Design, Synthesis, and Functional Analysis of Highly Specific Artificial Small RNAs with Antiviral Activity in Plants

: Artificial microRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs) are two classes of artificial small RNAs (sRNAs) that have been broadly used to confer antiviral resistance in plants. However, methods for designing, synthesizing and functionally analyzing antiviral artificial sRNAs have not been optimized for time and cost-effectiveness and high-throughput applicability since recently. Here we present a systematic methodology for the simple and fast-forward design, generation, and functional analysis of large numbers of artificial sRNA constructs engineered to induce high levels of antiviral resistance in plants. Artificial sRNA constructs are transiently expressed in Nicotiana benthamiana plants, which are subsequently inoculated with the virus of interest. The antiviral activity of each artificial sRNA construct is assessed by monitoring viral symptom appearance, and through molecular analysis of virus accumulation in plant tissues. This approach is aimed to easily identify artificial sRNAs with high antiviral activity that could be expressed in transgenic plants for highly durable antiviral resistance.