dsRNAs composed of multiple functionally characterized, efficient siRNAs provide effective protection against Cucumber mosaic virus. A new level of RNA-based plant protection - dsRNAs designed from functionally characterized siRNAs highly effective against Cucumber mosaic virus

In response to viral infection of a plant, Dicer-like enzymes (DCLs) recognize viral double-stranded RNA and process it into a large number of small interfering RNAs (siRNAs). However, only a few of these siRNAs actually interfere with viral replication by forming a functional RNA-induced silencing complex (RISC). The activity of a certain siRNA depends mainly on its efficient loading into an antiviral Argonaute (AGO) protein, the core component of RISC, and on the accessibility of its complementary target site within the viral RNA. Here, we used a novel in vitro screening approach to identify functionally effective siRNAs (esiRNAs) targeting Cucumber mosaic virus (CMV, strain Fny). The approach is essentially based on a cytoplasmic extract from Nicotiana tabacum BY-2 protoplasts (BY-2 lysate, BYL), which exhibits DCL activity and facilitates the assembly of active RISCs with an in vitro translated AGO protein of choice. We exposed double-stranded versions of CMV RNA 2 and RNA 3 to BYL to generate a pool of viral siRNAs. Total RNA was isolated from the reactions and DCL-generated CMV siRNAs were identified by small RNA-seq. In another approach, AGO1/RISC- or AGO2/RISC-associated siRNAs were isolated from the pool by FLAG-AGO immunoprecipitation (AGO-IP) and again analyzed by small RNA-seq. The antiviral activity of siRNAs with high affinity to AGO proteins was characterized in vitro and in vivo. Finally, double-stranded RNAs composed of multiple esiRNA sequences were designed. The DCL-mediated processing of these edsRNAs into siRNAs in BYL was analyzed by small RNA-seq and the antiviral activity was characterized in vitro and in vivo.