Measuring PTI Response in N. benthamiana

The experiment was performed as previously reported [33 (link)]. Briefly, leaves from four N. benthamiana plants infiltrated by A. tumefaciens cells carrying LtCSEP1 or the GFP were sprayed with 1 µM flg22 (Sangon Biotech, Shanghai, China) 12 h post the initial infiltration. The expression of three PTI-associated genes NbAcre31, NbGras2, and NbPti5 was evaluated by qRT-PCR. Four Nicotiana benthamiana leaves were collected 12 and 24 h after infiltration of flg22. For chemiluminescence detection of ROS burst, leaf disks (5 mm in diameter) were collected and suspended into a 96-well plate containing double-distilled water overnight. Then, the water was withdrawn and replaced with 100 mL of luminol solution containing 200 mM luminol (Sigma), 10 mg/mL horseradish peroxidase (Sigma). Chemiluminescence was continuously measured using a microplate reader (Tecan, Männedorf, Switzerland).