Figure 6. Secretion of T6SS effector Hcp in Acidovorax avenae subsp. avenae strain RS-1 under exposure to Amp and mutation of T6SS genes.

(a) Hcp detection in four different components using polyclonal anti-Hcp rabbit serum generated by Huaan company (Hangzhou, China) using Hcp-His fusion protein (about 23 kDa), which was obtained by amplification of hcp gene (483 bp) using the designed primers (Table S10), digestion with both BamH I and EcoR I, and ligation to plasmid pET-28a, overexpression by IPTG induction in Escherichia coli BL21 (DE3), purification using Ni-NTA-Sepharose Beads (Sangon Biotech, China), and size-fractionated on a 15% SDS-PAGE gel as described by Yang et al.⁴⁴. The tested proteins including Hcp-His fusion protein were subsequently validated by western blot and ELISA analysis. Western blot was performed using primary polyclonal antibody (1:5000) followed by a secondary antibody using Horseradish Peroxidase-Conjugated goat anti-rabbit immunoglobulin G (Sangon Biotech, China), and then visualized using ImageQuant LAS 4000 (GE Health). The indirect ELISA was performed according to the method of Slutzki et al.⁴⁵ by coating each well in the immunoassay plates (Maxisorp, Nunc, Denmark) using 1.0 μg/ml of the tested proteins, charging with rabbit poly-antibody at a dilution of 1:5000 according to the protocol of EL-TMB colored reagents (Sangon Biotech, China), and then measuring the OD 450 nm value in an ELISA reader (Multiscan Ex. Lab systems, Thermo Fisher Scientific Inc., USA). M: marker; IM: inner membrane; OM: outer membrane. (b) ELISA detection of Hcp from secretory proteins using polyclonal antibody (1:5000) under Amp (+) condition. (c) ELISA detection of Hcp from cytoplasmic proteins and secretory proteins in T6SS mutants using polyclonal antibody (1:5000). The purified Hcp-His fusion protein of 1.0 μg/ml was used as the positive control, while the BSA was used as the negative control. Each experiment was repeated three times independently with similar results.