Protein Expression Vector Construction and Characterization

The protein expression vectors pH1-hrpG::FLAG, pH3-hrpX::FLAG, and pH3-hrpB1::FLAG were constructed in our previous study [6 (link)], then were electroporated into the Xoo PXO99^A, PΔminC, PΔminD, and PΔminCDE, respectively. Overnight, Xoo strains were grown in NB medium at 28 °C and collected by centrifugation. Bacterial cells were rinsed with sterile water and resuspended at an OD₆₀₀ of 2.0 in a type III-inducing XOM3 medium. These XOM3 suspensions were incubated in the shaken culture at 28 °C for 12 h. Protein samples were extracted from XOM3 suspension and separated by 10% SDS-PAGE. The proteins were then transferred to a PVDF membrane for immunoblotting using the Flag tag and anti-mouse IgG antibody (TransGen, Beijing, China). The membrane was visualized with the EasySee Western Kit (TransGen, Beijing, China). We used the E. coli RNA polymerase subunit (RNAP) antibody as the loading control.