miR398-Dirigent Module Negatively Regulates Cassava Resistance to Cassava Common Mosaic Virus by Affecting the Cell Wall Lignification and Thickness.

Abstract

Studies have demonstrated microRNA398 (miR398) hyperaccumulation in plant species infected with viruses; however, its role in a viral context remains unexplored. Here, we identified cassava (Mes)-miR398 as a regulatory miRNA responsive to cassava common mosaic virus (CsCMV) infection through small-RNA analysis. Mes-miR398 targets Dirigent (Dir; Manes.05G165700.1) associated with disease resistance. Transgenic cassava lines overexpressing Mes-miR398 (OE398) showed downregulation of the Dir gene during CsCMV infection. A dual-luciferase reporter assay confirmed the direct targeting of the Dir gene by miR398. Interestingly, OE398 lines exhibited enhanced vegetative growth. However, CsCMV accumulation was significantly higher in OE398-CsCMV and Dir-silenced (SDir)-CsCMV lines and reduced in Dir-overexpressing (OEDir-CsCMV) lines, indicating a negative regulatory role of the Mes-miR398/Dir module in antiviral defence. Transmission electron microscopy revealed a reduction in cell wall thickness in CsCMV-infected plants compared with healthy controls. A similar decrease was observed in SDir lines, whereas OEDir lines maintained greater wall thickness. Furthermore, lignin content was significantly reduced in SDir lines relative to OEDir lines. Histochemical staining with Toluidine Blue O and Safranin O-Fast Green supported these findings, showing increased lignification in OEDir lines and reduced lignification in SDir lines. Subcellular localisation demonstrated that the Dir protein localises to the cell membrane, suggesting a role in regulating the cell wall membrane. These results revealed that the mes-miR398/Dir regulatory module negatively affects cassava resistance to CsCMV by altering cell wall lignification. This study is the first to identify miR398 as a regulator of a Dir gene and provides critical insights into the molecular interactions between cassava and CsCMV.