Epigenetics, Gene Silencing, RNAi

Plant Genome Diversity Volume 1: Plant Genomes, Their Residents, and Their Evolutionary Dynamics (2012)
Chap. 16: Evolutionary Significance of Epigenetic Variation
Christina L. Richards, Koen, J.F. Verhoeven, and Oliver Bossdorf
Environmental induction of epigenetic variation that is stably inherited across generations seems to be particularly important in plant biology. One explanation for this might be that in plants, unlike in mammals, the germ line is separated from somatic tissue at a much later developmental stage (Jablonka and Lamb 1989,  Wessler 1996). As a result, there might be more opportunities for plants to transmit epigenetic modifications to offspring that were acquired in somatic tissue during the plant's life, including environmentally-induced epigenetic modifications. Several studies have used multi-generation experiments to show that parental exposure to biotic or abiotic stresses resulted in modified DNA methylation in unexposed offspring in tobacco (Boyko et al. 2007), dandelion (Verhoeven et al. 2010a) and A. thaliana (Boyko et al. 2010). For example, Verhoeven et al. (2010a) used methylation sensitive amplified fragment length polymorphism (MS-AFLP) to show that plants with identical genotypes exposed to different stresses had more changes in methylation sensitive markers compared to control (Fig. 16.5). In particular, chemical induction of herbivore and pathogen defenses triggered considerable methylation variation throughout the genome. Although some of these methylation differences reverted back to the original in the next generation of plants grown in common environment. Verhoeven et al. (2010a) provide some of the first evidence that the majority of the stress-induced changes in methylation are inherited in the next generation.

Mol Gen Genet. 1987 Oct ;209 (3):552-62
Cloning of DNA fragments complementary to tobacco nitrate reductase mRNA and encoding epitopes common to the nitrate reductases from higher plants.
R Calza, E Huttner, M Vincentz, P Rouzé, F Galangau, H Vaucheret, I Chérel, C Meyer, J Kronenberger, M Caboche
Messenger RNAs encoding the nitrate reductase apoenzyme from tobacco can be translated in a cell-free system. Poly(A)+ mRNA fractions from the 23-32 S area of a sucrose gradient were used to build a cDNA library in the expression vector gt11 with an efficiency of cloning of approximately 10(4) recombinants/ng mRNA. Recombinant clones were screened with a rabbit polyclonal antibody directed against the corn nitrate reductase, which cross reacts specifically with the nitrate reductases from dicotyledons. Among 240000 recombinant plaques, eight clones were isolated containing inserts of sizes ranging from 1.6 kb to 2.1 kb and sharing sequence homologies. Seven of these clones contained a common internal 1.6 kb EcoRI fragment. The identity of these clones was confirmed as follows. A fusion protein of 170 kDa inducible by IPTG and recognized by the rabbit nitrate reductase antibody was expressed by a lysogen derived from one of the recombinants. The antibodies binding the fused protein were eluted and shown to be inhibitory to the catalytic activity of tobacco nitrate reductase. Two monoclonal antibodies directed against nitrate reductase were also able to bind the hybrid protein. The 1.6 kb EcoRI fragment was sequenced by the method of Sanger. The open reading frame corresponding to a translational fusion with the galactosidase coding sequence of the vector shared strong homology at the amino acid level with the heme-binding domain of proteins of the cytochrome b5 superfamily and with human erythrocyte cytochrome b5 reductase. When the 1.6 kb EcoRI fragment was used as a probe for Northern blot experiments a signal corresponding to a 3.5 kb RNA was detected in tobacco and in Nicotiana plumbaginifolia mRNA preparations but no cross-hybridization with corn mRNAs was detected. The probe hybridized with low copy number sequences in genomic blots of tobacco DNA.

Plant Genome IV Conference; Town & Country Conference Center, San Diego, CA, January, 1995.
Homology-Dependent Gene Silencing in Transgenic Plants
M.A. Matzke, Y.D. Park, I. Papp, V.A. Iglesias, E.A. Moscone, H. Vaucheret and A.J.M. Matzke
Homology-dependent gene silencing can be observed when multiple copies of a transgene or a transgene with homology to an endogenous plant gene are present in a plant genome. Both linked and unlinked copies can be affected. Two general types of silencing, involving either transcriptional inactivation or a post-transcriptional process, have been identified. The former has been associated with homology in promoter regions, increased methylation, and meiotically heritable reductions in gene activity, while the latter requires homology in the protein coding region and does not usually lead to heritable alterations in either methylation or gene expression. Different silencing and target loci that exemplify each of these silencing effects will be described. Comparisons will be made with nontransgenic plant and fungal systems that exhibit similar behavior. The possibility that homology-dependent interactions between noncoding repeats play a role in regulating eukaryotic gene expression by silencing large portions of genomes will be discussed.

