleotide primers: fwGFP-Hop: 5′-CACCGCCGACGAAGCTAAGGC-3′; revGFP-Hop: 5′-CTATCATTGGACAATTCCTGCATTAATCAACTTTTG-3′) and subcloned into pENTR/D-TOPO^®(Thermo Fisher Scientific) according to the manufacturer’s advice. The final construct was generated using the LR-Clonase^® Enzyme Mix (Thermo Fisher Scientific) and the destination vector pK7WGF2 (Karimi et al., 2002 (link)).
The plasma membrane-GFP marker as well as the ER-mCherry marker generated by Nelson et al. (2007) (link) were obtained from the Arabidopsis Biological Resource Center¹ as stock # CD3-1003 and CD3-959, respectively.
The FLS2p::FLS2-3xmyc-mCherry construct was kindly provided by Prof. Silke Robatzek (The Sainsbury Laboratory, Norwich, United Kingdom).

The plasmid for the doxycycline-induced overexpression was constructed based on the Gateway System (Thermo Fisher Scientific). The sequences of human SOX17 cDNA were amplified from pENTR-SOX17 (Vigene Bioscience, CH803137), and human NANOG cDNA was amplified from pENTR-NANOG (Vigene Bioscience, CH832818) and DHFR was amplified by PCR from pBMN-DHFR-YFP (Addgene, #29,325). The PCR products were first cloned into pENTR1A no ccDB (Adegene, #17,398) by Gibson Assembly Mix (NEB, #E2611) according to the manufacturer’s protocol and were then recombined into the pInducer20 vector (Addgene, #44,012) or pLEX_307 (Addgene, #41,392) with LR Clonase Enzyme Mix (Thermo Fisher, 11,791,020). The destination vector was designed as shown in Additional file 5: Fig. S5C. All sgRNA were cloned into the px330 vector (Addgene, #42,230), sgRNAs used for the construction are shown in Additional file 9: Tables.
1 × 10⁶ hESCs were then infected by the lentiviruses. 100 μg/ml Geneticin (Thermo Fischer, 10,131,027), and 0.25 μg/ml puromycin (Thermo Fisher, A1113803), were added 7 ~ 10 days after the infection. The induction of the transgene upon 1 μg/ml doxycycline (Sigma-Aldrich, D9891) and 10 μM Trimethoprim (TMP) (Sigma-Aldrich, T7883) administration of the selected hESC clones was assessed at hPGCLC induction period.