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American Society of Plant Biologists RNA Silencing and the Mobile Silencing Signal
a Department of Biological Sciences, University of South Carolina, Columbia, South Carolina 29208 1 To whom correspondence should be addressed E-mail vance{at}biol.sc.edu; fax 803-777-4002
RNA silencing is a sequence-specific RNA degradation mechanism that occurs in a broad range of eukaryotic organisms including fungi (quelling), animals (RNA interference [RNAi]), and plants (post-transcriptional gene silencing). In all these organisms, the process is triggered by double-stranded RNA (dsRNA) and requires a conserved set of gene products (for recent reviews of RNA silencing in plants, see Matzke et al., 2001  ; Vance and Vaucheret, 2001; Voinnet, 2001; Waterhouse et al., 2001; Baulcombe, 2002; in fungi or animals, see Cogoni and Macino, 2000; Bernstein et al., 2001a; Carthew, 2001; Zamore, 2001). The mechanism for RNA silencing involves an initial processing of the inducing dsRNA into small interfering RNAs (siRNAs) of 21 to 25 nucleotides, corresponding to both sense and antisense strands of the target gene (Hamilton and Baulcombe, 1999). These siRNAs become associated with a protein complex referred to as the RNA-induced silencing complex (RISC), where they serve as guides to select the target RNAs and effect their degradation (Hammond et al., 2000; Zamore et al., 2000). In plants, RNA silencing is typically correlated with methylation within the transcribed regions of the transgene that correspond to target RNA (reviewed in Wassenegger, 2000; Bender, 2001). Methylation of genomic DNA occurs even when the silencing is induced by an RNA virus that replicates exclusively in the cytoplasm (Jones et al., 1998), suggesting communication between the cytoplasm and the nucleus. A good deal of evidence suggests that RNA silencing plays a natural role in defense against foreign nucleic acids, including virus resistance in plants (Covey et al., 1997; Ratcliff et al., 1997, 1999; Mourrain et al., 2000; reviewed in Voinnet, 2001) and in control of transposons in a number of other organisms (Ketting et al., 1999; Tabara et al., 1999; Grishok et al., 2000; Djikeng et al., 2001; Elbashir et al., 2001b; Takeda et al., 2001). Consistent with the antiviral nature of RNA silencing in plants, many plant viruses have evolved proteins that suppress RNA silencing (reviewed in Li and Ding, 2001).
One of the most intriguing aspects of RNA silencing is that it is non-cell-autonomous: in both plants and Caenorhabditis elegans it can be induced locally and then spread to distant sites throughout the organism (Palauqui et al., 1997
The first clue that RNA silencing is non-cell-autonomous came from studies of transgenic plants that silenced developmentally to produce a visible phenotype that was acquired in distinctive but nonclonal spatial patterns (Boerjan et al., 1994 ; Palauqui et al., 1996). Soon afterward, independent experiments in two different laboratories provided direct evidence for a systemic silencing signal (Palauqui et al., 1997; Voinnet and Baulcombe, 1997). In grafting experiments, systemic silencing was transmitted across a graft junction from spontaneously silenced transgenic tobacco rootstocks to isogenic scions that had not silenced spontaneously (Figure 1; Palauqui et al., 1997). Lines transgenic for one of three different transgenes were examined: the endogenous loci Nia and Nii, encoding nitrate and nitrite reductase respectively, and the exogenous locus uidA, encoding the reporter protein  -glucuronidase (GUS). Similar results were obtained with all, indicating that systemic RNA silencing is not a peculiarity of one particular genetic locus. In all cases, silencing in the scion was specific for the coding sequences that were silenced in the rootstock, demonstrating that the mobile signal is sequence specific. This sequence specificity suggested that the mobile signal is a nucleic acid or includes a nucleic acid.
