Primer star max premix

Using cDNA from CaLas-infected Wanjincheng orange plants as a template, the gene encoding for CaLasSDE115 (CLIBASIA_05115) was amplified with the primer pair T-SDE115-F/R (Supplementary Table 1). The PCR was performed in a final volume of 50 μL, containing 25 μL of Primer STAR Max Premix (TaKaRa, Ojin, Japan), 1.5 μL of each primer (10 mM⋅L^–1), 19 μL of H₂O and 3 μL of cDNA (0.5 × 10^–5 ng⋅L^–1). The PCR conditions were as follows: pre-denaturation at 96°C for 5 min, followed by 35 amplification cycles (96°C for 30 s, 56°C for 30 s and 72°C for 30 s), before a final extension at 72°C for 3 min. The PCR product was T-cloned into the pGEM-Teasy vector (Promega, WI, United States). The sequence of CaLasSDE115 was then determined by Sanger sequencing.
The signal peptide (SP) and transmembrane structure of CaLasSDE115 were predicted using the signalP5.0 tool¹ and TMHMM Server (v.2.0)², respectively. The secondary as well as tertiary structures of its mature protein were analyzed using SOPMA³, SWISS-MODEL⁴ and Phyre⁵ tools, respectively. Multiple sequence alignment of the CaLasSDE115 protein with homologs from other selected species was also performed using ClustalW2 tool⁶. A phylogenetic tree was constructed based on the neighbor-joining method using MEGA7.0 software (Kumar et al., 2016 (link)).

Conventional PCR amplifications were performed in 50-µl reactions. Each reaction included 25 µl of Primer STAR Max Premix (2 × Takara), 21 µl of nuclease-free H₂O, 2 µl of purified gDNA (100 ng), and 1 µl of each forward and reverse primer (10 µM). Thermal cycling began at 94°C for 3 min, followed by 33 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 45 s; this was followed by 10 min at 72°C for 10 min. PCR was performed on Applied Biosystems Veriti Dx 96-Well Thermal Cycler (Thermo Fisher Scientific, Massachusetts, USA). Each set of reactions included a non-template control (NTC) to eliminate false positives. Following amplification, PCR products were detected on a 1.5% agarose gel, separated by electrophoresis at 130 V for approximately 25 min, and then visualized with a UV transilluminator. For nested PCR assays, we initially amplified the target gene of P. cinnamomi using specific primers (Pcinn13739-nest-F and Pcinn13739-nest-R). The thermal cycling program was the same as for conventional PCR (Table 1). For the second round of amplification, the PCR products arose from the first round, and then we added 2 μl of PCR products to each reaction and performed amplifications with Pcinn13739-F and Pcinn13739-R. All PCR and nested PCR reactions were repeated at least three times.

Human DNMT1 and human DNMT3A cloned into pCAG-EGFP were kindly provided by Dr. Isao Suetake^{45 (link),46 (link)}. Human DNMT3B1 cloned into p3 × FLAG-CMV10 was kindly provided by Drs. Motoka Unoki and Hiroyuki Sasaki^{47 (link)}. Mutants of DNMT3B were generated by substituting serine for cysteine using the QuickChange Site-Directed Mutagenesis Kit (Agilent Technologies) according to the manufacturer’s instructions using the following primers: C651S mutant 5′-GAT TGG CGG AAG CCC AAG CAA CGA TCT CTC AAA TG-3′ and 5′-CAT TTG AGA GAT CGT TGC TTG GGC TTC CGC CAA TC-3′, C716S mutant 5′-CAT CTC ACG GTT CCT GGA GAG TAA TCC AGT GAT TG-3′ and 5′-CAA TCA CTG GAT TAC TCT CCA GGA ACC GTG AGA TG-3′. A DNMT3B Q772A/F809A double mutant was generated by the megaprimer method using PrimerStar MAX Premix (Takara). F809A mutant 5′-CTC GAA AGG ATC GCT GGC TTT CCT GTG-3′ and 5′-GGG AGA TCT CTA TTC ACA TGC AAA G-3′, Q772A mutant 5′-CCC CTC GAG CTG CAG GAC TGC TTG GAA TAC AAT AGG ATA GCC AAG TTA AAG AAA GTA GAG ACA ATA ACC AAG-3′. All constructs were verified by sequencing. pcDNA3/Myc-DNMT1 (Addgene plasmid # 36939) and pcDNA3/Myc-DNMT3B1 (Addgene plasmid # 35522) were a gift from Arthur Riggs⁴⁸ . Each plasmid was transiently introduced into cells with polyethylene imine (PEI)-max (Polysciences) or Lipofectamine 2000 (Thermo Fisher Scientific), and the S-nitrosylation of proteins was analyzed after 24 h.