The ROS burst assay was performed as described previously (Tian et al., 2020 (link)). In brief, rice leaf discs of 7-day-old seedlings were cut and suspended in 100 μL water in a 96-well plate overnight. To detect ROS production, water was replaced with 100 μL reaction solution (20 μM luminol and 2.5 μg/mL peroxidase) containing 400 nM chitin (hexa-N-acetylchitohexaose). Time-dependent quantification of ROS production was recorded on a Mithras luminometer (Berthold) every 2 min for 1 h. Sixteen replicates were performed for each sample. For observing and quantifying chitin-induced callose deposition, the leaves of 7-day-old seedlings were detached and incubated with 400 nM chitin. The assay was performed as described previously (Yang et al., 2019 (link)). The callose deposits were observed using UV light (excitation 405 nm, emission 498 nm; Zeiss LSM880). The numbers of deposits were counted according to all fields of vision using ImageJ version 1.43U software (Schneider et al., 2012 (link)).