Simultaneous silencing of ten essential Sclerotinia sclerotiorum genes via spray- and host-induced gene silencing enhances against Sclerotinia stem rot resistance in oilseed rape

A 1,250-bp chimeric dsRNA simultaneously targeting ten Sclerotinia sclerotiorum genes effectively suppresses fungal development and pathogenicity. Both spray-induced (SIGS) and host-induced (HIGS) gene silencing confer enhanced resistance to Sclerotinia stem rot in oilseed rape. HIGS transgenic lines show stable, heritable resistance across multiple generations without compromising yield performance. Sclerotinia stem rot (SSR) is caused by the necrotrophic fungus Sclerotinia sclerotiorum and threatens global oilseed rape ( Brassica napus ) production. Moreover, researchers have not yet identified a gene that confers complete resistance. Here, we developed a multi-target RNA interference (RNAi) strategy to enhance plant resistance by simultaneously silencing eight fungal genes involved in development ( SsChsI–VII , SsGas1 ) and two involved in pathogenicity ( SsPG1 , SsOAH1 ) of S . sclerotiorum . Accordingly, we designed a 1,250-bp chimeric double-stranded RNA (dsRNA) consisting of ten 125-bp fragments each targeting a different gene, and evaluated its effectiveness using spray-induced gene silencing (SIGS) and host-induced gene silencing (HIGS) via stable transformation. In vitro application of the chimeric dsRNA resulted in >50% downregulation of nine target genes, indicating efficient uptake and processing by S . sclerotiorum . Both lesion area and fungal biomass were significantly lower in Nicotiana benthamiana and oilseed rape plants following SIGS. Moreover, stable transgenic plants for HIGS effectively generated gene-specific short interfering RNAs and exhibited an increase in resistance from the T 2 to T 5 generations, with lesions that were 38.9–59.1% smaller in leaves and 43.2–65.8% smaller in stems in the T 5 generation compared with the control plants. Gene silencing resulted in lower oxalic acid accumulation, decreased polygalacturonase activity, and impaired hyphal development, suggesting interference with multiple fungal infection pathways. Notably, HIGS conferred stable, heritable resistance without yield penalty, whereas SIGS provided rapid, nontransgenic protection. This study demonstrates the effectiveness of long chimeric dsRNAs for multi-target gene silencing and highlights a promising RNAi-based strategy for improving disease resistance in oilseed rape, possibly in combination with natural quantitative resistance loci.