Independent evidence for the involvement of a systemic signal in RNA silencing has come from the demonstration that systemic silencing can be induced in transgenic tobacco species by using infiltration with Agrobacterium tumefaciens (agro-infiltration) or particle bombardment to deliver exogenous DNA homologous to the transgene (Figure 1; Voinnet and Baulcombe, 1997
The patterns of systemic silencing suggest that the signal moves both cell-to-cell and through the phloem, mimicking patterns of viral movement through the plant. In 35S green fluorescent protein (GFP) plants, stomatal guard cells that have lost the plasmodesmatal connections to other cells before induction of systemic silencing do not silence, providing evidence that the signal moves cell-to-cell through plasmodesmata (Voinnet et al., 1998 ). Movement of the signal through the phloem has been most evident from the establishment of systemic silencing along major and minor veins prior to subsequent spread into mesophyll cells (Figures 2 and 3
; Palauqui et al., 1997; Voinnet et al., 1998). Phloem movement of the signal is further supported by data showing that systemic silencing initiated from a single leaf is restricted to shoots that emerge from the same side of the stem as the initiating leaf (Voinnet et al., 1998), a pattern reminiscent of phloem transport of dyes and systemic virus (Roberts et al., 1997). The silencing signal can travel relatively long distances in plants: at least several centimeters as shown by propagation of silencing through leafless grafted spacers that cannot silence because homologous sequences are absent (Palauqui et al., 1997; Voinnet et al., 1998). Experiments using agro-infiltration or particle bombardment to induce silencing show that the systemic signal is produced and translocated rapidly, moving out of a leaf within 2 to 3 days (Voinnet et al., 1998; Palauqui and Balzergue, 1999). Although reported to move only upward (Palauqui et al., 1997), the signal moves bidirectionally, but more efficiently upward than downward (Voinnet et al., 1998; Sonoda and Nishiguchi, 2000). Viruses are excluded from meristems after systemic infection of plants (Matthews, 1991), and this is also true for systemic silencing; extreme meristematic zones of shoots, flowers, and roots remain green fluorescent subsequent to extensive and persistent systemic silencing of GFP transgenes (Voinnet et al., 1998). Similarly, silencing is not observed in meristems in Nia or GUS-silenced plants (Beclin et al., 1998). It has not been shown whether the mobile silencing signal fails to get into the meristem or whether meristematic tissue is unable to silence in response to the signal.
Movement of the signal into distant tissues is not sufficient to induce silencing. The same types of experiment that provided direct evidence for the mobile signal have also shown that the nature of the target is important in determining whether—or to what degree—systemic silencing is elicited when signal is provided. In particular, transgenes are much better targets for systemic silencing than are endogenous genes, and the gene must be transcriptionally active. For example, scions expressing a Nia transgene became uniformly silenced for Nia when grafted onto rootstocks silenced for a Nia transgene, whereas the endogenous Nia mRNA in nontransgenic scions and transcriptionally silenced Nia transgenic scions was unaffected (Palauqui et al., 1997 ). When the level of Nia mRNA in scions lacking a Nia transgene was increased by eliminating feedback inhibition of Nia transcription, the scions became uniformly silenced (Palauqui and Vaucheret, 1998), suggesting that the amount of target RNA is important in establishing systemic silencing in response to the mobile signal. Agro-infiltration experiments using a transgene that produces a replicating RNA were also able to induce systemic silencing of endogenous genes, but—unlike the widespread, persistent silencing observed for a GFP transgene—the silencing was transient and limited to regions near the veins of the affected leaves (Voinnet et al., 2000). This limited degree of systemic silencing was induced whether the targeted RNA was expressed at high levels (small subunit of rubisco) or low levels (phytoene desaturase). Thus, requirements for establishment of systemic silencing by the mobile signal are not well understood, but a favored current hypothesis is that amplification of the signal is necessary for efficient systemic silencing (Palauqui and Vaucheret, 1998; Voinnet et al., 1998).
