Highly efficacious antiviral protection of plants by siRNAs identified in vitro

In response to a viral infection, the plant’s RNA silencing machinery processes viral RNAs into a huge number of small interfering RNAs (siRNAs). However, very few of these siRNAs actually interfere with viral replication. A reliable approach to define the characteristics underlying the activity of these immunologically effective siRNAs (esiRNAs) has not been available so far. We developed a novel screening approach that enables a rapid functional identification of antiviral esiRNAs. The approach is essentially based on the use of a cytoplasmic extract from Nicotiana tabacum BY-2 protoplasts (BY-2 lysate, BYL), that shows Dicer-like (DCL) activity and facilitates the assembly of active RNA-induced silencing complexes (RISC) with an in vitro-translated Argonaute (AGO) protein of choice. We exposed double-stranded (ds) RNA of Tomato bushy stunt virus (TBSV) to the BYL to generate viral siRNAs (DCL assay). Total RNA was isolated from the reactions and DCL-generated TBSV siRNAs were identified by NGS. In another approach, AGO1/RISC- or AGO2/RISC-associated siRNAs were isolated using FLAG-AGO immunoprecipitation (AGO-IP) and analyzed by NGS. Subsequently, the antiviral activity of siRNAs with high affinity to AGO proteins was characterized in vitro and in vivo. siRNA sequence data was obtained from three different DCL assays, two AGO1-IPs and three AGO2-IPs