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bioRxiv · 10.1101/2025.11.17.688593

Rice NH2 Functions as a Positive Regulator of Salicylic Acid Mediated Defense Responses Against Sheath Blight and Bacterial Blight

Ponnurangan, V.·Ashokkumar, S.·KK, K.·Eswaran, k.·Loganathan, A.·Duraialagara, S.·Chellappan, G.·Vaikuntavasan, P.·Maduraimuthu, D.·Shanmugam, V.
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Abstract

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 critical 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 dense mycelial growth, wider hyphae, and elevated 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 (WRKY4, WRKY45, WRKY80, TGA2 and TGA3), pathogenesis-related (PR) genes (PR1, PR3 and PR5), and SA biosynthesis genes (PAL and ICS1) in the mutants. Additionally, OsNH2 mutants in both cultivars exhibited reduced endogenous SA levels upon Rhizoctonia solani infection. Exogenous SA treatment partially restored resistance and upregulated OsNH1/3 expression in mutants, though not to wild-type 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. HighlightsCRISPR/Cas9-edited OsNH2 knockout mutants, along with in-frame deletion mutants lacking 15-17 amino acids in the BTB/POZ domain, exhibited increased susceptibility to sheath blight disease in rice. OsNH2 disruption led to reduced endogenous salicylic acid (SA) levels and significant downregulation of OsNH1, OsNH3, key WRKY and TGA transcription factors, and pathogenesis-related (PR) genes. Exogenous SA treatment partially restored resistance and upregulated OsNH1/3 expression in mutants, though not to wild-type levels--highlighting OsNH2s essential role in sustaining SA-mediated defense signaling. OsNH2 mutants also showed increased susceptibility to bacterial leaf blight (BLB), emphasizing its coordination with OsNH1 and OsNH3 in defense against multiple rice pathogens. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/688593v1_ufig1.gif" ALT="Figure 1000"> View larger version (51K): org.highwire.dtl.DTLVardef@f0c18forg.highwire.dtl.DTLVardef@33b59eorg.highwire.dtl.DTLVardef@d8bf27org.highwire.dtl.DTLVardef@1edc5fc_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Ponnurangan, V., Ashokkumar, S., KK, K., Eswaran, k., Loganathan, A., Duraialagara, S., Chellappan, G., Vaikuntavasan, P., Maduraimuthu, D., Shanmugam, V.. 2025-11-18. Rice NH2 Functions as a Positive Regulator of Salicylic Acid Mediated Defense Responses Against Sheath Blight and Bacterial Blight. https://doi.org/10.1101/2025.11.17.688593

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