Annu. Rev. Plant Physiol. Plant Mol. Biol. 2000. 51: 167-94.
(Trans)gene silencing in plants: How many mechanisms?
M Fagard and H Vaucheret
Epigenetic silencing of transgenes and endogenous genes can occur at the transcriptional level (TGS) or at the posttranscriptional level (PTGS). Because they can be induced by transgenes and viruses, TGS and PTGS probably reflect alternative (although not exclusive) responses to two important stress factors that the plant's genome has to face: the stable integration of additional DNA into chromosomes and the extrachromosomal replication of a viral genome. TGS, which results from the impairment of transcription initiation through methylation and/or chromatin condensation, could derive from the mechanisms by which transposed copies of mobile elements and T-DNA insertions are tamed. PTGS, which results from the degradation of mRNA when aberrant sense, antisense, or double-stranded forms of RNA are produced, could derive from the process of recovery by which cells eliminate pathogens (RNA viruses) or their undesirable products (RNA encoded by DNA viruses). Mechanisms involving DNA-DNA, DNA-RNA, or RNA-RNA interactions are discussed to explain the various pathways for triggering (trans)gene silencing in plants.

J Cell Sci. 2001 Sep;114(Pt 17):3083-91.
Post-transcriptional gene silencing in plants.
Vaucheret H, Beclin C, Fagard M.
Post-transcriptional gene silencing (PTGS) in plants is an RNA-degradation mechanism that shows similarities to RNA interference (RNAi) in animals. Indeed, both involve double-stranded RNA (dsRNA), spread within the organism from a localised initiating area, correlate with the accumulation of small interfering RNA (siRNA) and require putative RNA-dependent RNA polymerases, RNA helicases and proteins of unknown functions containing PAZ and Piwi domains. However, some differences are evident. First, PTGS in plants requires at least two genes—SGS3 (which encodes a protein of unknown function containing a coil-coiled domain) and MET1 (which encodes a DNA-methyltransferase)—that are absent in C. elegans and thus are not required for RNAi. Second, all Arabidopsis mutants that exhibit impaired PTGS are hypersusceptible to infection by the cucumovirus CMV, indicating that PTGS participates in a mechanism for plant resistance to viruses. Interestingly, many viruses have developed strategies to counteract PTGS and successfully infect plants—for example, by potentiating endogenous suppressors of PTGS. Whether viruses can counteract RNAi in animals and whether endogenous suppressors of RNAi exist in animals is still unknown.

Genes Dev. 2006 Dec 15;20 (24):3407-25
A diverse and evolutionarily fluid set of microRNAs in Arabidopsis thaliana.
Ramya Rajagopalan, Hervé Vaucheret, Jerry Trejo, David P Bartel
To better understand the diversity of small silencing RNAs expressed in plants, we employed high-throughput pyrosequencing to obtain 887,000 reads corresponding to Arabidopsis thaliana small RNAs. They represented 340,000 unique sequences, a substantially greater diversity than previously obtained in any species. Most of the small RNAs had the properties of heterochromatic small interfering RNAs (siRNAs) associated with DNA silencing in that they were preferentially 24 nucleotides long and mapped to intergenic regions. Their density was greatest in the proximal and distal pericentromeric regions, with only a slightly preferential propensity to match repetitive elements. Also present were 38 newly identified microRNAs (miRNAs) and dozens of other plausible candidates. One miRNA mapped within an intron of DICER-LIKE 1 (DCL1), suggesting a second homeostatic autoregulatory mechanism for DCL1 expression; another defined the phase for siRNAs deriving from a newly identified trans-acting siRNA gene (TAS4); and two depended on DCL4 rather than DCL1 for their accumulation, indicating a second pathway for miRNA biogenesis in plants. More generally, our results revealed the existence of a layer of miRNA-based control beyond that found previously that is evolutionarily much more fluid, employing many newly emergent and diverse miRNAs, each expressed in specialized tissues or at low levels under standard growth conditions.

