The Histidine-25-Arginine Mutation in the Rice MACPF Protein OsCAD1 Induces Cell Death and Activates Defense...

Plants defend themselves against pathogens through pattern-triggered immunity (PTI) and effector-triggered immunity (ETI), with the latter often inducing a hypersensitive response (HR) characterized by localized programmed cell death (PCD). Lesion mimic mutants (LMMs), which spontaneously form HR-like lesions in the absence of pathogen infection, have served as valuable genetic resources for dissecting the molecular mechanisms underlying cell death and immune signaling in plants. In this study, we characterize the rice lesion mimic mutant spl17, derived from the IR64 cultivar, and identify the mutation responsible for its phenotype. We demonstrate that the spl17 mutation leads to the accumulation of reactive oxygen species (ROS), induces light-dependent cell death and lesion formation, elevates levels of salicylic acid (SA) and jasmonic acid (JA), activates defense-related genes, and confers enhanced resistance to Xanthomonas oryzae pv. oryzae. Using map-based cloning, we identified a single Histidine-25-Arginine substitution (OsCAD1^H25R) in OsCAD1, a gene encoding a membrane attack complex/perforin (MACPF) domain-containing protein in rice, as the causal mutation. CRISPR/Cas9 genome editing revealed that a knockout of OsCAD1 (OsCAD1^KO) results in seedling lethality, whereas a weak allele (OsCAD1^D8) leads to a viable lesion mimic phenotype and enhances resistance to X. oryzae pv. oryzae. Subcellular localization studies demonstrated that eGFP-OsCAD1 is broadly distributed in Nicotiana benthamiana cells. Transcriptome analyses, including RNA-Seq and Gene Set Enrichment Analysis (GSEA), indicate that differentially expressed genes (DEGs) in spl17 are enriched in catalytic activity, metabolic processes, and membrane functions. Together, these results suggest that OsCAD1 is indispensable for rice growth and development, and that its mutation triggers cell death and defense responses.