<scp>RNAi</scp> Strategies Against Downy Mildews: Insights Into <scp>dsRNA</scp> Uptake and Silencing

Downy mildew (DM) diseases are caused by destructive obligate pathogens with limited control options, posing a significant threat to global agriculture. RNA interference (RNAi) has emerged as a promising, environmentally sustainable strategy for disease management. We evaluated the efficacy of dsRNA-mediated RNAi in suppressing key biological functions in DM pathogens of Arabidopsis thaliana, pea and lettuce: Hyaloperonospora arabidopsidis (Hpa), Peronospora viciae f. sp. pisi (Pvp) and Bremia lactucae (Bl), respectively. Conserved genes, cellulose synthase 3 (CesA3) and beta-tubulin (BTUB), were targeted. Silencing these genes significantly impaired spore germination and infection across species and reduced gene expression correlated with suppressed sporulation, confirming silencing efficacy. We tested dsRNAs from chemical synthesis, in vitro transcription, and Escherichia coli expression. Uptake and silencing efficiency varied with dsRNA length and concentration. In Hpa, short dsRNAs (21-25 bp) produced a variable spore germination rate, with 25 bp dsRNA causing a 247.10% increase, whereas longer dsRNAs (≥ 30 bp) completely inhibited germination. Similarly, in Pvp, dsRNAs of 21-25 bp resulted in a 73.05%-77.46% germination rate, while 30-75 bp dsRNAs abolished germination. Confocal microscopy using Cy-5-labelled short-synthesised dsRNA (SS-dsRNA) confirmed uptake by spores. Sequence specificity influenced efficacy, highlighting the need for precise target design. Multiplexed RNAi impacted silencing synergistically, further reducing germination and sporulation in Hpa. Importantly, SS-dsRNA-mediated silencing was durable, with reduced gene expression sustained at 4, 7, 10 and 11 days post-inoculation. Taken together, our findings demonstrate the potential of dsRNA-mediated gene silencing as a precise, sustainable tool for managing DM pathogens in multiple crop species.