Multi‐targeting of viral RNAs with synthetic trans‐acting small interfering RNAs enhances plant antiviral resistance

RNA interference (RNAi)‐based tools are used in multiple organisms to induce antiviral resistance through the sequence‐specific degradation of target RNAs by complementary small RNAs. In plants, highly specific antiviral RNAi‐based tools include artificial microRNAs (amiRNAs) and synthetic trans‐acting small interfering RNAs (syn‐tasiRNAs). syn‐tasiRNAs have emerged as a promising antiviral tool allowing for the multi‐targeting of viral RNAs through the simultaneous expression of several syn‐tasiRNAs from a single precursor. Here, we compared in tomato plants the effects of an amiRNA construct expressing a single amiRNA and a syn‐tasiRNA construct expressing four different syn‐tasiRNAs against Tomato spotted wilt virus (TSWV), an economically important pathogen affecting tomato crops worldwide. Most of the syn‐tasiRNA lines were resistant to TSWV, whereas the majority of the amiRNA lines were susceptible and accumulated viral progenies with mutations in the amiRNA target site. Only the two amiRNA lines with higher amiRNA accumulation were resistant, whereas resistance in syn‐tasiRNA lines was not exclusive of lines with high syn‐tasiRNA accumulation. Collectively, these results suggest that syn‐tasiRNAs induce enhanced antiviral resistance because of the combined silencing effect of each individual syn‐tasiRNA, which minimizes the possibility that the virus simultaneously mutates all different target sites to fully escape each syn‐tasiRNA.     

RNA-binding proteins in RNA interference

Short RNAs (21–27 nt) silence genes that contain homologous nucleotide sequences; this is known as RNA silencing. This review considers the generation of short RNAs from their precursors: double-stranded RNAs, capable of inducing RNA interference, and hairpin RNAs, whose processing yields microRNAs, as well as the properties of RNA-binding domains that were initially identified in proteins operating in RNA interference. The interactions between these domains and known RNA-binding modules within proteins involved in RNA interference and microRNA generation are described.     

Comparative analysis of non-autonomous effects of tasiRNAs and miRNAs in Arabidopsis thaliana

In plants, small interfering RNAs (siRNAs) can trigger a silencing signal that may spread within a tissue to adjacent cells or even systemically to other organs. Movement of the signal is initially limited to a few cells, but in some cases the signal can be amplified and travel over larger distances. How far silencing initiated by other classes of plant small RNAs (sRNAs) than siRNAs can extend has been less clear. Using a system based on the silencing of the CH42 gene, we have tracked the mobility of silencing signals initiated in phloem companion cells by artificial microRNAs (miRNA) and trans-acting siRNA (tasiRNA) that have the same primary sequence. In this system, both the ta-siRNA and the miRNA act at a distance. Non-autonomous effects of the miRNA can be triggered by several different miRNA precursors deployed as backbones. While the tasiRNA also acts non-autonomously, it has a much greater range than the miRNA or hairpin-derived siRNAs directed against CH42, indicating that biogenesis can determine the non-autonomous effects of sRNAs. In agreement with this hypothesis, the silencing signals initiated by different sRNAs differ in their genetic requirements.     

The Phaseolus vulgaris miR159a precursor encodes a second differentially expressed microRNA

Plant microRNAs originate from a stem-loop structured single-stranded RNA precursor. Each stem-loop is processed to generate a mature microRNA that is recruited to an ARGONAUTE-containing multi-protein complex to direct silencing of its target mRNA. Here we report that the conserved plant miR159a precursor produces a second 21-nt long RNA with the properties of a microRNA. Its presence in different plant species is supported by its conservation in the stem-loop position and expression as determined by northern blot analysis. We show that successive processing by DCL1 produces this novel microRNA from the same precursor as miR159a. In contrast to the low levels observed in other plant models for the equivalent of miR159.2, in P. vulgaris, the accumulation of miR159.2 is easily detectable and when compared to miR159a, their expression patterns are distinct in different organs and growth conditions. Further evidence of the functionality of miR159.2 comes from its association with silencing complexes as demonstrated by co-immunoprecipitation experiments using an AGO1-specific antibody and processing of an artificial GFP reporter construct containing a complementary target sequence. These results indicate that the second small RNA corresponds to a microRNA, at least partially independent of miR159 activity, and that in plants a miRNA precursor may encode multiple regulatory small RNAs.     

