Functional Characterization of Rice Spotted-Leaf Mutant HM113 Reveals an Amino Acid Substitution in a Cysteine-Rich Receptor-like Kinase

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.