E coli top10 competent cells

The Aequorea coerulescens GFP gene was inserted into the plasmid pET28a(+) (Novagen), between the NdeI and BamHI restriction sites conserving the plasmid N-terminal hexa-histidine (His)-tag sequence and originating the pET_GFP plasmid. Primers were designed (Table 1) to obtain different fragments of the PlyPl23 lysin C-terminus (further referred as the cell binding containing fragment - CBCF). These primers enabled to truncate the CBCF, first on the N-terminus (conserving the C-terminus) and then at the C-terminus (conserving the N-terminus). Primer melting temperatures were calculated using OligoCalc^{35 (link)}. The fragments were amplified with Phusion DNA Polymerase (ThermoFisher Scientific) with the PlyPl23 plasmid^{17 (link)} as DNA template and digested with the restriction enzymes SacI and XhoI. The digested fragments were inserted into the pET_GFP (in order to fuse them with the GFP upstream, at the N-terminus) and ligated with the T4 ligase (ThermoFisher Scientific) to obtain the different constructions (pET_GFP-CBCFx), further used to transform E. coli TOP10 competent cells (Invitrogen). Colonies were screened through colony PCR and positives used for plasmid extraction and further confirmation through Sanger sequencing. Correct pET_GFP-CBCFx plasmids were used to transform competent E. coli BL21 (DE3).

The ZBED (LOC_Os01g36670) cDNA, cloned into pDONR207 plasmid was used to generate point mutations in the three BED domains in order to determine whether BED domains were responsible of DNA binding. BED point mutations were done by substituting the two cysteines of the zinc finger to glycines in each of the three domains present in ZBED (Supplementary Figure 2). To this end, we used QuickChange lightning site-directed mutagenesis kit (Agilent Technologies) following the manufacturer’s instructions. Primers used are provided in Supplemental Table 1. Briefly, PCR conditions were initial denaturation: 95°C 2 min followed by 20 cycles of 95°C 15 s denaturation, 60°C 10 s annealing, and 68°C 4 min (30 s per kb) of extension step, with a final 68°C for 5 min. E. coli Top10 competent cells (Invitrogen™) were transformed with these plasmids and plated in LB agar with gentamicin selection and sent for sequencing to verify point mutation. Once the point mutation on each BED domain was confirmed, we proceeded to clone these constructs into a gateway plasmid 35S-eGFP-GWY (pB7FWG2 destination vector, https://gateway.psb.ugent.be) in order to transform Agrobacterium tumefaciens GV3101 cells for transient expression assays in Nicotiana benthamiana.