The complex interplay between plant viruses and host RNA-silencing pathways

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RNA silencing was originally identified as an immune system targeted against transposons and viruses, but is now also recognized as a major regulatory process that affects all layers of host gene expression through the activities of various small RNA species. Recent work in plants and animals indicates that viruses not only suppress, but can also exploit, endogenous RNA silencing pathways to redirect host gene expression. There are also indications that cellular, as opposed to virus-derived small RNAs, might well constitute an unsuspected defense layer against foreign nucleic acids. This complex interplay has implications in the context of disease resistance and evolution of viral genomes.

Introduction

In most eukaryotes, the perception of RNA that has double-stranded (ds) features triggers its conversion, by homologs of the RNaseIII enzyme Dicer, into 21–24 nt-long RNAs [1, 2]. These small RNAs guide RNA-induced silencing complexes (RISCs) that act at the level of RNA to inhibit transcript stability or translation, or at the level of DNA to promote epigenetic modifications [3, 4]. These various processes are all manifestations of ‘RNA silencing’. In Arabidopsis, four Dicer-like enzymes (DCL1–4) account for the production of endogenous small RNA species that mediate RNA silencing [5••]. These small RNA species can be sub-divided into at least two major classes. Small interfering RNAs (siRNAs) are produced as populations from long dsRNAs that result from read-through or bi-directional transcription of DNA repeats or transposon loci, and from the action of host-encoded RNA-dependent RNA polymerases that synthesize complementary strands from cellular RNAs [6, 7]. siRNAs are also generated from transgenic pan-handled transcripts that are used to provide experimental RNA interference (RNAi) [8]. Endogenous siRNAs either direct the endonucleolytic cleavage of homologous transcripts (trans-acting siRNAs [9, 10]) or promote DNA methylation and heterochromatin formation at the genetic loci from which they originate (cis-acting siRNAs [5••]), often resulting in transcriptional gene silencing (TGS). Cis-acting siRNAs are produced in the nucleus by DCL3, whereas trans-acting siRNAs require DCL1 for their biogenesis. The DCL(s) that are involved in the execution of experimental RNAi remain unknown [5••].

MicroRNAs (miRNAs) constitute the second class of endogenous small RNAs. Those molecules are excised by DCL1 from nuclear and non-coding precursor transcripts, of approximately 70–200 nt in length, which acquire a partial stem-loop structure. Mature miRNAs are cytoplasmic and direct the cleavage or translational inhibition of mRNAs that carry discrete complementary target sites [11, 12]. Work with animal miRNAs indicates that the degree of complementarity between small RNAs and their target sequences largely influences the outcome of their interaction [13]. Inhibition of translation is favored by incomplete pairing (prevalent in animals), whereas cleavage is instigated by a perfect or near-perfect complement (prevalent in plants). The first plant miRNA targets to be identified were a series of evolutionarily conserved transcription factors that control important developmental fates [14], but recent work indicates that miRNAs regulate many other biological processes [15, 16]. Moreover, gene inversion or duplication events can generate species-specific miRNAs that probably contribute to the ability of plants to adapt to their environment [17•, 18].

The dsRNA features of plant viruses are also recognized by the host RNA-silencing machinery, such that the presence of virus-derived small RNAs and the consequent silencing of viral genes dampens the accumulation of the pathogens in a process referred to as virus-induced gene silencing (VIGS) [2, 19, 20]. Accordingly, viruses have evolved strategies to avoid or suppress this defense, one of which is the production of highly diverse suppressor proteins that target many steps of the silencing machinery (reviewed in [21]). In the most simplistic view, antiviral RNA-silencing can be perceived as yet another illustration of the continuing arms race between hosts and pathogens. In this review, we provide insights into the complex interactions between plant viruses and cellular RNA silencing pathways, and discuss how viruses not only interfere with but also exploit silencing-based regulatory networks. We also highlight recent work in plants and animals that suggest that the interplay between viruses, host RNA-silencing pathways and classical disease resistance networks might be extremely sophisticated and might have consequences that are beyond the simple scope of defense, including effects on the evolution of pathogen and, perhaps, host genomes.

Section snippets

Triggering and executing VIGS: things are not always what they seem

The small RNAs that are produced by plant viruses are usually thought to promote the endonucleolytic cleavage of pathogen RNAs upon their incorporation into a RISC complex. However, this assumption has never been strictly experimentally validated, because steady-state transcript levels rather than quantitative RNA-cleavage assays [22] are used to measure the impact of viral small RNAs. It is in fact possible that translational repression, as opposed to RNA turnover, also contributes to

Viruses not only interfere with but also exploit plant RNA-silencing pathways

A common response of plant viruses to RNA silencing is the production of suppressor proteins [30]; but by inhibiting RNA silencing of their own genomes, viruses often (although not always) also interfere with endogenous silencing pathways that regulate host gene expression. In Arabidopsis, the transgenic expression of several viral-encoded silencing suppressors causes a set of recurrent developmental abnormalities. These abnormalities have variable penetrance correlating to the degree of

Cellular small RNAs have direct and indirect antiviral functions

Several cloned small RNAs, including some with features of miRNAs, do not show sequence homology to cellular protein-encoding genes in Arabidopsis [36]. Although it is possible that these small RNAs have protein-encoding targets that have evaded computer prediction because of imperfect base-pairing, this observation has prompted the idea that some of the orphan small RNAs could constitute a reservoir of defensive molecules owing to their complementarity to invading viral genomes [37]. Recent

Host and viral small RNAs could direct the evolution of viral genomes

The recent findings highlighted in this review have profound implications for our current understanding of host–virus interactions and provide a new frame with which to investigate the impact of cellular or host polymorphisms on viral susceptibility and viral genome evolution. First, the antiviral potential of cellular small RNAs ([38••]; Figure 2) could explain, at least in part, why specific tissues are often more permissive to viruses than others: the repertoire of expressed small RNAs is

Conclusions

Although several of the ideas evoked in this review remain to be formally tested through experimentation, they provide a glimpse of the extraordinary complexity that can be expected as a result of viral interference and usurpation of host silencing pathways. In addition, the concepts discussed here might not be restricted to viruses but could, in principle, apply to other types of pathogens that exploit foreign nucleic acids as part of their infection strategy. The challenge now will be to

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

We thank Peter Brodersen for critical reading of the manuscript and Lionel Navarro for fruitful discussions. Work in our laboratory is supported by the Centre National de la Recherche Scientifique (CNRS), the Federation of European Biochemical Societies (FEBS) and the European Molecular Biological Organization (EMBO).

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