The maintenance of RNA silencing after an exogenous source of signal is removed also depends on the nature of the target. Grafted scions in which the targeted locus is a transgene that never silences spontaneously (referred to as class I transgenes; Palauqui and Vaucheret, 1998
In principle, the nucleic acid component of the mobile silencing signal(s) involved in transgene silencing could either be DNA or RNA derived from the transgene. Indirect evidence for an RNA component comes from the likely antiviral function of the signal in nontransgenic plants. This antiviral function was suggested in experiments in which RNA silencing was activated in upper leaves of wild-type Nicotiana benthamiana by inoculating lower leaves with movement-defective mutants of Potato virus X (PVX) (Voinnet et al., 2000 ). Systemic spread of silencing could be monitored in planta because the modified PVX contained fragments of endogenous genes. Thus, the silencing signal generated by localized virus replication was not only primed against the viral genome but also against the corresponding endogenous genes, therefore generating a visual systemic silencing phenotype. Systemic silencing was only apparent when replication-competent PVX was used as an inoculum. Because PVX has an RNA genome that is replicated via RNA intermediates, this result suggests that the signal, or part of it, is an RNA. This suggestion is reinforced by the fact that >90% of plant viruses, many of which are likely to induce systemic silencing, are RNA viruses that replicate without any DNA intermediate.
siRNAs Small RNAs associated with RNA silencing were discovered in plants (Hamilton and Baulcombe, 1999 ), and similar RNAs have since been associated with the related RNA degradation processes in animal systems, where they are referred to as siRNAs (Hammond et al., 2000; Parrish et al., 2000; Yang et al., 2000; Zamore et al., 2000; Elbashir et al., 2001b). Indeed, these RNAs are considered a hallmark of RNA silencing. The possibility that siRNAs are involved in systemic signaling is an attractive and popular model (Hamilton and Baulcombe, 1999). The characteristics of siRNAs make them ideal candidates for the mobile signal. They are consistently associated with RNA silencing; they are long enough to convey sequence specificity, yet small enough to move easily through plasmodesmata. In addition, they are sufficient to induce RNA silencing in vitro in Drosophila (Elbashir et al., 2001b) and in vivo in mammalian cell lines and in C. elegans (Caplen et al., 2001; Elbashir et al., 2001a). However, direct evidence that siRNAs are mobile or that they play a role in systemic silencing is lacking.
Two lines of evidence argue against a role for siRNAs in systemic signaling of silencing. One line of evidence deals with the viral suppressor of RNA silencing, helper component–proteinase (HC-Pro; discussed in detail in a later section). When an HC-Pro–expressing tobacco line was crossed to a transgenic line silenced for GUS, the GUS silencing was suppressed, GUS mRNA accumulated, and the GUS siRNAs were eliminated. Strikingly, these plants, despite the absence of detectable levels of GUS siRNA, remained capable of producing and sending the mobile silencing signal when used as rootstocks in grafting experiments (Mallory et al., 2001
Recent work points to the complexity of small RNAs in eukaryotic cells: the siRNAs may serve roles other than as guides for the RISC complex, and some populations of small RNAs are not involved in RNA silencing. Evidence from animal RNAi systems has raised the possibility that siRNAs are involved in production of new dsRNA to feed into the silencing pathway (Lipardi et al., 2001 ; Sijen et al., 2001a). Lipardi and co-workers have shown that siRNAs added to an in vitro silencing system become incorporated into long dsRNA via the activity of a cellular RNA-dependent RNA polymerase (RdRP). This result suggests that siRNAs