Protease inhibitor 1x

Infiltrated leaves expressing the protein of interest were powdered in a mortar under liquid N 2 . About 2 g of tissue was weighed, and to it, 3 volumes of lysis buffer (50 mM Tris-Cl pH 7.4, 150 mM KCL, 1 % Triton X100, Protease inhibitor 1 X [Roche], NEM 20uM) was added. The supernatant was collected after a spin at 16000 g for 30 min and incubated with GFP-Trap (Chromtek) or MBP magnetic beads (NEB) for 3 h at 4°C. Beads were magnetically separated from the lysate and washed 5 times in wash buffer (50 mM Tris-Cl, pH 7.4; 150 mM KCL, 1 mM PMSF) until the green colour completely disappeared. The final pull-down beads were transferred to a 1.5 ml tube and again twice with wash buffer. The 3X SDS sample dye was added to the beads and the sample was heated at 70°C for 10 min. The pull-down products were resolved in 4-20 % Tris-Glycine SDS gradient gels (Bio Rad). IP beads used are listed in Supplemental Table 2.

Immuno-precipitation was performed as previously described (Nair et al., 2020) .
Briefly, Infiltrated or transgenic plant leaves expressing the protein of interest were finely powdered under liquid nitrogen. Three volumes of lysis buffer (50 mM Tris-Cl pH 7.4, 150 mM KCl, 1% Triton-X100, Protease inhibitor 1 X [Roche], NEM 20 μM) was added to 2 g of powdered tissue. The lysate was clarified by high-speed centrifugation and incubated with GFP-Trap (Chromtek) for 3h at 4°C. Beads were magnetically separated from the lysate and washed 5 times in wash buffer (50 mM Tris-Cl, pH 7.4; 150 mM KCl, 1 mM PMSF) until the green colour completely disappeared. The washed beads were transferred to a 1.5 ml tube and again washed twice with wash buffer. The buffer was completely removed and 3X SDS sample dye was added to the beads and incubated at 70°C for 10 min. The pulldown products were resolved in 4-20% Tris-Glycine SDS gradient gels (Bio-Rad).