Complete edta free proteinase inhibitor cocktail

Protein extraction for western blotting with RIPA buffer was performed following the manufacturer guideline’s (Thermo Fisher). RIPA buffer was supplemented with PhosSTOP inhibitor (Merck) at 10X concentration and cOmplete EDTA-free Proteinase Inhibitor Cocktail (Roche) at 25X concentration. Trypsinised cells were washed twice with PBS and cold complete RIPA buffer was added to each vessel and kept on ice for 5 min. The lysate was collected with a scrapper and centrifuged at 4 °C at maximum speed for 15 min. The supernatant was collected and quantified with the Pierce BCA Protein Assay Kit (Thermo Fisher) as explained by the manufacturer guidelines. The standard curve was prepared in triplicate with albumin standard (BSA) diluted in PBS, working concentrations are 2, 1.5, 1, 0.5, 0.2 and 0 μg/μL. The BCA working reagent was prepared by mixing 50 parts of BCA Reagent A with 1 part of BCA reagent B. All the standards and samples were prepared by triplicate, and 10 μL of each standard or sample were added to a 96-well plate with 200 μL of BCA working reagent and mixed by pipetting. The plate was incubated for 30 min at 37 °C. Absorbance was measured at 562 nm on a plate reader and the standard curve was used to determine the protein concentration of each sample.

For isolation of the soluble FGs (SS^ExtHis(SP)₁₀L_SrtA-mIFNγ or SS^ExtHis(SP)₁₀L_TEV-mIFNγ), the infiltrated leaves were harvested at 5 dpi. Leaf homogenates were prepared with the use of 1:2 (w/v) extraction buffer A [50 mM Tris–HCl, pH 7.6, 120 mM KCl, 15 mM MgCl₂, 0.1 mM PMSF, 20% glycerol, 0.1% β-mercaptoethanol, and one tablet of cOmplete™ EDTA-free proteinase inhibitor cocktail (Roche Life Science, Penzberg, Germany)] (Osman and Buck, 1996 (link)). The homogenized extracts were filtered through a layer of Miracloth and centrifuged at 30,000 × g for 30 min to separate the pellet (designated P30) and supernatant (designated S30) fractions. To remove the abundant RuBisCO protein contaminations, the S30 fraction was treated with ultrapure acetic acids to achieve a pH value of 5.1 and then subjected to centrifugation at 30,000 × g for 30 min to obtain the P30-treated (P30t) and S30-treated (S30t) fractions as described previously (Park et al., 2015 (link); Jiang et al., 2020 (link)). After the removal of most RuBisCO protein contaminations, the pH of soluble FGs in the S30t fraction was adjusted to 7.0 by adding 1 M NaOH to avoid protein degradation.

Cell lysates were prepared in Laemmli buffer containing benzonase nuclease (Sigma), complete EDTA-free proteinase inhibitor cocktail (Roche), and PhosStop phosphatase inhibitor cocktail (Roche) and size-fractionated by 4–20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were blotted to Immobilon-P nylon membrane (Millipore). Membranes were blocked with 10% Blocker BSA (bovine serum albumin) buffer (Thermo Fisher Scientific) and incubated with the primary antibodies overnight at 4 °C. After incubation with each appropriate secondary antibody, bands were detected with ECL (GE Healthcare) using X-ray films. Antibodies were diluted in 10% Blocker BSA buffer (Thermo Fisher Scientific). Antibodies used in this study are listed in Supplementary Data 4.