Lr recombinase

Casp1^KO and GSDMD^KO cell lines, U937 cell lines expressing WT, p.S208C, p.S242R, p.M694V, p.M680I under the control of a doxycycline-inducible promoter, have been previously described (Lagrange et al., 2018 (link); Magnotti et al., 2019 (link)). p.[V726A], p.[E244K], ΔPYD, ΔPLD, ΔB-Box, ΔCoiled-coil, ΔB30.2 MEFV were generated by mutagenesis of the pENTR1A-3xFlag MEFV using primers presented in Table S3, pfu ultra II Fusion high fidelity polymerase (Agilent) followed by digestion of the parental plasmid using Dpn1 restriction enzyme. The resulting plasmids were validated by sequencing and the mutated MEFV constructs were transferred into the GFP-expressing plasmid pINDUCER21 (Meerbrey et al., 2011 (link)) under the control of a doxycycline-inducible promoter using LR recombinase (Invitrogen). Lentiviral particles were produced in 293T cells using pMD2.G and psPAX2 (from Didier Trono, Addgene plasmids #12259 and #12260), and pINDUCER-21 plasmids. U937 cells were transduced by spinoculation and selected at day 4 post-transduction based on GFP expression on an Aria cell sorter and maintained polyclonal. Pyrin expression was induced by treatment with doxycycline (1 μg.mL⁻¹) for 16 h before stimulation. All parental cell lines were tested for mycoplasma contamination.

For the spr1 mutant of the Arabidopsis complement test and AtSPR1 overexpression assay, SmSPR1 and AtSPR1 cDNA was amplified and introduced into pDONR221 via BP reaction and to pEarleyGate104 of Gateway vectors via LR recombinase (Invitrogen) (Earley et al., 2010 (link)). All primers are listed in Supplementary Table 4. The resulting constructs were transformed into Arabidopsis using A. tumefaciens (GV3101) via Arabidopsis floral dip method as described elsewhere (Zhang et al., 2006 (link)). The homozygous T3 seedlings were used for further analyses.

For co-immunoprecipitation experiments, we used GFP-trap (ChromoTek, Martinsried, Germany) for the immunoprecipitation of green fluorescent protein (GFP)-fusion proteins. OsPYL/RCAR7 and OsPP2C51 were inserted into pENTR/D-topo vectors (Invitrogen, CA, USA) and then recombined with pGEM-gw-GFP vectors for GFP fusion, and pGEM-gw-3xHA vector for HA tagging using LR recombinase (Invitrogen, CA, USA) [21 (link)]. The indicated constructs were introduced into rice protoplasts using the polyethylene glycol (PEG)-mediated method, and the transformed protoplasts were incubated at 28 °C. Cellular extracts from transformed protoplasts in immunoprecipitation buffer (150 mM NaCl, 50 mM Tris-HCl at Ph 7.5, 1 mM EDTA, 2 mM EGTA, 2 mM MgCl₂, 0.5% NP40, 0.5% Triton X-100, and 1× protease inhibitor cocktail (complete ULTRA tablet, Roche, IN, USA) were incubated with pre-cleaned GFP-trap beads at 4 °C for 2 h. After washing five times with immunoprecipitation buffer, the precipitated proteins, together with GFP-trap, were subjected to SDS-PAGE and immunoblot analysis. Precipitated GFP or HA tagged proteins were detected with anti-GFP rabbit antibody (Life Technologies, OR, USA) or anti-HA rat antibody (Roche, IN, USA), respectively.

