RNAI-based strategies and nanomaterial-mediated delivery for green control of clubroot disease in rapeseed

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• Fluorescence-labeling experiments showed that Plasmodiophora brassicae can efficiently take up exogenous dsRNA. • PbPK1 (a glycolytic pyruvate kinase gene in P. brassicae ) was identified as an effective RNAi target. Host-induced gene silencing (HIGS) of PbPK1 in Arabidopsis and rapeseed significantly reduced clubroot disease severity, suppressed pathogen biomass, and disrupted its life cycle. RNAi targeting PbPK2 showed no efficacy, confirming the specificity of PbPK1 for intervention. • PbPK1 -dsRNA complexed with mesoporous silica nanoparticles (MSNs) could transport dsRNA to rapeseed roots via clathrin − mediated endocytosis. In pot and field trials, the PbPK1 -dsRNA-MSNs complex silenced the PbPK1 gene, decreased the clubroot disease index, and enhanced rapeseed’s disease resistance. • There was no negative impact on rapeseed germination, root growth, or non-target organisms (e.g., earthworms, zebrafish, Pseudomonas spp, S. sclerotiorum ), demonstrating environmental safety. Clubroot, caused by Plasmodiophora brassicae , threatens crucifers globally. RNAi shows promise for disease control, yet its use against clubroot is unexplored. This study aimed to evaluate RNAi-based strategies for combating P. brassicae infection in rapeseed ( Brassica napus ), focusing on two pyruvate kinase genes ( PbPK1 and PbPK2 ) involved in glycolysis. We identified PbPK1 and PbPK2 from P. brassicae genomic and transcriptomic data. The uptake of exogenous dsRNA by the pathogen was confirmed through fluorescence-labeling experiments. Host-induced gene silencing (HIGS) transgenic Arabidopsis and rapeseed lines targeting PbPK1 or PbPK2 were generated, and small RNA sequencing confirmed the production of 21-nt siRNAs. We then evaluated their resistance to clubroot disease. Additionally, dsRNA targeting PbPK1 and PbPK2 was complexed with mesoporous silica nanoparticles (MSNs) for root delivery, and their efficacy was assessed in both pot and field trials. A safety assessment and off-target analysis were also conducted. Upon P. brassicae inoculation, PbPK1 and PbPK2 were silenced by 41%-56% in HIGS plants. The PbPK1 -RNAi lines in Arabidopsis and rapeseed significantly reduced the clubroot disease index, suppressed pathogen biomass accumulation in roots, and disrupted pathogen development, whereas the PbPK2 -RNAi lines exhibited no resistance. The dsRNA- PbPK1 -MSN complex, which enters cells via clathrin-mediated endocytosis, effectively silenced PbPK1 in rapeseed roots, reduced disease severity in trials, and enhanced resistance, while the dsRNA- PbPK2 -MSNs had no therapeutic effect. The safety assessment showed that there was no influence on rapeseed seed germination and root growth, and no harm to non-target organisms such as earthworms and zebrafish. Our findings demonstrate that both HIGS and nanomaterial-mediated dsRNA delivery are viable, eco-friendly strategies for clubroot control. PbPK1, a key glycolytic enzyme, emerges as a promising RNAi target. This study provides novel genetic tools and approaches for sustainable clubroot management and disease-resistant crop breeding.

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