Transcript-Specific Editing of Overlapping Genes Reveals Functional Divergence Between OsRLCK280/OsPUB52 and its Antisense Transcript in Rice Immunity and Development

Abstract

Sense‒antisense overlapping gene pairs are widespread in plant genomes and constitute an important layer of gene regulation. However, only a few natural antisense transcripts (NATs) have been functionally characterized, primarily as regulators of their sense counterparts via small RNAs or long noncoding RNAs. Here, we employed genome editing to dissect an overlapping gene locus in rice, designated R883 , which comprises a U-box containing receptor-like cytoplasmic kinase ( OsRLCK280 , also known as OsPUB52 ) and its natural antisense transcript NAT1 . Targeted mutagenesis of the shared exon resulted in pleiotropic phenotypes, including reduced blast resistance, diminished brassinosteroid (BR) sensitivity, increased leaf angle, and elongated grains. Using transcript-specific editing, we demonstrated that OsRLCK280 positively regulates BR signaling and blast resistance but does not affect grain length. RNA-seq analysis revealed that OsRLCK280 reprograms the expression of 514 genes, most of which respond to BR treatment and/or Magnaporthe oryzae infection, positioning OsRLCK280 as a novel node integrating BR signaling and fungal immunity. In contrast, NAT1 encodes a micropeptide conserved within the Oryza genus. Specific editing of NAT1 increased grain length without compromising blast resistance. Together, our findings demonstrate that sense and antisense transcripts at a single locus can independently control distinct traits. This study highlights the necessity of transcript-specific editing for dissecting complex loci and provides insights into the coordinated regulation of growth and defense in rice.