Control of tomato brown rugose fruit virus (ToBRFV) in tomato plants using <i>in vivo</i> synthesized dsRNA

The tomato brown rugose fruit virus (ToBRFV) is an increasingly prevalent pathogen that poses a threat to the global tomato industry. Topical application of dsRNA has shown promise as an effective tool to control many pathogens, including viruses; however, it this has not yet been demonstrated for ToBRFV. In this study, ToBRFV-specific long dsRNA molecules were synthesized in vivo by incorporating parts of its genome into that of bacteriophage phi6, thereby enabling the amplification of the chimeric dsRNA in Pseudomonas syringae. Co-inoculation of ToBRFV and purified, high-quality (hq)-dsRNA onto tomato (Solanum lycopersicum) plants resulted in reduction of both viral RNA levels and disease symptoms. Functional analysis of the hq-dsRNA response against the virus revealed its independence of RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) and SUPPRESSOR OF GENE SILENCING 3 (SGS3). In addition, non-infected plants showed a mild activation of innate immune responses upon hq-dsRNA treatment, including accumulation of callose at plasmodesmata. Overall, our results provide evidence for hq-dsRNA as a tool for controlling ToBRFV in tomato plants, and demonstrate the potential of in vivo produced dsRNA in the battle against crop pathogens.