act as primers for the cellular RdRP, resulting in a cycle of dsRNA generation and degradation that amplifies the siRNA (Lipardi et al., 2001). It is not clear if the same siRNA molecules serve dual roles in silencing or if there are subpopulations of siRNA with distinct functions: some serving as guides in the RISC complex and others serving as primers in amplification of siRNA via RdRP. The siRNAs derive from longer dsRNA by the activity of a ribonuclease III (RNase III) called DICER (Hammond et al., 2000; Bernstein et al., 2001b; Knight and Bass, 2001). In Drosphila and C. elegans, DICER also produces another population of small RNAs called small temporal RNAs (stRNAs) (Grishok et al., 2001; Hutvagner et al., 2001; Ketting et al., 2001). Although produced by the activity of the same enzyme, stRNAs differ from siRNAs both structurally and functionally: they are single-stranded (rather than double-stranded), and they interact with the 3' untranslated region of the target mRNA and inhibit its translation (rather than triggering its degradation). In addition, a novel class of abundant and diverse stRNA-like RNAs, termed microRNAs, have recently been uncovered not only in worms and flies but also in humans (Lagos-Quintana et al., 2001; Lau et al., 2001; Lee and Ambros, 2001), suggesting that stRNA species may be more widespread than previously envisioned. Finally, the small RNAs that accumulate during dsRNA-induced transcriptional gene silencing (TGS) are double-stranded and siRNA-like in size (Mette et al., 2000; Sijen et al., 2001b). However, unlike siRNAs, the TGS-associated small RNAs are not involved in RNA degradation (Mette et al., 2001), and their function, if any, is unknown. Because of the structural and functional diversity of small RNAs, it remains a viable possibility that a subpopulation of small RNAs serves as the mobile silencing signal in RNA silencing.
Aberrant RNAs Another likely candidate for the mobile silencing signal is the RNA transcript from a silenced locus (or a derivative of that mRNA), which is in some way aberrant so that it triggers RNA silencing upon arrival in a new cell. Cell-to-cell and systemic movement of mRNA in plants is not unprecedented, and in fact, some evidence suggests that it is commonplace (reviewed in Jorgensen et al., 1998 ; Oparka and Santa Cruz, 2000). Thus, although the paradigm for RNA trafficking in plants is virus infection, it is likely that viruses have simply usurped host systems of macromolecular trafficking for their own purposes. Plant viruses encode special proteins to facilitate movement of viral RNA (reviewed in Lazarowitz and Beachy, 1999), but there is good evidence that there are endogenous plant proteins that function as RNA movement proteins. For instance, viroids—plant pathogens with a noncoding RNA genome composed of a circular RNA duplex—move cell to cell and systemically, probably by recruiting specific host factors (Gomez and Pallas, 2001; Owens et al., 2001; Zhu et al., 2001). There are also several examples of endogenous mRNAs, such as the maize KNOTTED1 (Lucas et al., 1995) and SUT1 in tobacco, tomato, and potato (Kuhn et al., 1997), which move through plasmodesmata presumably using endogenous mechanisms for RNA trafficking. Systemic movement of endogenous RNAs across graft junctions also occurs. Pumpkin NACP mRNA moves from a pumpkin rootstock into the apex of cucumber scions (Ruiz-Medrano et al., 1999). In grafting experiments with tomato, an mRNA derived from a dominant negative mutant locus in the rootstock moves into a scion that is wild type for the locus and confers the mutant phenotype in the scion (Kim et al., 2001). The mechanism of RNA trafficking may involve proteins that function in a manner analogous to viral movement proteins such as CmPP16 from Cucurbita maxima (Xoconostle-Cazares et al., 1999). Thus, RNA trafficking, both local and systemic, may be a normal aspect of plant gene expression.
