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.