Na931

Western blotting was performed as previously described10 (link). In brief, overnight cultures of the respective E. coli strains were diluted 1:100 using fresh LB medium (w/o antibiotics) and grown for 2.5 h at 37 °C with agitation (220 r.p.m.). The OD₆₀₀ of the cultures was monitored during bacterial growth and all samples were taken when the cultures had OD₆₀₀ readings between 0.69 and 0.79. For each sample, equal volumes of the liquid cell culture were pelleted by centrifugation (10,000g, 1 min) and the cell pellets were re-suspended in one-tenth of the original culture volume using a 1 × SDS loading buffer (80 mM Tris-HCl pH 6.8, 2% (w/v) SDS, 10% (v/v) glycerol, 5% (w/v) ß-mercaptoethanol, 0.01% (w/v) brophenol blue). The samples were boiled for 5 min at 95 °C. Ten microlitres of each sample was loaded on a Tris-glycine SDS–polyacrylamide gel electrophoresis containing 10% (v/v) acrylamide. The primary antibody was the monoclonal anti-RpoS antibody (Neoclone, W0009) and used at a 1:1,000 dilution. The secondary antibody was an anti-mouse horseradish peroxidase-coupled antibody (GE Healthcare, NA931), which was diluted 1:5,000 in 1 × TBST with 4% (w/v) milk powder, prior to use. The protein bands were visualized using a homemade ECL reagent and standard film.

Samples were solubilized in sample buffer (3X) and boiled at 95 °C for 5 min for immunoblotting. All samples were electrophoresed on 10% (w/v) sodium dodecyl sulphate-polyacrylamide (SDS-PAGE) gels. Separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes. For all immunoblot experiments, membranes were blocked for 1 h at ambient temperature in blocking buffer composed of 5% (w/v) skim milk powder in TBS. PVDF membranes were incubated overnight at 4 °C with primary antibodies. A complete list of primary antibodies and the appropriate methodology used is shown in Additional file 1: Table S1. Membranes were then washed with TBS containing 0.05% Tween 20 and incubated for 1 h at ambient temperature with the following secondary antibodies diluted in blocking buffer: either α-mouse horseradish peroxidase (HRP)-conjugated (NA931, GE Healthcare; 1:5000), α-rabbit HRP-conjugated (NA934, GE Healthcare, 1:5000), or α-goat HRP-conjugated (sc-2433, Santa Cruz, 1:5000) antibody. Visualization of immunoblot labelling was achieved using chemiluminescence with the Western Lighting Plus ECL kit (Perkin Elmer).

Equal amounts of protein were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Sigma-Aldrich, St. Louis, MO, USA) with Mini-PROTEAN^® Tetra Handcast Systems (Bio-Rad, Hercules, CA, USA). The membranes were incubated with primary antibodies (for anti-GFP (Abcam, AB6556), 1:5000; anti-HA (Roche, 11867423001), 1:5000; anti-PR1, 1:5000; anti-RbCL (Clontech 632475), 1:2000; anti-GST (Virogen, 101-A-100), 1:5000; or anti-His (Thermo Fisher, PA1-23024), 1:5000) in 1× TBS with 5% w/v skim milk at 4 °C overnight. Treated membranes were washed with 1× TBS and incubated with a secondary horse radish peroxidase (HRP)-conjugated antibody (anti-rabbit HRP (Santa Cruz, sc-2004), 1:10,000; anti-rat (Santa Cruz, sc-2006), 1:10,000; anti-mouse (GE Healthcare Life Science; NA931), 1:10,000) at room temperature for 2 h. Signals were visualized using the Pierce ECL and ChemiDoc MP (Bio-Rad) systems. Protein band intensity was measured by using ImageJ software. Relative intensity protein band under Pto infection condition was calculated by normalizing to the intensity of protein band under mock condition, which was set as 1.

Serum antibody titer was measured by enzyme-linked immunosorbent assay (ELISA) as in our previous report^{25 (link)}. In brief, 96-well ELISA plates were coated with 2 µg/ml rHA at 4 °C overnight. After blocking with 5% non-fat milk, two-fold serial dilutions of immune sera were added and incubated at room temperature for 90 min. After washing in PBS supplemented with 0.05% Tween 20 (PBST), horseradish peroxidase (HRP)-conjugated sheep anti-mouse IgG secondary antibodies (1:2500, NA931, GE Healthcare Life Sciences) were added and incubated at room temperature for 1 h. After washing in PBST, TMB substrates were added and reactions were then stopped by addition of 3 N H₂SO₄. Optical absorbance (OD_450nm) was determined using a microplate reader (Molecular Device). Serum antibody titer was defined as the reciprocal dilution factor that resulted in OD_450nm ~ 3 times higher than the background values.