Highly efficient virus resistance mediated by artificial microRNAs that target the suppressor of PVX and PVY in plants

Abstract

MicroRNAs (miRNAs) processed from nuclear-encoded transcripts control expression of target transcripts by directing cleavage or translational inhibition. Artificial miRNAs (amiRNAs) that exploit this endogenous gene silencing mechanism can be designed to target any gene of interest and provide a highly specific approach for effective post-transcriptional gene silencing (PTGS) in plants. Here, using Arabidopsis thaliana miR159a, miR167b and miR171a precursors as backbones, we designed two types of amiRNA targeting sequence that encode the silencing suppressor HC-Pro of Potato virus Y (PVY) and the TGBp1/p25 (p25) of Potato virus X (PVX). The detected amiRNAs efficiently inhibited HC-Pro and p25 gene expression and conferred highly specific resistance against PVY or PVX infection in transgenic Nicotiana tabacum; this resistance was also maintained under conditions of increased viral pressure. Moreover, resistance was strongly influenced by the complementarity between the target sequence and amiRNA, and was well correlated to amiRNA expression level; the expression level of amiRNAs was also well related to the precursor backbones. We further showed that transgenic N. tabacum developed highly effective resistance to both PVY and PVX through expression of a dimeric amiRNA precursor. Together, our findings indicate that transgenic plants with multiple virus-specific resistance can be obtained through co-expression of several amiRNAs targeting multiple viruses.

Strategic RNA Silencing for Plant Viral Resistance

“…Furthermore, co-expression of two amiRNA precursors on the same construct to generate a dimeric amiRNA could be used to produce two amiRNAs and provide resistance to two different viruses (Niu et al 2006). Another study has also reported that transgenic N. tabacum expressing amiRNAs targeting HC-Pro and p25 from A. thaliana pre-miR159a showed highly specific resistance against PVY and PVX (Ai et al 2011). Therefore, chimeric hpRNA, shRNA, and multimeric amiRNA strategies are all extremely useful to address the issue of multiple virus infections under field condition.…”

“…The miRNA precursor backbones that are highly expressed in organisms are the best for amiRNA function. By comparison of the three different amiRNA-mediated resistances, the different resistance levels of transgenic plants were positively correlated with the expression levels of amiRNAs (Ai et al 2011). Concerning the study of targeting viral sequence, targeting 2a and 2b RNAs of CMV induced a higher antiviral ability than only targeting 2b (Qu et al 2007;Zhang et al 2011a).…”

“…Subsequently, the amiRNA approach was reported to confer high resistance against two different strains of CMV (Shandong and Q strains) (Duan et al 2008). Recently, PVY or Potato virus X (PVX) resistance was obtained in N. tabacum expressing amiRNAs targeting HC-Pro of PVY or TGBp1/ p25 of PVX (Ai et al 2011).…”

Host-Derived Artificial MicroRNA as an Alternative Method to Improve Soybean Resistance to Soybean Cyst Nematode

“…The amiRNA based approach for antiviral resistance was first demonstrated by targeting virus silencing suppressor P69 and HC-Pro of turnip yellow mosaic virus and turnip mosaic virus, respectively, in Arabidopsis [19]. Additional examples of antiviral resistance using the amiRNA-based approach to target different viral genes including 2b gene, 3’ untranslated region (UTR), coat protein (CP), nuclear inclusion protein a and b, and silencing suppressors have been documented for cucumber mosaic virus (CMV), potato virus X (PVX), and potato virus Y (PVY) [20,21,22,23]. More recently, improved plant resistance was achieved by targeting the nucleoprotein (N) but not silencing suppressor genes (NSs) in tomato spotted wilt virus (TSWV) using a shortened miR159a precursor backbone [24].…”

Small Non-Coding RNAs in Plant Immunity

“…Indeed several studies demonstrated that artificial miRNAs (amiRNAs) targeting key components of the viral replication machinery can efficiently impair viral growth upon infection [59][60][61][62][63][64][65]. These efforts have revealed that such resistance is cell-autonomous, inheritable, more efficient than siRNA-mediated strategies and successful in blocking viral replication and movement [59,66].…”