Artificial microRNAs and synthetic trans‐acting small interfering RNAs interfere with viroid infection

Artificial microRNAs (amiRNAs) and synthetic trans‐acting small interfering RNAs (syn‐tasiRNAs) are two classes of artificial small RNAs (sRNAs) engineered to silence endogenous transcripts as well as viral RNAs in plants. Here, we explore the possibility of using amiRNAs and syn‐tasiRNAs to specifically interfere with infections by viroids, small (250–400‐nucleotide) non‐coding circular RNAs with compact secondary structure infecting a wide range of plant species. The combined use of recent high‐throughput methods for artificial sRNA construct generation and the Potato spindle tuber viroid (PSTVd)–Nicotiana benthamiana pathosystem allowed for the simple and time‐effective screening of multiple artificial sRNAs targeting sites distributed along PSTVd RNAs of (+) or (–) polarity. The majority of amiRNAs were highly active in agroinfiltrated leaves when co‐expressed with an infectious PSTVd transcript, as were syn‐tasiRNAs derived from a construct including the five most effective amiRNA sequences. A comparative analysis showed that the effects of the most effective amiRNA and of the syn‐tasiRNAs were similar in agroinfiltrated leaves, as well as in upper non‐agroinfiltrated leaves in which PSTVd accumulation was significantly delayed. These results suggest that amiRNAs and syn‐tasiRNAs can be used effectively to control viroid infections in plants.