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

Alberto Carbonell,José‐Antonio Daròs

doi:10.1111/mpp.12529

Abstract

SummaryArtificial 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.

Highlights

Potato spindle tuber viroid (PSTVd) progenies from all samples including anti-PSTVd amiRNAs were sequenced and, in all cases, target sites did not present mutations. These results indicate that the majority of anti-PSTVd amiRNAs tested are active, with different efficacies
Northern blot analysis at 2 dpa of RNA preparations from agroinfiltrated leaves expressing the 35S:PSTVd construct showed that PSTVd accumulated to significantly lower levels in samples including 35S:syn-tasiR-PSTVd and 35S:MIR173a compared with control samples including 35S:syn-tasiR-GUS and 35S:MIR173a (Fig. 3B, lanes 3 and 5, respectively; P < 0.03 in Student’s t-test comparison) or 35S:syn-tasiR-PSTVd only (Fig. 3B, lanes 4 and 5, respectively; P < 0.01 in Student’s t-test comparison)
We studied the antiviroid effects of 35S:amiR-PSTVd(1)-2, the most effective anti-PSTVd amiRNA construct, and 35S:syn-tasiR-PSTVd compared with 35S:GUS, when co-agroinfiltrated with 35S:PSTVd

Summary

Introduction

Selected amiRNA sequences (Figs 1B, 2B and S2, Text S2, see Supporting Information) were transferred to pMDC32BAtMIR390a-B/c vector (Methods S1, Table S5, see Supporting Information), previously validated for efficient, one-step cloning of amiRNA inserts and high expression of amiRNAs in different plant species, including N. benthamiana (Carbonell et al, 2014). We generated the 35S:syn-tasiR-PSTVd construct by introducing five syn-tasiRNA sequences corresponding to the five most effective anti-PSTVd amiRNA sequences in the pMDC32BAtTAS1c-B/c vector (Fig. 3A, Methods S1), previously validated for efficient, one-step cloning of syn-tasiRNA inserts and high expression of TAS1c-based syn-tasiRNAs in different plant species, including N. benthamiana (Carbonell et al, 2014).

Results

Conclusion