Transient Expression and Flg22-Induced ROS Assay

The full-length cDNA sequence of aopV was cloned and fused with pBI121-eGFP vector [46 (link)]. All primers used for PCR are listed in Table S2. Agrobacterium tumefaciens GV3101 was used to transiently express EV (pBI121-eGFP empty vector) and AopV at an optical density (OD₆₀₀) of 0.5. A Flg22-induced ROS assay was conducted with N. benthamiana leaves 36 h after inoculation. Twelve leaf discs (4 mm in diameter) were collected from each inoculation area and floated on 100 μL sterilized distilled water in a 96-well plate. The water was then removed, and 100 μL of a solution containing 100 nM flg22, 20 μg/mL horseradish peroxidase, and 100 μM luminol was added to each well. The luminescence was recorded immediately for 1 h using Tecan Infinite F200 luminometer (Tecan, Männedorf, Switzerland) [47 (link)].