Edited by: Yiguo Hong, Hangzhou Normal University, China
Reviewed by: Dominique Loqué, Lawrence Berkeley National Laboratory, USA; Alexis Maizel, Heidelberg University, Germany
*Correspondence: Hervé Vaucheret, Institut Jean-Pierre Bourgin, INRA Centre de Versailles-Grignon, 78026 Versailles Cedex, France. e-mail:
This article was submitted to Frontiers in Plant Physiology, a specialty of Frontiers in Plant Science.
This is an open-access article distributed under the terms of the
Given their sessile condition, land plants need to integrate environmental cues rapidly and send signal throughout the organism to modify their metabolism accordingly. Small RNA (sRNA) molecules are among the messengers that plant cells use to carry such signals. These molecules originate from fold-back stem-loops transcribed from endogenous loci or from perfect double-stranded RNA produced through the action of RNA-dependent RNA polymerases. Once produced, sRNAs associate with Argonaute (AGO) and other proteins to form the RNA-induced silencing complex (RISC) that executes silencing of complementary RNA molecules. Depending on the nature of the RNA target and the AGO protein involved, RISC triggers either DNA methylation or chromatin modification (leading to transcriptional gene silencing, TGS) or RNA cleavage or translational inhibition (leading to post-transcriptional gene silencing, PTGS). In some cases, sRNAs move to neighboring cells and/or to the vascular tissues for long-distance trafficking. Many genes are involved in the biogenesis of sRNAs and recent studies have shown that both their origin and their protein partners have great influence on their activity and range. Here we summarize the work done to uncover the mode of action of the different classes of sRNA with special emphasis on their movement and how plants can take advantage of their mobility. We also review the various genetic requirements needed for production, movement and perception of the silencing signal.
Plants produce miRNAs and siRNAs, but no piRNAs. The biogenesis pathways responsible for the production of miRNA and siRNA molecules share a few similarities. They all derive from double-stranded RNA (dsRNA) molecules that are cleaved into one or many small RNA (sRNA) duplexes by one of the four dicer-like enzymes (DCL) found in plants (Baulcombe,
Most of the sRNA duplexes are methylated at their 3′ extremities by the methyl-transferase HUA ENHANCER 1 (HEN1) (illustrated in Figure
RNA silencing was discovered in plants as a mechanism whereby invading nucleic acids such as transgenes and viruses are silenced through the action of small homologous RNA molecules (Ding and Voinnet,
Transgene reliable expression has been one of the major challenges of plant molecular biology, and RNA silencing has been one of its major obstacles. One of the first examples of PTGS was encountered while trying to over-express
Expressing part of a viral genome into plants sometimes lead to virus resistance. The underlying mechanism was revealed when plants were shown to produce siRNAs corresponding to the infecting virus (Hamilton and Baulcombe,
In addition to plant antiviral defense responses that rely on siRNAs derived directly from the genome of the pathogen, there are now indications that host-, as opposed to parasite-encoded sRNAs might also participate in antiviral defense. Indeed, two miRNAs, bra-miR158, and bra-miR1885, are significantly upregulated during
Genome-encoded miRNAs have also been shown to contribute to resistance against bacteria. One notable example is miR393, which, in Arabidopsis, is induced by treatment with the Flg22 peptide, derived from the bacterial flagellin, a well-known pathogen-associated molecular pattern mimicking bacterial infection (Navarro et al.,
Although a large portion of the plant genome is actively transcribed into RNA, only a small fraction encodes proteins. In many cases, non-protein coding RNAs produce sRNAs which direct either transcriptional or post-transcriptional repression of genes with conserved cellular functions and serve as a flexible sequence-specific source of regulation that promotes adaptability (Dunoyer et al.,
The importance of miRNAs is illustrated by the fact that several miRNAs regulates the functioning of the miRNA pathway. Indeed, feedback loops control the expression of
Plants have further adapted RNA silencing to regulate protein-coding genes through a class of siRNA known as
Plant genomes contain massively abundant and unstable transposable elements (TE), most of which are inactivated or silent because of epigenetic suppression (Wang et al.,
On the other hand, TEs are an important source of epigenetic novelty. Indeed, some of the genetic changes caused by TEs, including alterations in gene expression, gene deletion and insertion, and chromosome rearrangements, can be beneficial at the population scale. This requires the movement of TEs to new positions within the host genome under certains circumstances, which requires escaping from silencing by sRNAs. Supporting this model, Arabidopsis plants impaired in 24-nt siRNA production showed stress-dependent activation of retrotransposons in germinal cells (Ito et al.,
Early on, grafting experiments have showed that PTGS can propagate to different parts of the plant (Palauqui et al.,
Within cells, RNA silencing can spread from an inducing locus to homologous targets present at unlinked loci, a phenomenon generally referred to as
