Ndei restriction enzyme

The I-DsqI HEG, containing or lacking the I-47, was introduced into expression vector pTH1 (Addgene). pTH1 generates N-terminal maltose binding protein (MBP) fusions. Briefly, the I-DsqI HEG was PCR amplified using cDNA made from total RNA from D. squamulosum and the first-strand oligonucleotide primer OP41, using the primer pair OP1422 and OP1423. The resulting entry clone pDONR221-I-DsqI was used to introduce the HEG into the pTH1 expression vector in an LR reaction. Final construct for N-terminal MBP fusion was named pI-DsqI-MBP. Target DNA for homing endonuclease cleavage was made by insertion of a 1.59 kb fragment of a L2066-lacking Didymium LSU rDNA (amplified by OP122 and OP747) into the pGEM-T easy vector. The plasmid was linearized with NdeI restriction enzyme (New England Biolabs) prior to activity studies. Primer information: OP122, 5′-CGC GCA TGA ATG GAT TA-3′; OP747, 5′-TCC AAC ACT TAC TGA ATT CT-3′; OP1422, 5′-GGG GAC AAG TTT GTA CAA AAA AGC AGG CTT CAT GAA CAA CTA CCA GCA GGC A-3′; OP1423, 5′-GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC CTA CCA GCA TGC TGG GGT GTG GTT-3′.

All total RNAs purified in this study were pooled to be used for the double-strand cDNA pools constructed using the SMART^™ cDNA Library Construction Kit (Clontech, Mountain view, CA, USA) according to the manufacturer’s instruction. The predicted open reading frame (ORF) sequences were obtained from RAP-DB (https://rapdb.dna.affrc.go.jp/) and amplified from the ds-cDNA pools by Phusion^® High-Fidelity DNA polymerase (Toyobo, Osaka, Japan) with the primers containing an attB1 or attB2 sequence for creating the Gateway Entry clones (Supplementary Table S2). To identify their ORF sequences, the amplified PCR products were cloned into pDONR221 using Gateway^® BP Clonase^® II Enzyme Mix according to the manufacturer’s instructions. Then, those entry clones were further amplified by KOD FX polymerase (Toyobo, Osaka, Japan) with corresponding primers containing the recognition site of a NdeI restriction enzyme (Supplementary Table S2) to allow them to be cloned into pDONR221-Kz-NdeI-sGFP [38 (link)] for the analyses of their subcellular localizations in rice protoplasts. The amplified PCR products were digested with NdeI restriction enzyme (NEB, Ipswich, MA, USA) and cloned into pDONR221-Kz-NdeI-sGFP.

Total genomic DNA (5 µg) of N. benthamiana was digested with Dra I (Nippon Gene), Hind III-HF, or Nde I restriction enzymes (New England BioLabs, Ipswich, MA, USA). Southern blot hybridization was performed using the DIG application kit (Roche, Basal, Switzerland) according to the manufacturer’s instructions. Specific DNA probes for the detection of NbEXA1 were synthesized using the PCR DIG Probe Synthesis Kit (Roche) according to the manufacturer’s protocols using the primer sets Xh-NbEXA1-268F/Bm-NbEXA1-567R or NbEXA1g-5015F/NbEXA1g-5500R, and with N. benthamiana genomic DNA as a template.

Standard and nested PCR mix contained 1x Taq Polymerase buffer, 1.5 to 2.5 mM MgCl₂ (MgCl₂ concentration was optimized for every PCR), 200 μM deoxynucleotide triphosphate (dNTP), 2.5 U of Taq Polymerase (all from Thermo Fisher Scientific, Waltham, MA), and 200 nM of each primer (IDT Technologies). For standard PCR, 100 ng of DNA from samples or control cell lines or 5.5 × 10⁻⁵ ng from control plasmid (equivalent to the number of moles contained in 100 ng of genomic DNA) were used. Plasmids were linearized with restriction enzyme NdeI (New England BioLabs) before use and mixed with DNA from Daudi cell line in order to run the amplification reaction under identical mass/volume DNA concentration for both samples and controls. For the nested PCR, 0.5 or 0.05 μL (1 : 100 or 1 : 1000 dilution resp.) of product of first round PCR was used as template. All PCR reactions were carried out in a final volume of 50 μL. All primer sequences and cycling conditions used are detailed in Table 2. PCR products were analyzed by electrophoresis in 1.8% agarose gels stained with ethidium bromide and photographed under ultraviolet light using the Quantum ST4 System (VilberLourmat, Torcy, Marne-la-Vallée, FR). The viral identity of PCR amplified fragments was confirmed by sequencing.

Viral targets were PCR amplified using primers described in Table 1 and cloned individually into the commercial vector pGEM-T Easy (Promega, Madison, WI, USA), according to the manufacturer’s instructions. Plasmids containing viral fragments were digested with restriction enzyme EcoRI (New England BioLabs, Ipswich, MA, USA) to release the insert. The expected size of every viral fragment was confirmed by agarose gel electrophoresis and then identified by Sanger sequencing. Plasmids containing viral fragments were also digested with restriction enzyme NdeI (New England BioLabs) to produce linear plasmids, which were then used in the multiplex qPCRs. From here, these plasmids containing viral fragments will be mentioned throughout the text as standard plasmids.