Planta. 2007 Jan;225(2):365-79.
A single transgene locus triggers both transcriptional and post-transcriptional silencing through double-stranded RNA production.
Philippe Mourrain, Rik van Blokland, Jan Kooter, Hervé Vaucheret
Silencing of a target locus by an unlinked silencing locus can result from transcription inhibition (transcriptional gene silencing; TGS) or mRNA degradation (post-transcriptional gene silencing; PTGS), owing to the production of double-stranded RNA (dsRNA) corresponding to promoter or transcribed sequences, respectively. The involvement of distinct cellular components in each process suggests that dsRNA-induced TGS and PTGS likely result from the diversification of an ancient common mechanism. However, a strict comparison of TGS and PTGS has been difficult to achieve because it generally relies on the analysis of distinct silencing loci. We describe a single transgene locus that triggers both TGS and PTGS, owing to the production of dsRNA corresponding to promoter and transcribed sequences of different target genes. We describe mutants and epigenetic variants derived from this locus and propose a model for the production of dsRNA. Also, we show that PTGS, but not TGS, is graft-transmissible, which together with the sensitivity of PTGS, but not TGS, to RNA viruses that replicate in the cytoplasm, suggest that the nuclear compartmentalization of TGS is responsible for cell-autonomy. In contrast, we contribute local and systemic trafficking of silencing signals and sensitivity to viruses to the cytoplasmic steps of PTGS and to amplification steps that require high levels of target mRNAs.

EMBO J. 2006 Jul 26;25 (14):3347-56
An antagonistic function for Arabidopsis DCL2 in development and a new function for DCL4 in generating viral siRNAs.
Nicolas Bouché, Dominique Lauressergues, Virginie Gasciolli, Hervé Vaucheret
Plants contain more DICER-LIKE (DCL) enzymes and double-stranded RNA binding (DRB) proteins than other eukaryotes, resulting in increased small RNA network complexities. Analyses of single, double, triple and quadruple dcl mutants exposed DCL1 as a sophisticated enzyme capable of producing both microRNAs (miRNAs) and siRNAs, unlike the three other DCLs, which only produce siRNAs. Depletion of siRNA-specific DCLs results in unbalanced small RNA levels, indicating a redeployment of DCL/DRB complexes. In particular, DCL2 antagonizes the production of miRNAs and siRNAs by DCL1 in certain circumstances and affects development deleteriously in dcl1 dcl4 and dcl1 dcl3 dcl4 mutant plants, whereas dcl1 dcl2 dcl3 dcl4 quadruple mutant plants are viable. We also show that viral siRNAs are produced by DCL4, and that DCL2 can substitute for DCL4 when this latter activity is reduced or inhibited by viruses, pointing to the competitiveness of DCL2. Given the complexity of the small RNA repertoire in plants, the implication of each DCL, in particular DCL2, in the production of small RNAs that have no known function will constitute one of the next challenges.

Nat Genet. 2006 Jul ;38 (7):850
Erratum: Functions of microRNAs and related small RNAs in plants.
Allison C Mallory, Hervé Vaucheret
In the version of this article initially published, two labels depicting methylation (CH3) were inadvertently omitted from the miRNA duplex shown below HEN1 in Figure 1. In addition, the arcs accompanying the pie chart in Figure 2 were misaligned. Corrected figures are shown here. These errors have been corrected in the HTML and PDF versions of the article.

Nat Genet. 2006 Jun ;38 Suppl 1 :S31-6
Functions of microRNAs and related small RNAs in plants.
Allison C Mallory, Hervé Vaucheret
MicroRNAs (miRNAs) and short interfering RNAs (siRNAs), 20- to 27-nt in length, are essential regulatory molecules that act as sequence-specific guides in several processes in most eukaryotes (with the notable exception of the yeast Saccharomyces cerevisiae). These processes include DNA elimination, heterochromatin assembly, mRNA cleavage and translational repression. This review focuses on the regulatory roles of plant miRNAs during development, in the adaptive response to stresses and in the miRNA pathway itself. This review also covers the regulatory roles of two classes of endogenous plant siRNAs, ta-siRNAs and nat-siRNAs, which participate in post-transcriptional control of gene expression.

