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Olivier Voinnet

Publications and source records attributed to Olivier Voinnet.

At least 19 recordsLinked to original sources
PubMed2006

RNA silencing of host transcripts by cauliflower mosaic virus requires coordinated action of the four Arabidopsis Dicer-like proteins.

RNA silencing is an ancient mechanism of gene regulation with important antiviral roles in plants and insects. Although induction of RNA silencing by RNA viruses has been well documented in plants, the interactions between DNA viruses and the host silencing machinery remain poorly understood. We investigate this question with cauliflower mosaic virus (CaMV), a dsDNA virus that expresses its genome through the polycistronic 35S RNA, which carries an unusually extensive secondary structure known as translational leader. We show that CaMV-derived siRNAs accumulate in turnip- and Arabidopsis-infected plants and that the leader is a major, albeit not exclusive, source for those molecules. Biogenesis of leader-derived siRNA requires the coordinated and hierarchical action of the four Arabidopsis Dicer-like (DCL) proteins. Our study also uncovers a "facilitating" role exerted by the microRNA biosynthetic enzyme DCL1 on accumulation of DCL2-, DCL3-, and DCL4-dependent siRNAs derived from the 35S leader. This feature of DCL1 defines a small RNA biosynthetic pathway that might have relevance for endogenous gene regulation. Several leader-derived siRNAs were found to bear near-perfect sequence complementarity to Arabidopsis transcripts, and, using a sensor transgene, we provide direct evidence that at least one of those molecules acts as a bona fide siRNA in infected turnip. Extensive bioinformatics searches identified >100 transcripts potentially targeted by CaMV-derived siRNAs, several of which are effectively down-regulated during infection. The implications of virus-directed silencing of host gene expression are discussed.

Arabidopsis↗
PubMed2006

Hierarchical action and inhibition of plant Dicer-like proteins in antiviral defense.

The mechanisms underlying induction and suppression of RNA silencing in the ongoing plant-virus arms race are poorly understood. We show here that virus-derived small RNAs produced by Arabidopsis Dicer-like 4 (DCL4) program an effector complex conferring antiviral immunity. Inhibition of DCL4 by a viral-encoded suppressor revealed the subordinate antiviral activity of DCL2. Accordingly, inactivating both DCL2 and DCL4 was necessary and sufficient to restore systemic infection of a suppressor-deficient virus. The effects of DCL2 were overcome by increasing viral dosage in inoculated leaves, but this could not surmount additional, non-cell autonomous effects of DCL4 specifically preventing viral unloading from the vasculature. These findings define a molecular framework for studying antiviral silencing and defense in plants.

Arabidopsis↗
PubMed2006

A plant miRNA contributes to antibacterial resistance by repressing auxin signaling.

Plants and animals activate defenses after perceiving pathogen-associated molecular patterns (PAMPs) such as bacterial flagellin. In Arabidopsis, perception of flagellin increases resistance to the bacterium Pseudomonas syringae, although the molecular mechanisms involved remain elusive. Here, we show that a flagellin-derived peptide induces a plant microRNA (miRNA) that negatively regulates messenger RNAs for the F-box auxin receptors TIR1, AFB2, and AFB3. Repression of auxin signaling restricts P. syringae growth, implicating auxin in disease susceptibility and miRNA-mediated suppression of auxin signaling in resistance.

Arabidopsis↗
PubMed2006

The diversity of RNA silencing pathways in plants.

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 (20-26 nt) homologous RNA molecules. Our understanding of small RNA biology has significantly improved in recent years, and it is now clear that there are several cellular silencing pathways in addition to those involved in defense. Endogenous silencing pathways have important roles in gene regulation at the transcriptional, RNA stability and translational levels. They share a common core of small RNA generator and effector proteins with multiple paralogs in plant genomes, some of which have acquired highly specialized functions. Here, we review recent developments in the plant RNA silencing field that have identified components of specific silencing pathways and have shed light on the mechanisms and biological roles of RNA silencing in plants.

Gene Silencing↗
PubMed2006

Induction, suppression and requirement of RNA silencing pathways in virulent Agrobacterium tumefaciens infections.

Regulation of gene expression through microRNAs (miRNAs) and antiviral defense through small interfering RNAs (siRNAs) are aspects of RNA silencing, a process originally discovered as an unintended consequence of plant transformation by disarmed Agrobacterium tumefaciens strains. Although RNA silencing protects cells against foreign genetic elements, its defensive role against virulent, tumor-inducing bacteria has remained unexplored. Here, we show that siRNAs corresponding to transferred-DNA oncogenes initially accumulate in virulent A. tumefaciens-infected tissues and that RNA interference-deficient plants are hypersusceptible to the pathogen. Successful infection relies on a potent antisilencing state established in tumors whereby siRNA synthesis is specifically inhibited. This inhibition has only modest side effects on the miRNA pathway, shown here to be essential for disease development. The similarities and specificities of the A. tumefaciens RNA silencing interaction are discussed and contrasted with the situation encountered with plant viruses.

