A new level of RNA-based plant protection: dsRNAs designed from functionally characterized siRNAs highly effective against <i>Cucumber mosaic virus</i>

RNA-mediated crop protection increasingly becomes a viable alternative to agrochemicals that threaten biodiversity and human health. Pathogen-derived double-stranded RNAs (dsRNAs) are processed into small interfering RNAs (siRNAs), which can then induce silencing of target RNAs, e.g. viral genomes. However, with currently used dsRNAs, which largely consist of undefined regions of the target RNAs, silencing is often ineffective: processing in the plant generates siRNA pools that contain only a few functionally effective siRNAs (esiRNAs). Using an in vitro screen that reliably identifies esiRNAs from siRNA pools, we identified esiRNAs against Cucumber mosaic virus (CMV), a devastating plant pathogen. Topical application of esiRNAs to plants resulted in highly effective protection against massive CMV infection. However, optimal protection was achieved with newly designed multivalent 'effective dsRNAs' (edsRNAs), which contain the sequences of several esiRNAs and are preferentially processed into these esiRNAs. The esiRNA components can attack one or more target RNAs at different sites, be active in different silencing complexes, and provide cross-protection against different viral variants-important properties for combating rapidly mutating pathogens such as CMV. esiRNAs and edsRNAs have thus been established as a new class of 'RNA actives' that significantly increase the efficacy and specificity of RNA-mediated plant protection.