The CUT&Tag assay was conducted on protoplasts that had been transiently transformed with OsDOF3-GFP and then sorted using a flow cytometer.

Crop production is persistently threatened by a multitude of destructive diseases. In rice, the Ca2+ sensor RESISTANCE OF RICE TO DISEASES1 (ROD1) is a key regulator of defense against major pathogens, including those causing blast, sheath blight, and bacterial blight. However, the molecular mechanisms underlying ROD1-mediated immune suppression remain largely elusive. Here, we characterize an immune signalling cascade comprising the B3-domain transcription factor OsVAL2, the DOF transcription factor OsDOF3, as well as their downstream target OsCSE encoding a caffeoyl shikimate esterase. We demonstrate that the OsVAL2-OsDOF3-OsCSE module promotes lignin deposition and cell wall fortification to confer broad-spectrum disease resistance. ROD1 interacts with and sequesters OsVAL2 in the cytoplasm, thereby preventing its nuclear translocation and subsequent initiation of this immune program. Our findings reveal a spatiotemporal regulatory mechanism that maintains immune homeostasis, highlighting the crucial role of cell wall remodelling in crop disease resistance. Overall design: Protoplasts were isolated from 14-day-old hydroponically grown wild-type rice seedlings (TP309). These protoplasts were then transiently transformed with the OsDOF3-GFP construct. Approximately 200,000 GFP-positive cells were collected using fluorescence-activated cell sorting (FACS). After centrifugation, the cell pellets were harvested, and chromatin fragmentation was performed using the Vazyme TD904-C2 kit. The experimental group (OsDOF3_CUTTAG) was incubated with a GFP antibody, while the no-antibody control (OsDOF3_control_CUTTAG) was processed in parallel. Finally, libraries were constructed following the kit's protocol and sent for sequencing.