Agrobacterium tumefaciens↗
PubMed2005

DICER-LIKE 4 is required for RNA interference and produces the 21-nucleotide small interfering RNA component of the plant cell-to-cell silencing signal.

In RNA interference, the RNase-III enzyme Dicer processes exogenous double-stranded RNA into small interfering RNAs (siRNAs). siRNAs guide RNA-induced silencing complexes to cleave homologous transcripts, enabling gene-specific knock-down. In plants, double-stranded RNA is processed into siRNA species of 21 nucleotides (nt) and 24 nt (ref. 5), but, unlike in nematodes, the Dicer enzymes involved in this processing have not been identified. Additionally, in both plants and nematodes, systemic signals with RNA components convey the sequence-specific effects of RNA interference between cells. Here, we describe Arabidopsis thaliana mutants with altered silencing cell-to-cell movement beyond the vasculature. At least three SILENCING MOVEMENT DEFICIENT genes (SMD1, SMD2 and SMD3) are required for trafficking, the extent of which correlates with siRNA levels in the veins. Five alleles defective in synthesis of 21-nt, but not 24-nt, siRNAs carry mutations in Dicer-like 4 (DCL4) that are involved in biogenesis of trans-acting siRNAs. We show that the biogenesis and function of trans-acting siRNA can be genetically uncoupled from a bona fide DCL4-dependent pathway that accounts for RNA interference and for production of the 21-nt siRNA component of the plant cell-to-cell silencing signal.

Alleles↗
PubMed2005

Non-cell autonomous RNA silencing.

In plants and in some animals, the effects of post-transcriptional RNA silencing can extend beyond its sites of initiation, owing to the movement of signal molecules. Although the mechanisms and channels involved are different, plant and animal silencing signals must have RNA components that account for the nucleotide sequence-specificity of their effects. Studies carried out in plants and Caenorhabditis elegans have revealed that non-cell autonomous silencing is operated through specialized, remarkably sophisticated pathways and serves important biological functions, including antiviral immunity and, perhaps, developmental patterning. Recent intriguing observations suggest that systemic RNA silencing pathways may also exist in higher vertebrates.

Animals↗
PubMed2005

A cellular microRNA mediates antiviral defense in human cells.

In eukaryotes, 21- to 24-nucleotide-long RNAs engage in sequence-specific interactions that inhibit gene expression by RNA silencing. This process has regulatory roles involving microRNAs and, in plants and insects, it also forms the basis of a defense mechanism directed by small interfering RNAs that derive from replicative or integrated viral genomes. We show that a cellular microRNA effectively restricts the accumulation of the retrovirus primate foamy virus type 1 (PFV-1) in human cells. PFV-1 also encodes a protein, Tas, that suppresses microRNA-directed functions in mammalian cells and displays cross-kingdom antisilencing activities. Therefore, through fortuitous recognition of foreign nucleic acids, cellular microRNAs have direct antiviral effects in addition to their regulatory functions.

Animals↗
PubMed2005

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

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.

Biological Evolution↗
PubMed2005

Induction and suppression of RNA silencing: insights from viral infections.

In eukaryotes, small RNA molecules engage in sequence-specific interactions to inhibit gene expression by RNA silencing. This process fulfils fundamental regulatory roles, as well as antiviral functions, through the activities of microRNAs and small interfering RNAs. As a counter-defence mechanism, viruses have evolved various anti-silencing strategies that are being progressively unravelled. These studies have not only highlighted our basic understanding of host-parasite interactions, but also provide key insights into the diversity, regulation and evolution of RNA-silencing pathways.

Animals↗
PubMed2004

In vivo investigation of the transcription, processing, endonucleolytic activity, and functional relevance of the spatial distribution of a plant miRNA.

We show, with miR171, that plant miRNA genes are modular independent transcription units in which the fold-back pre-miRNA is sufficient for miRNA processing, and that the upstream region contains highly specific promoter elements. Processing depends on flanking sequences within the miRNA stem-loop precursor rather than the miRNA sequence itself, and mutations affecting target pairing at the center and 5' but not 3' region of the miRNA compromise its function in vivo. Inactivation of the SDE1 RNA-dependent-RNA-polymerase was mandatory for accurate representation of miRNA activity by sensor constructs in Arabidopsis. Work in sde1 background revealed a near-perfect spatial overlap between the patterns of miR171 transcription and activity, supporting the idea that plant miRNAs enable cell differentiation.

Arabidopsis↗
PubMed2004

Probing the microRNA and small interfering RNA pathways with virus-encoded suppressors of RNA silencing.