Curr Biol. 2006 May 9;16 (9):927-32
DRB4-Dependent TAS3 trans-Acting siRNAs Control Leaf Morphology through AGO7.
Xavier Adenot, Taline Elmayan, Dominique Lauressergues, Stéphanie Boutet, Nicolas Bouché, Virginie Gasciolli, Hervé Vaucheret
trans-acting siRNAs (ta-siRNAs) are endogenous RNAs that direct the cleavage of complementary mRNA targets . TAS gene transcripts are cleaved by miRNAs; the cleavage products are protected against degradation by SGS3, copied into dsRNA by RDR6, and diced into ta-siRNAs by DCL4 . We describe hypomorphic rdr6 and sgs3 Arabidopsis mutants, which do not exhibit the leaf developmental defects observed in null mutants and which, like null alleles, are impaired in sense-transgene-induced posttranscriptional gene silencing and virus resistance. Null rdr6 and sgs3 mutants lack TAS1, TAS2, and TAS3 ta-siRNAs and overaccumulate ARF3/ETTIN and ARF4 mRNAs, which are TAS3 ta-siRNA targets. A hypomorphic rdr6 mutant accumulates wild-type TAS3 ta-siRNA levels but not TAS1 and TAS2 ta-siRNAs, suggesting that TAS3 is required for proper leaf development. Consistently, tas3 but not tas1 or tas2 mutants exhibits leaf morphology defects, and ago7/zip and drb4 mutants, which exhibit leaf morphology defects, lack TAS3 but not TAS1 and TAS2 ta-siRNAs in leaves. These results indicate that the dsRNA binding protein DRB4 is required for proper ta-siRNA production, presumably by interacting with DCL4, an interaction analogous to that of HYL1 with DCL1 during miRNA production, and that TAS3 ta-siRNAs are required for proper leaf development through the action of AGO7/ZIPPY.

Genes Dev. 2006 Apr 1;20 (7):759-771
Post-transcriptional small RNA pathways in plants: mechanisms and regulations.
Hervé Vaucheret
Small RNAs are riboregulators that have critical roles in most eukaryotes. They repress gene expression by acting either on DNA to guide sequence elimination and chromatin remodeling, or on RNA to guide cleavage and translation repression. This review focuses on the various types of post-transcriptional small RNA-directed pathways in plants, describing their roles and their regulations.

Mol Cell. 2006 Apr 7;22 (1):129-36
AGO1 Homeostasis Entails Coexpression of MIR168 and AGO1 and Preferential Stabilization of miR168 by AGO1.
Hervé Vaucheret, Allison C Mallory, David P Bartel
Arabidopsis ARGONAUTE1 (AGO1) encodes the RNA slicer enzyme of the microRNA (miRNA) pathway and is regulated by miR168-programmed, AGO1-catalyzed mRNA cleavage. Here, we describe two additional regulatory processes required for AGO1 homeostasis: transcriptional coregulation of MIR168 and AGO1 genes, and posttranscriptional stabilization of miR168 by AGO1. Disrupting any of these regulatory processes by using mutations or transgenes disturbs a proper functioning of the miRNA pathway. In contrast, minor perturbation leads to fine-tuned posttranscriptional adjustment of miR168 and AGO1 levels, thereby maintaining a proper balance of other miRNAs, which, together with AGO1, control the mRNA levels of miRNA targets. We suggest that miR168 stabilization occurs at the level of silencing-complex assembly and that modulating the efficiency of assembling miRNA-programmed silencing complexes will also be important in other contexts.

Curr Biol. 2005 Nov 8;15 (21):1919-25
Arabidopsis RPA2: A Genetic Link among Transcriptional Gene Silencing, DNA Repair, and DNA Replication.
Taline Elmayan, Florence Proux, Hervé Vaucheret
Transcriptional gene silencing (TGS) controls the expression of transposable elements and of endogenous genes containing promoter repeats, and it is associated with increased DNA methylation. TGS-deficient mutants impaired in siRNA accumulation and/or chromatin modification (ago4, bru1, cmt3, dcl3, ddm1, drd1, drm2, fas1, fas2, hda6, hog1, met1, mom1, nrpd1a, nrpd1b, nrpd2a, rdr2, suvh2, and suvh4) have been identified, but not all mutations affect the same subset of targets []. Here, we identify Arabidopsis RPA2, a conserved protein with DNA replication and DNA repair motifs [], as a novel TGS component that is dispensable for endogenous small RNA accumulation. bru1, cmt3, ddm1, fas1, fas2, hda6, hog1, met1, mom1, and rpa2 mutants are impaired in TGS of dispersed Athila/TSI retrotransposons and of the transgene repeat locus L5, but unlike bru1, cmt3, ddm1, fas1, fas2, hda6, hog1, and met1, the rpa2 and mom1 mutants do not affect the accumulation of 5S-derived siRNAs. Like BRU1, FAS1, FAS2, and MOM1, RPA2 is dispensable for DNA methylation, and rpa2, bru1, fas1, and fas2, but not mom1, mutants are hypersensitive to the DNA damage agent MMS. These results suggest a coordination of the TGS machinery with DNA replication, repair, or recombination machinery at some loci, and they emphasize the diversification of the TGS pathway.

