Gfp trap

N. benthamiana leaves or Arabidopsis seedlings were ground to a fine powder in liquid nitrogen with sand (Sigma-Aldrich). Proteins were extracted in buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% glycerol, 10 mM DTT, 1 mM NaF, 1 mM Na2MoO4.2H2O, 1% Phosphatase Inhibitor Cocktails 2 and 3 (Sigma-Aldrich), 1% (v/v) P9599 Protease Inhibitor Cocktail (Sigma-Aldrich), 100 μM phenylmethylsulphonyl fluoride and 0.5% (v/v) IGEPAL CA-630 (Sigma-Aldrich). Extracts were incubated 30 min at 4°C and centrifuged for 20 min at 16,000 g at 4°C. Supernatants were incubated for 1–2 h at 4°C with GFP-Trap (ChromoTek) or ANTI-FLAG M2 Affinity Gel (Sigma-Aldrich), and washed 3–4 times with extraction buffer containing 0.1–0.5% (v/v) IGEPAL CA-630 (Sigma-Aldrich). For GFP-Trap immunoprecipitation, beads were boiled in NuPAGE LDS sample buffer (Thermo Scientific) to release proteins. FLAG peptide was used for specific elution of anti-FLAG immunoprecipitated proteins. For immunoprecipitation in Arabidopsis protoplasts, protoplasts were transfected with indicated plasmids, incubated overnight and then treated with H₂O or 1 μM flg22 or elf18 for 15–30 min. Proteins were extracted with extraction buffer (50 mM HEPES [pH 7.5], 150 mM KCl, 1 mM EDTA, 0.5% Trition-X 100, 1 mM DTT, proteinase inhibitor cocktail) at 4°C. Immunoprecipitation was then carried out as described above.

GFP-recombinant virions or VLPs were immunopurified using an anti-GFP single domain antibody conjugated to agarose beads (GFP-Trap; Chromotek). Briefly, 300 μL of purified virions/VLPs were added to 25 μL of equilibrated bead slurry and mixed/rotate end-over-end for 1 hour at 4 °C. Virions/VLPs bound to the GFP-Trap agarose beads were separated from the unbound proteins through Pierce Spin Columns (Thermo Fisher, Rockford, IL, USA), following the manufacturer's instructions. Bound virions/VLPs were eluted from the beads by adding 50 μL of 0.2 M glycine (pH 2.5), incubating it for 10 min at RT followed by centrifugation at 2.500 rpm for 2 min. The eluted samples were neutralized with 5 μl of 1 M Tris base (pH 10.4) and directly used for western blotting for detection of the CSDaV CPp21 protein as described above.