In plants, small interfering RNAs (siRNAs) and microRNAs (miRNAs) are effectors of RNA silencing, a process involved in defense through RNA interference (RNAi) and in development. Plant viruses are natural targets of RNA silencing, and as a counterdefensive strategy, they have evolved highly diverse silencing suppressor proteins. Although viral suppressors are usually thought to act at distinct steps of the silencing machinery, there had been no consensus system so far that allowed a strict side-by-side analysis of those factors. We have set up such a system in Arabidopsis thaliana and used it to compare the effects of five unrelated viral silencing suppressors on the siRNA and miRNA pathways. Although all the suppressors inhibited RNAi, only three of them induced developmental defects, indicating that the two pathways are only partially overlapping. These developmental defects were remarkably similar, and their penetrance correlated with inhibition of miRNA-guided cleavage of endogenous transcripts and not with altered miRNA accumulation per se. Among the suppressors investigated, the tombusviral P19 protein coimmunoprecipitated with siRNA duplexes and miRNA duplexes corresponding to the primary cleavage products of miRNA precursors. Thus, it is likely that P19 prevents RNA silencing by sequestering both classes of small RNAs. Moreover, the finding here that P19 binds siRNAs and suppresses RNAi in Hela cells also suggests that this factor may be useful to dissect the RNA silencing pathways in animals. Finally, the differential effects of the silencing suppressors tested here upon other types of Arabidopsis silencing-related small RNAs revealed a surprising variety of biosynthetic and, presumably, functional pathways for those molecules. Therefore, silencing suppressors are valuable probes of the complexity of RNA silencing.

Arabidopsis↗
PubMed2004

RNA silencing: no mercy for viruses?

'RNA silencing' is a highly conserved mechanism leading to suppression of gene expression through nucleotide sequence-specific interactions that are mediated by 21-24 nucleotide-long RNAs. This process was first discovered as an unexpected consequence of transgenesis in plants, and similarly, it was subsequently identified in animals as an anomaly of antisense gene inhibition. We summarize the progressive steps that paved the way to our current understanding of the molecular basis and fundamental biological roles of RNA silencing in both plants and animals. In particular, we describe the general antiviral function of this mechanism in higher plants where it forms the basis of a highly elaborate immune system. All defense systems show some level of fallibility, and RNA silencing is no exception to this rule, as plant viruses have developed sophisticated ways to counteract various steps of the process. Recent work indicates that viruses are also engaged into a similar arms race in insects, but it remains unclear if RNA silencing plays a defensive role against virus infection of higher vertebrates. We also discuss some biotechnological applications of RNA silencing in mammalian cells that have fueled optimism that this mechanism may hold a promising future in antiviral human therapy.

Animals↗
PubMed2003

Transitivity-dependent and -independent cell-to-cell movement of RNA silencing.

One manifestation of RNA silencing, known as post-transcriptional gene silencing (PTGS) in plants and RNA interference (RNAi) in animals, is a nucleotide sequence-specific RNA turnover mechanism with the outstanding property of propagating throughout the organism, most likely via movement of nucleic acids. Here, the cell-to-cell movement of RNA silencing in plants is investigated. We show that a short-distance movement process, once initiated from a small group of cells, can spread over a limited and nearly constant number of cells, independent of the presence of homologous transcripts. There is also a long-range cell-to-cell movement process that occurs as a relay amplification, which requires the combined activity of SDE1, a putative RNA-dependent RNA polymerase, and SDE3, a putative RNA helicase. Extensive and limited cell-to-cell movements of silencing are triggered by the same molecules, occur within the same tissues and likely recruit the same plasmodesmata channels. We propose that they are in fact manifestations of the same process, and that extensive cell-to-cell movement of RNA silencing results from re-iterated short-distance signalling events. The likely nature of the nucleic acids involved is presented.

Animals↗
PubMed2003

RNA silencing bridging the gaps in wheat extracts.

In plants, RNA silencing plays important roles in antiviral defence, genome integrity and development. This process involves nucleotide sequence-specific interactions that are mediated by small RNA molecules of 21-25 nucleotides. Although the core biochemical reactions of RNA silencing have been well characterized in animals, such information was crucially missing in plants. Recent work now addresses this question and reveals an overall similarity between the plant and animal RNA-silencing pathways, as well as some intriguing plant-specific aspects.

Animals↗
PubMed2003

An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus.

Transient gene expression is a fast, flexible and reproducible approach to high-level expression of useful proteins. In plants, recombinant strains of Agrobacterium tumefaciens can be used for transient expression of genes that have been inserted into the T-DNA region of the bacterial Ti plasmid. A bacterial culture is vacuum-infiltrated into leaves, and upon T-DNA transfer, there is ectopic expression of the gene of interest in the plant cells. However, the utility of the system is limited because the ectopic protein expression ceases after 2-3 days. Here, we show that post-transcriptional gene silencing (PTGS) is a major cause for this lack of efficiency. We describe a system based on co-expression of a viral-encoded suppressor of gene silencing, the p19 protein of tomato bushy stunt virus (TBSV), that prevents the onset of PTGS in the infiltrated tissues and allows high level of transient expression. Expression of a range of proteins was enhanced 50-folds or more in the presence of p19 so that protein purification could be achieved from as little as 100 mg of infiltrated leaf material. The effect of p19 was not saturated in cells that had received up to four individual T-DNAs and persisted until leaf senescence. Because of its simplicity and rapidity, we anticipate that the p19-enhanced expression system will have value in industrial production as well as a research tool for isolation and biochemical characterisation of a broad range of proteins without the need for the time-consuming regeneration of stably transformed plants.

Gene Expression↗