Plant Cell. 2002 ;14 Suppl :S289-301
RNA silencing and the mobile silencing signal.
Sizolwenkosi Mlotshwa, Olivier Voinnet, M Florian Mette, Marjori Matzke, Herve Vaucheret, Shou Wei Ding, Gail Pruss, Vicki B Vance
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).

Plant J. 2004 Jun ;38 (6):1004-14
Geminivirus VIGS of endogenous genes requires SGS2/SDE1 and SGS3 and defines a new branch in the genetic pathway for silencing in plants.
Nooduan Muangsan, Christophe Beclin, Herve Vaucheret, Dominique Robertson
Summary Virus-induced gene silencing (VIGS) is a sequence-specific RNA degradation process that can be used to downregulate plant gene expression. Both RNA and DNA viruses have been used for VIGS, but they differ in their mode of replication, gene expression, and cellular location. This study examined silencing mediated by a DNA virus, cabbage leaf curl virus (CaLCuV), in several silencing-deficient Arabidopsis mutants. A DNA VIGS vector derived from CaLCuV, which silenced chlorata42 (ChlI) needed for chlorophyll formation, was used to test endogenous gene silencing responses in suppressor of gene silencing (sgs)1, sgs2, sgs3, and Argonaute (ago)1 mutants defective in sense transgene-mediated post-transcriptional silencing (S-PTGS). SGS2/silencing defective (SDE)1, SGS3, and AGO1 are each dispensable for silencing mediated by transgenes containing inverted repeats (IR-PTGS), and SGS2/SDE1 is dispensable for RNA VIGS. We show that DNA VIGS requires both SGS2/SDE1 and SGS3, regardless of the orientation of 362 nt ChlI transcripts produced from the viral DNA promoter. Viral DNA accumulation is slightly higher, and viral symptoms increase in sgs2 and sgs3, whereas overexpression of SGS2/SDE1 mRNA results in decreased viral symptoms. Mutants affected in SGS1 and AGO1 function are only delayed in the onset of silencing, and have a small effect on chlorophyll accumulation. DNA VIGS is unaffected in defective DNA methylation (ddm)1/somniferous (som)8 and maintenance of methylation (mom)1 mutants, impaired for TGS. These results demonstrate that SGS2/SDE1 and SGS3 are needed for endogenous gene silencing from DNA viruses, and suggest that SGS2/SDE1 may reduce geminivirus symptoms by targeting viral mRNAs.

PLoS One. 2012 ;7 (1):e29785
RDR2 Partially Antagonizes the Production of RDR6-Dependent siRNA in Sense Transgene-Mediated PTGS.
Vincent Jauvion, Maud Rivard, Nathalie Bouteiller, Taline Elmayan, Hervé Vaucheret
Institut Jean-Pierre Bourgin, INRA, Versailles, France.
RNA-DEPENDENT RNA POLYMERASE6 (RDR6) and SUPPRESSOR of GENE SILENCING 3 (SGS3) are required for DNA methylation and post-transcriptional gene silencing (PTGS) mediated by 21-nt siRNAs produced by sense transgenes (S-PTGS). In contrast, RDR2, but not RDR6, is required for DNA methylation and TGS mediated by 24-nt siRNAs, and for cell-to-cell spreading of IR-PTGS mediated by 21-nt siRNAs produced by inverted repeat transgenes under the control of a phloem-specific promoter. PRINCIPAL FINDINGS In this study, we examined the role of RDR2 and RDR6 in S-PTGS. Unlike RDR6, RDR2 is not required for DNA methylation of transgenes subjected to S-PTGS. RDR6 is essential for the production of siRNAs by transgenes subjected to S-PTGS, but RDR2 also contributes to the production of transgene siRNAs when RDR6 is present because rdr2 mutations reduce transgene siRNA accumulation. However, the siRNAs produced via RDR2 likely are counteractive in wildtype plants because impairement of RDR2 increases S-PTGS efficiency at a transgenic locus that triggers limited silencing, and accelerates S-PTGS at a transgenic locus that triggers efficient silencing. CONCLUSIONS/SIGNIFICANCE These results suggest that RDR2 and RDR6 compete for RNA substrates produced by transgenes subjected to S-PTGS. RDR2 partially antagonizes RDR6 because RDR2 action likely results in the production of counteractive siRNA. As a result, S-PTGS efficiency is increased in rdr2 mutants.

