Gol, D. | bioRxiv Science

bioRxiv2025

Advancing RNAi-Based Strategies Against Downy Mildews: Insights Into dsRNA Uptake and Gene Silencing

Downy mildew (DMs) dieases 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. In this study, we evaluated the efficacy of dsRNA-mediated RNAi in suppressing key biological functions in DM pathogens of Arabidopsis thaliana, pea and lettuce DM pathogens, Hyaloperonospora arabidopsidis (Hpa), Peronospora viciae f. sp. pisi (Pvp) and Bremia lactucae (Bl), respectively. We specifically targeted the cellulose synthase 3 (CesA3) and the beta tubulin (BTUB) genes. Silencing CesA3 impaired spore germination and infection across multiple species, while BTUB silencing reinforced the potential of dsRNA-mediated inhibition. Reduction in gene expression levels correlated well with the sporulation assays confirming the effectiveness of dsRNA-mediated gene silencing. We used dsRNAs that were chemically synthesized, in vitro transcribed (IVT) or produced in E. coli. We found that the length and concentration of these dsRNAs significantly affected uptake efficiency, spore germination, and sporulation, with higher concentrations enhancing inhibitory effects. Confocal microscopy using Cy-5-labelled short-synthesized dsRNA (SS-dsRNA) provided direct evidence of spore uptake, confirming the potential of SS-dsRNA for pathogen control. However, species-specific sequence variations influenced dsRNA efficacy, underscoring the importance of target sequence design. Multiplexed RNAi impacted silencing synergisticly, further reducing germination and sporulation in Hpa. Additionally, we demonstrated that SS-dsRNA-mediated gene silencing is sustained over time, with a significant reduction in gene expression level at 4, 7, 10 and 11dpi. This indicates the durability and efficacy of this approach. Taken together, these findings demonstrate the potential of dsRNA-mediated gene silencing as a precision tool for managing DM pathogens.

plant biology↗

bioRxiv2024

Synchronization of Circadian Clock Gene Expression in Arabidopsis and Hyaloperonospora arabidopsidis and its Impact on Host-Pathogen Interactions

Organisms across all kingdoms have an internal circadian clock running in 24h cycles. This clock affects a variety of processes, including innate immunity in plants. However, the role of pathogen circadian clocks had not been extensively explored. We previously showed that light can influence infection of the oomycete Hyaloperonospora arabidopsidis (Hpa, downy mildew disease) on its natural host Arabidopsis thaliana. Here, we identified Hpa orthologs of known circadian clock genes (CCGs) Drosophila TIMELESS (TIM) and Arabidopsis Sensitive to Red Light Reduced 1 (AtSRR1) genes. Expression of both HpaTIM and HpaSRR1 showed a circadian rhythm when Hpa was exposed to constant light. Contrastingly, these two genes were negatively regulated by constant dark exposure. Furthermore, the expression patterns of HpaTIM and HpaSRR1 correlate with those of AtCCA1 and AtLHY, indicating a synchronisation of biological clock genes between the host and the pathogen. In addition, screening mutants of Arabidopsis Clock Regulated Genes (AtCRGs) with three virulent Hpa isolates revealed that mutations in AtCRGs influenced HpaTIM and HpaSRR1 expression and Hpa development, indicating a functional link between the plant biological clock and virulence. Moreover, sporulation of Hpa was reduced by targeting HpaTIM and HpaSRR1 with short synthesized small interfering RNAs, indicating that the pathogen clock is also relevant to virulence. We propose that plant and pathogen clocks are synchronized during infection and that proper regulation of both clocks are genetically necessary for pathogen virulence.

plant biology↗