Rice spotted leaf mutants are helpful to investigate programmed cell death (PCD) and defense response pathways in plants. Using a map-based cloning strategy, we characterized novel rice spotted leaf mutation spl^HM143 that encodes a 7-hydroxymethyl chlorophyll a reductase (OsHCAR). The wild-type (WT) allele could rescue the mutant phenotype, as evidenced by complementation analysis. OsHCAR was constitutively expressed at all rice tissues tested and its expression products localized to chloroplasts. The mutant exhibited PCD and leaf senescence with increased H₂O₂ (hydrogen peroxide) accumulation, increased of ROS (reactive oxygen species) scavenging enzymes activities and TUNEL (terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling) -positive nuclei, upregulation of PCD related genes, decreased chlorophyll (Chl) contents, downregulation of photosynthesis-related genes, and upregulation of senescence-associated genes. Besides, the mutant exhibited enhanced bacterial blight resistance with significant upregulation of defense response genes. Knockout lines of OsHCAR exhibited spotted leaf phenotype, cell death, leaf senescence, and showed increased resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo) coupled with upregulation of five pathogenesis-related marker genes. The overexpression of OsHCAR resulted in increased susceptibility to Xoo with decreased expression of pathogenesis-related marker genes. Altogether, our findings revealed that OsHCAR is involved in regulating cell death and defense response against bacterial blight pathogen in rice.

The spotted-leaf mutant, characterized by spontaneous lesion formation resembling pathogen-induced hypersensitive cell death, serves as an ideal model for studying the molecular mechanisms behind rice (Oryza sativa) disease resistance and programmed cell death, as these plants display hypersensitive responses that mimic those triggered by pathogen infection. In this study, we generated a knockout line using CRISPR/Cas9 technology in homologous mutant HM113-induced calli. LOC_Os07g30510 encodes a cysteine-rich receptor kinase with a DUF26 domain, consisting of 688 amino acids. HM113 was localized to the cytosol and expressed in most rice tissues at various growth stages. A single nucleotide substitution from A to T was observed at the 847th base of LOC_Os07g30510, causing an amino acid change from serine to cysteine. Our results demonstrated that the A847T mutation was responsible for the spotted-leaf phenotype in the HM113 mutant through gene editing technology, as new frameshift mutations were introduced upstream of the A847T site in the HM113 gene. The mutation phenotype of HM113 was eliminated and resistance to bacterial blight was also lost, indicating that it is a gain-of-function gene.