These results raise the possibility that the mobile silencing signal is an mRNA or mRNA/protein complex that moves across the graft junction into the scion via normal pathways for macromolecular trafficking. Upon arrival in the scion, the translocated RNA becomes a template for a cellular RdRP, leading to production of dsRNA and thus initiating RNA silencing. What distinguishes this particular translocated RNA and marks it as a template for RdRP? One possibility is that the RNA is tagged in some way, perhaps by the set of proteins that bind to it. Interestingly, one of the genes required for transgene-induced gene silencing, SDE3 (Dalmay et al., 2001
dsRNA
Early experiments with transgenic plants revealed that gene silencing could be mediated at either the transcriptional level (TGS or transcriptional gene silencing) or at the post-transcriptional level (originally called post-transcriptional gene silencing, or PTGS, but now commonly referred to as RNA silencing) (Vaucheret and Fagard, 2001 ). Whereas RNA silencing involves sequence-specific RNA degradation in the cytoplasm, TGS involves a block in RNA synthesis in the nucleus. Both types of silencing are correlated with de novo DNA methylation: in transcribed regions in RNA silencing and in promoter regions in TGS. The sequence-specific methylation in silencing suggested the involvement of RNA–DNA or DNA–DNA interactions. Because promoter regions are not normally transcribed, DNA–DNA pairing was a favored model for TGS, and the mechanisms of RNA silencing and TGS were thought to be distinct. In the past few years, however, it has become clear that the two kinds of silencing share additional features that suggest a mechanistic link: both are induced by dsRNA and associated with the accumulation of small RNA species with homology to the inducing dsRNA (Mette et al., 2000; Sijen et al., 2001b). This possibility of a common mechanism raises the question of whether there is also a systemic signal in TGS.
Transgene-Induced TGS
The H9NP line was constructed in experiments designed to deliberately transcribe promoter sequences (Mette et al., 1999
Similar grafting experiments using transgenic line 271 suggest that the inability of TGS to move systemically is a general feature of trans-TGS. The 271 locus contains multiple copies of a plasmid carrying an nptII selectable marker under the control of the Cauliflower mosaic virus (CaMV) 19S promoter and an Nii gene in the antisense orientation under the control of the CaMV 35S promoter (Vaucheret, 1993 It is not clear why TGS is not graft-transmissible. One possibility is that dsRNA and small RNAs associated with TGS are confined to the nucleus and thus unable to move cell to cell and into the phloem. A second possibility is that TGS-associated RNAs can move but that they are not amplified in the scion and thus do not induce detectable silencing there. If amplification requires transcription of the target sequence, for example, TGS would not induce amplification because the target is a promoter, and promoter sequences are not ordinarily transcribed. A third possibility is that the dsRNA and the small RNAs that accumulate during TGS are not the mobile signal and that this signal is produced at a step in the RNA silencing pathway that is downstream of any steps in common between TGS and RNA silencing.
Virus-Induced TGS
In virus-induced TGS, it is unlikely that a systemic signal for either silencing or methylation is produced from the inactivated transgene. Indeed, Jones et al. (2001)
The discovery in 1998 that certain plant viruses encode proteins that suppress RNA silencing (Anandalakshmi et al., 1998 ; Beclin et al., 1998; Brigneti et al., 1998; Kasschau and Carrington, 1998) provided a new approach for dissecting the steps in the RNA silencing pathway. The first indication that a viral suppressor might interfere specifically with systemic RNA silencing came from reversal of silencing assays using the Cucumber mosaic virus (CMV) (Beclin et al., 1998) or the CMV 2b protein (Cmv2b) (Brigneti et al., 1998). In these assays, neither CMV nor Cmv2b was able to reverse established silencing in older tissues, but each completely suppressed the signal-mediated spread of silencing into newly emerging tissues. In contrast, HC-Pro, a viral suppressor encoded by potyviruses, was able to reverse silencing in both types of tissues (Brigneti et al., 1998). These results suggest that these two suppressors interfered with silencing at different points and that each might define a step in the silencing pathway. In this case, it appeared that Cmv2b might interfere specifically with systemic spread of silencing.