Another important form of intracellular movement occurs between the nucleus and the cytoplasm. Indeed, it was assumed that part of the silencing machinery (associated with TGS) is restricted to the nucleus while other factors (associated with PTGS) remain in the cytoplasm. This has important consequences for the silencing models; for instance, it was long thought that the PTGS machinery was localized exclusively in the cytoplasm, forbidding that sRNA could be generated against a pre-mRNA. However, a recent study has showed that an intron introduced in plants as an IR could cause the silencing of the corresponding gene (Hoffer et al.,
Early studies of transgene-mediated PTGS have demonstrated that RNA silencing is able to spread to about 10–15 cells away from where it was initiated (Himber et al.,
Even before the discovery of sRNAs, it became obvious that whatever the signaling molecule was, silencing was able to move throughout the plant (Palauqui et al.,
The ability of riboregulators to traffic throughout the plant provides obvious advantages to the organism. First, this signal could coordinate the adaptation to an environmental cue in the entire plant after the cue is perceived by a few cells. For example, regulating the intake of phosphate involves miR399, which is present in the phloem sap and moves from shoot to root where it downregulates its target (Pant et al.,
The exact form of the silencing signal(s) is still under debate. Obviously, it must contain nucleic acids, most certainly RNA, to ensure sequence-specificity. However, whether it is a long or short, single- or double-stranded molecule remains unclear. Moreover, it is possible that different forms of silencing signals exist (Melnyk et al.,
To address some of these questions, the movement of fluorescent 21- and 25-nt sRNAs was tested by microinjection in
Another approach used micro-grafting of Arabidopsis plants followed by sRNA deep sequencing. These experiments confirmed that both primary and secondary siRNAs of all sizes are able to move through the graft union (Melnyk et al.,
With the exception of a few miRNAs that are produced in companion cells and loaded into the phloem and also miR165/166 and miR390 that have been proposed to move at short distance (Carlsbecker et al.,
Given that most miRNAs and 21-nt siRNAs bind to AGO1 after duplex separation, it is tempting to speculate that the difference in mobility between miRNAs and 21-nt siRNAs is related to the DCL involved in their biogenesis, rather than the AGO to which they associate. However, association with other AGOs than AGO1 may also contribute to mobility. Indeed, miR165/166 and miR390, which were shown to move at short distance, associate with AGO10 and AGO7, respectively (Montgomery et al.,
It is surprising that no factor involved in the actual movement of silencing-associated molecules has been identified through forward genetic screens yet (Melnyk et al.,
In the hope of revealing the factors involved in sRNA silencing movement, two similar approaches have been used. They involved the companion cell-specific expression of an IR triggering the silencing of an endogenous gene of Arabidopsis, either the
The screens described above also confirmed that the 21-nt sRNAs are essential for the short distance propagation of PTGS. Indeed, the intensity of the photo-bleaching phenotype is directly correlated to the abundance of the 21-nt. It is therefore expected that any mutation influencing the abundance of these sRNAs would influence the overall phenotype. This is probably why mutations in
Lastly, it is interesting to note that silencing of endogenous genes does not propagate systemically in an RDR6-dependent manner, and remains localized to the 10–15 cells neighboring the veins. Indeed, mutations in
Obviously, the movement of RNA silencing signals is crucial for plants. What appeared primarily as a simple and straightforward mean to counter-attack RNA viruses has turned into a complex battery of traveling molecules insuring the coordinated development of the organism as well as genome stability and perhaps even the capacity for fast adaption to the environment. With such important implications in the various biological functions of the plant, it is not surprising that RNA interference raises high interest in the fields of agriculture and biotechnology. Indeed, sRNAs now appear as a promising mean to be able to modify the plant metabolism in any number of ways. The potential practical applications of gene silencing emphasize the importance of answering the leftover questions regarding the sRNA molecules. For instance, it remains to be confirmed if sRNAs are actually the silencing effectors of these processes. Also, the carriers allowing movement through plasmodesmata and/or into the phloem remain to be identified. Increasing our knowledge in this field will also be of vital importance for the understanding of cell fate, defense mechanisms, stress adaptation and evolution of plants.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The authors thank the lab members for fruitful discussions. Jean-Sébastien Parent was supported by a fellowship from the Fonds de Recherche du Québec—Nature et Technologies and a long-term fellowship from the European Molecular Biology Organization. Research in the Vaucheret lab is supported by the French Agence Nationale pour la Recherche (ANR) and a grant form the Fondation Louis D. de l'Institut de France.