4 nitroblue tetrazolium nbt

Ni²⁺‐NTA‐based affinity pulldown assays were performed as described previously (Deb et al., 2020 (link)). Briefly, xopU, xopV, and xopG were cloned with the EGFP tag in the pH7WGF2 vector. EGFP::gus was used from a previous study as a control (Deb et al., 2020 (link)). xopX was cloned with a 6×His tag in the pMDC7 vector (Table S2). pMDC7::xopQ‐6×His was used from a previous study (Deb et al., 2020 (link)). Roots of 4‐day‐old TN‐1 rice seedlings were cocultured with the respective bacterial suspensions of AGL1. Total protein was isolated, and in vivo pulldown was carried out using Ni²⁺‐NTA beads 24 h after cocultivation. Western blot analysis was performed using alkaline phosphatase‐conjugated anti‐His antibody (Sigma Aldrich) and anti‐GFP antibody (Abcam). For immunoblotting, alkaline phosphatase conjugated to anti‐rabbit immunoglobulin G secondary antibody (Sigma Aldrich), 4‐nitroblue tetrazolium (NBT; Roche), and 5‐bromo‐4‐chloro‐3‐indolyl‐phosphate 4‐toluidine salt (BCIP; Roche) were used.