Artificial microRNA-mediated virus resistance in plants

RNA silencing in plants is a natural defense system against foreign genetic elements including viruses. This natural antiviral mechanism has been adopted to develop virus-resistant plants through expression of virus-derived double-stranded RNAs or hairpin RNAs, which in turn are processed into small interfering RNAs (siRNAs) by the host's RNA silencing machinery. While these virus-specific siRNAs were shown to be a hallmark of the acquired virus resistance, the functionality of another set of the RNA silencing-related small RNAs, microRNAs (miRNAs), in engineering plant virus resistance has not been extensively explored. Here we show that expression of an artificial miRNA, targeting sequences encoding the silencing suppressor 2b of Cucumber mosaic virus (CMV), can efficiently inhibit 2b gene expression and protein suppressor function in transient expression assays and confer on transgenic tobacco plants effective resistance to CMV infection. Moreover, the resistance level conferred by the transgenic miRNA is well correlated to the miRNA expression level. Comparison of the anti-CMV effect of the artificial miRNA to that of a short hairpin RNA-derived small RNA targeting the same site revealed that the miRNA approach is superior to the approach using short hairpin RNA both in transient assays and in transgenic plants. Together, our data demonstrate that expression of virus-specific artificial miRNAs is an effective and predictable new approach to engineering resistance to CMV and, possibly, to other plant viruses as well.     

SINE RNA induces severe developmental defects in Arabidopsis thaliana and interacts with HYL1 (DRB1), a key member of the DCL1 complex

The proper temporal and spatial expression of genes during plant development is governed, in part, by the regulatory activities of various types of small RNAs produced by the different RNAi pathways. Here we report that transgenic Arabidopsis plants constitutively expressing the rapeseed SB1 SINE retroposon exhibit developmental defects resembling those observed in some RNAi mutants. We show that SB1 RNA interacts with HYL1 (DRB1), a double-stranded RNA-binding protein (dsRBP) that associates with the Dicer homologue DCL1 to produce microRNAs. RNase V1 protection assays mapped the binding site of HYL1 to a SB1 region that mimics the hairpin structure of microRNA precursors. We also show that HYL1, upon binding to RNA substrates, induces conformational changes that force single-stranded RNA regions to adopt a structured helix-like conformation. Xenopus laevis ADAR1, but not Arabidopsis DRB4, binds SB1 RNA in the same region as HYL1, suggesting that SINE RNAs bind only a subset of dsRBPs. Consistently, DCL4-DRB4-dependent miRNA accumulation was unchanged in SB1 transgenic Arabidopsis, whereas DCL1-HYL1-dependent miRNA and DCL1-HYL1-DCL4-DRB4-dependent tasiRNA accumulation was decreased. We propose that SINE RNA can modulate the activity of the RNAi pathways in plants and possibly in other eukaryotes.     

Processing of Drosophila endo-siRNAs depends on a specific Loquacious isoform

Drosophila melanogaster expresses three classes of small RNAs, which are classified according to their mechanisms of biogenesis. MicroRNAs are ∼22–23 nucleotides (nt), ubiquitously expressed small RNAs that are sequentially processed from hairpin-like precursors by Drosha/Pasha and Dcr-1/Loquacious complexes. MicroRNAs usually associate with AGO1 and regulate the expression of protein-coding genes. Piwi-interacting RNAs (piRNAs) of ∼24–28 nt associate with Piwi-family proteins and can arise from single-stranded precursors. piRNAs function in transposon silencing and are mainly restricted to gonadal tissues. Endo-siRNAs are found in both germline and somatic tissues. These ∼21-nt RNAs are produced by a distinct Dicer, Dcr-2, and do not depend on Drosha/Pasha complexes. They predominantly bind to AGO2 and target both mobile elements and protein-coding genes. Surprisingly, a subset of endo-siRNAs strongly depend for their production on the dsRNA-binding protein Loquacious (Loqs), thought generally to be a partner for Dcr-1 and a cofactor for miRNA biogenesis. Endo-siRNA production depends on a specific Loqs isoform, Loqs-PD, which is distinct from the one, Loqs-PB, required for the production of microRNAs. Paralleling their roles in the biogenesis of distinct small RNA classes, Loqs-PD and Loqs-PB bind to different Dicer proteins, with Dcr-1/Loqs-PB complexes and Dcr-2/Loqs-PD complexes driving microRNA and endo-siRNA biogenesis, respectively.