4.6. Flg22-Inudced ROS Burst Assay

The pBI121-3×FLAG vector inserted into 3 × FLAG tag to fuse with the pBI121 vector [43] was used for this assay. The full-length cDNA sequence of AopN was cloned into the pBI121-eGFP vector. The Flg22-elicited ROS assay was performed with the transient expression of EV (empty vector) or AopP fused FLAG using A. tumefaciens GV3101 at an optical density at 600 nm (OD₆₀₀) of 0.5. The 4–6-week-old N. benthamiana leaves were used in this assay. Ten leaf disks (4 mm in diameter) were collected from each inoculated site 24 h after inoculation and floated in 100 μL of sterile distilled water in a 96-well plate. Next, the water was replaced with 100 μL of solution containing 100 nM flg22, 20 μg/mL horseradish peroxidase, and 100 μM luminol. Luminescence was recorded immediately using a Tecan Infinite F200 luminometer (Tecan, Mannedorf, Switzerland) for 40 min. The experiment was independently repeated three times. All primers used in the PCR are listed in Table S2.