Gene silencing in plants by artificial small RNAs derived from minimal precursors and expressed via tobacco rattle virus

Highly specific, second-generation RNA interference tools are based on artificial small RNAs (art-sRNAs), such as artificial microRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs). Recent progress includes the use of minimal-length precursors to express art-sRNAs in plants. These minimal precursors retain the minimal structural elements for recognition and efficient processing by host enzymes. They yield high amounts of art-sRNAs and remain stable when incorporated into potato virus X-based viral vectors for art-sRNA-mediated virus-induced gene silencing (art-sRNA-VIGS). However, further adaptation to new viral vector systems with reduced symptomatology is needed to improve the versatility of art-sRNA-VIGS. Here, we developed a novel platform based on tobacco rattle virus (TRV)-a widely used viral vector inducing minimal or no symptoms-for the delivery of art-sRNAs into plants. TRV was engineered to express authentic amiRNAs and syn-tasiRNAs from minimal precursors in Nicotiana benthamiana, resulting in robust and highly specific silencing of endogenous genes. Notably, the expression of syn-tasiRNAs through TRV conferred strong resistance against tomato spotted wilt virus, an economically important pathogen. Furthermore, we established a transgene-free approach by applying TRV-containing crude extracts through foliar spraying, eliminating the need for stable genetic transformation. In summary, our results highlight the unique advantages of minimal precursors and extend the application of art-sRNA-VIGS beyond previously established viral vector systems, providing a scalable, rapid and highly specific tool for gene silencing.