Confirmation that Cmv2b interferes with systemic silencing comes from analyses using a well-characterized transgenic tobacco line silenced for the reporter gene encoding GUS (line 6b5; Elmayan and Vaucheret, 1996
The first viral protein shown to suppress systemic silencing was the 25-kD protein (p25) encoded by PVX (Voinnet et al., 2000 ). Interestingly, PVX-expressing p25 displayed no detectable activity in the reversal of silencing assay described above for Cmv2b and HC-Pro (Brigneti et al., 1998). However, p25 activity became apparent using a newly developed assay called Agrobacterium co-infiltration. In this assay, a GFP-expressing plant is infiltrated with a mixture of two Agrobacterium cultures: one expresses GFP and thereby induces RNA silencing of the GFP transgene; the other expresses a putative suppressor of silencing. In the co-infiltration assay, expression of the putative suppressor occurs prior to or at the same time as initiation of GFP silencing. PVX p25 prevented localized GFP silencing when co-infiltrated with a simple transgene (35S-GFP) but not when co-infiltrated with Agrobacterium expressing a transgene that encodes a replication-competent PVX-GFP vector. Importantly, p25 prevented systemic silencing in both cases (Voinnet et al., 2000). The Cmv2b protein also suppresses systemic silencing in this co-infiltration assay, confirming results obtained in transgenic plants (Guo and Ding, 2002) and in the reversal of silencing assay (Brigneti et al., 1998).
Inhibition of systemic silencing by p25 and Cmv2b might occur either before or after production of the silencing signal in the co-infiltration assay because the silencing inducer and suppressor are expressed in the same cells at the same time. However, in the grafting experiments, persistent export of the GUS-specific silencing signal from 6b5 rootstocks into 6b5 x Cmv2b scions failed to induce degradation of GUS mRNA in scions (Guo and Ding, 2002
Experiments in the 6b5 silencing system have revealed a correlation between a viral suppressor's effect on systemic silencing and its effect on transgene methylation. HC-Pro blocks RNA silencing in the 6b5 plants without interfering with either systemic signaling or transgene methylation (Mallory et al., 2001
The differential suppression of intra- and intercellular RNA silencing by HC-Pro and p25/Cmv2b suggests a basis for potyvirus synergism with PVX and CMV (Pruss et al., 1997
Although the phenomenon of systemic RNA silencing was demonstrated five years ago, the identity of the silencing signal remains largely elusive. To some extent, it is likely that the difficulty in investigating this aspect of RNA silencing lies in the inherent complexity of the silencing process. Hence, what is usually described as "signaling" in transgenic plants does not refer to the physical movement of the silencing factor per se but to the many consequences of its perception in recipient cells located outside the vasculature. This perception leads ultimately to transgene mRNA degradation, which translates into a systemic silencing phenotype. But perception of the signal is also accompanied by the generation of many additional nucleic acid species, including dsRNA and siRNA, that account for the amplification and mRNA degradation steps of RNA silencing. Induction of these new species in each cell makes it very difficult to identify the genuine signaling molecule.
A second difficulty is that signaling of RNA silencing may well involve several types of molecules and transport mechanisms. For instance, a recent report suggests that systemic movement of silencing, but not cell-to-cell spread, is inhibited by nontoxic concentrations of cadmium in tobacco and N. benthamiana (Ueki and Citovsky, 2001 A fundamental difficulty with understanding systemic silencing is that the mechanisms of intercellular trafficking of macromolecules remain largely unknown. Even if the molecular identity of the signal were known at this point, we still would not understand how and why this molecule moves systemically. Studies of the molecular basis of RNA trafficking should provide a framework for understanding the rules that govern the spread of RNA silencing.
We thank B. Roth and M. Endres for providing unpublished images of systemic silencing and B. Roth and A. Mallory for assistance with the figures. We are grateful to D. Baulcombe, M. Zerbini, and L. Bowman for helpful discussions and critical reading of the manuscript. We acknowledge financial support to O.V. from the Gatsby Charitable Foundation, to M.M. from the Austrian Fonds zur förderung der wissenschaftlichen Forschung, to H.V. from the French Ministry of Research, to S.W.D. from the U.S. Department of Agriculture, NRI Competitive Grants Program, and to V.B.V. from the National Institutes of Health and the U.S. Department of Agriculture, NRI Competitive Grants Program.
Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.001677. Received January 17, 2002; accepted March 17, 2002.
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-amanitin sensitivity of the potato spindle tuber viroid replication (Flores, 2001
