Rice NH2 functions as a positive regulator of salicylic acid-mediated defense responses against sheath blight disease

The rice genome encodes five non-expressors of pathogenesis-related (NPR) homologs, with OsNPR1/NH1 and OsNPR3/NH3 emerging as pivotal players in salicylic acid (SA)-mediated defense responses. Investigating the functional implications of the remaining NPR/NH genes is crucial for the development of disease-resistant rice cultivars. This study explores the role of OsNH2 in rice defense against sheath blight (ShB) using CRISPR/Cas9-edited mutants of the susceptible cultivar ASD16 and the moderately resistant CO51. OsNH2 knockout mutants showed increased susceptibility to ShB, as evidenced by denser mycelial growth, wider hyphae, and increased superoxide radical content. Two in-frame deletion mutants lacking 15-17 amino acids in the BTB/POZ domain also showed higher susceptibility, highlighting the importance of an intact OsNH2 protein for resistance. qRT-PCR analysis revealed significant downregulation of OsNH1, OsNH3, key transcription factors (OsWRKY4, OsWRKY45, OsWRKY80, OsTGA2, and OsTGA3), pathogenesis-related (PR) genes (OsPR1, OsPR3, and OsPR5), and SA biosynthesis genes (OsPAL and OsICS1) in the mutants. Additionally, OsNH2 mutants in both cultivars showed reduced endogenous SA levels upon Rhizoctonia solani AG1-1A infection. Exogenous SA treatment partially restored resistance and upregulated OsNH1/3 expression in mutants, though not to wild-type (WT) levels. These results suggest that OsNH2 is essential for maintaining SA-mediated defense signaling and optimal expression of NPR1 homologs. Moreover, OsNH2 mutants also showed increased susceptibility to bacterial leaf blight (BLB). Collectively, this research highlights the critical role of OsNH2 in coordinating with OsNH1 and OsNH3 in SA-mediated defense against ShB and BLB in rice.