To inhibit the activity of miR-497, a U6 sponge was used. Replication deficient adenoviruses for miR-497 sponge and null virus were prepared using the Getaway System (Invitrogen) according to the manufacturing's instructions. Briefly, the oligonucleotides for miR-497 were synthesized by Shanghai General Biotech with the following sequences: 5′-gtacaaaccacagtgtgctgctgcgtatacaaaccacagtgtgctgctggcatg-3′ (forward) and 5′-ccagcagcacactgtggtttgtatacgcagcagcacactgtggtttgtactgca-3′ (reverse), which contained two perfect miR-497 binding sites. Then the double-strand oligonucleotide was cloned into a shpDown-U6 RNA-eGFP plasmid (Cyagen, Beijing, China) and confirmed by sequencing. MiR-497 oligo/shpDwon-U6 RNA-eGFP plasmid was recombined with pAD/PL-DEST (Invitrogen) using LR recombinase (Invitrogen) which was confirmed by sequencing. The pEntr-U6-miR-497-sponge (Ad-miR-497-spong) was transfected to 293A cells using lipofectamine 2000. Control vector was prepared by transfecting 293A with pEntr-U6-scrambled oligonucleotide-eGFP (control vector). Viruses were purified using the Adenovirus purification mini Kit (V1160-01, Biomiga, San Diego, USA). The end-point dilution assay was used to measure virus titer.

Total RNA was extracted from A. thaliana Col-0 seedlings using an RNeasy Plant Mini Kit (Qiagen) and RNA was reverse transcribed using the iScript cDNA Synthesis Kit (BioRad) according to the manufacturers’ instructions. The PCR products of full-length coding sequences and C-terminally truncated sequences (see Supplemental Table 3 for primer sequences) were cloned into the pENTR/D-TOPO vectors (Invitrogen) by TOPO cloning, according to the manufacturer’s instructions, and the inserts were fully sequenced. Using LR recombinase (Invitrogen), the inserts were subsequently transferred into the destination vector pMP4409. pMP4409 was generated by cutting pMP625 containing the promoter and terminator of PMA1 (yeast endogenous plasma membrane H⁺-ATPase^{79 (link)}) with XhoI and SpeI, removing AHA2, and inserting the gene into Gateway Cassette A (Invitrogen); the correct orientation of the Gateway Cassette A was verified by sequencing.

Sequences from RRSVs6gp1 (AF020337), RRSVs9gp1 (GQ329711), and RRSVs10gp1 (U66712) were used to choose conserved 150-nt fragments. First, a BLAST search was performed to evaluate the nucleotide diversity among related sequences. Due to high conservation, 150-nt fragments were randomly chosen in each of the three genes. These fragments (450-nt tandem) were de novo synthetized in a pUC18 vector by Genecust Company (Boynes, France) with att recombination sites for further gateway cloning (Luxembourg). Then, the tandem sequence was first inserted in the gateway cassette of a pDONR207 vector at the BP recombinase site (Invitrogen, Carlsbad, CA, USA). Then, using LR recombinase (Invitrogen), the tandem sequence was transferred into the two gateway cassettes of the pANDA vector in sense and antisense orientations [53 (link)]. The resulting construct, specifically pANDA:siRRSV, was transformed into Agrobacterium strain EHA105 by electroporation.

The coding regions of mouse Aid (NM_009645.2, NCBI) and mouse Apobec1 (NM_001134391, NCBI) were cloned from mouse ES cells by RT-PCR. The PCR products were sequenced and subcloned into pENTR-D-TOPO (Invitrogen) and recombined with pMXs-gw [1] (link) using LR recombinase according to manufacturer’s instructions (Invitrogen). Mouse dominant-negative Apobec1 (H61K/C93S/C96S) [20] (link) was generated by PCR-based site-directed mutagenesis. To generate lentivirus vectors encoding doxycycline-inducible reprogramming factors, TRE, the Gateway cassette (Invitrogen) and rtTA2s-M2 (Clontech) were introduced into a pLKO.1 backbone (#10878, Addgene). Then coding sequences of Oct3/4, Sox2, Klf4 and c-Myc were inserted by the LR reaction to make pLV-TRE-rtTA2s-M2-Oct3/4, -Sox2, -Klf4 and -c-Myc. psPAX2 (#12260) and pMDG.2 (#12259) were obtained from Addgene. The primers used for the construction of plasmids are listed in Table S7.