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 (<i>Oryza sativa</i>) 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 <i>HM113</i>-induced calli. <i>LOC_Os07g30510</i> encodes a cysteine-rich receptor kinase with a DUF26 domain, consisting of 688 amino acids. <i>HM113</i> 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 <i>LOC_Os07g30510</i>, 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 <i>HM113</i> mutant through gene editing technology, as new frameshift mutations were introduced upstream of the A847T site in the <i>HM113</i> gene. The mutation phenotype of <i>HM113</i> was eliminated and resistance to bacterial blight was also lost, indicating that it is a gain-of-function gene.