PLoS One. 2011 ;6 (12):e28729
AGO1 and AGO2 Act Redundantly in miR408-Mediated Plantacyanin Regulation.
Nicolas Maunoury, Hervé Vaucheret
Institut Jean-Pierre Bourgin, INRA, Versailles, France.
In Arabidopsis, AGO1 and AGO2 associate with small RNAs that exhibit a Uridine and an Adenosine at their 5' end, respectively. Because most plant miRNAs have a 5'U, AGO1 plays many essential roles in miRNA-mediated regulation of development and stress responses. In contrast, AGO2 has only been implicated in antibacterial defense in association with miR393*, which has a 5'A. AGO2 also participates in antiviral defense in association with viral siRNAs. This study reveals that miR408, which has a 5'A, regulates its target Plantacyanin through either AGO1 or AGO2. Indeed, neither ago1 nor ago2 single mutations abolish miR408-mediated regulation of Plantacyanin. Only an ago1 ago2 double mutant appears compromised in miR408-mediated regulation of Plantacyanin, suggesting that AGO1 and AGO2 have redundant roles in this regulation. Moreover, the nature of the 5' nucleotide of miR408 does not appear essential for its regulatory role because both a wildtype 5'A-MIR408 and a mutant 5'U-MIR408 gene complement a mir408 mutant. These results suggest that miR408 associates with both AGO1 and AGO2 based on criteria that differ from the 5' end rule, reminiscent of miR390-AGO7 and miR165/166-AGO10 associations, which are not based on the nature of the 5' nucleotide.

Cell Res. 2011 Oct 25;: 22025251
Ingested plant miRNAs regulate gene expression in animals.
Hervé Vaucheret, Yves Chupeau
Institut Jean-Pierre Bourgin, INRA, 78000 Versailles, France.
The incidence of genetic material or epigenetic information transferred from one organism to another is an important biological question. A recent study demonstrated that plant small RNAs acquired orally through food intake directly influence gene expression in animals after migration through the plasma and delivery to specific organs.

Nucleic Acids Res. 2011 Nov;39(21):9339-44. Epub 2011 Aug 3.
The miRNA pathway limits AGO1 availability during siRNA-mediated PTGS defense against exogenous RNA.
Angel Emilio Martínez de Alba, Vincent Jauvion, Allison C Mallory, Nathalie Bouteiller, Hervé Vaucheret
Institut Jean-Pierre Bourgin, UMR1318, INRA, 78026 Versailles Cedex, France.
In plants, most microRNAs (miRNAs) and several endogenous small interfering RNAs (siRNAs) bind to ARGONAUTE1 (AGO1) to regulate the expression of endogenous genes through post-transcriptional gene silencing (PTGS). AGO1 also participates in a siRNA-mediated PTGS defense response that thwarts exogenous RNA deriving from viruses and transgenes. Here, we reveal that plants supporting transgene PTGS exhibit increased levels of AGO1 protein. Moreover, increasing AGO1 levels either by mutating miRNA pathway components or, more specifically, by impairing miR168-directed regulation of AGO1 mRNA leads to increased PTGS efficiency, indicating that the miRNA pathway dampens the efficiency of PTGS, likely by limiting the availability of AGO1. We propose that during the transgene PTGS initiation phase, transgene siRNAs and endogenous siRNAs and miRNA compete to bind to AGO1, leading to a transient reduction in AGO1-miR168 complexes and a decline in AGO1 mRNA cleavage. The concomitant increase in AGO1 protein levels would facilitate the formation of AGO1-transgene siRNA complexes and the entry into the PTGS amplification phase. We suggest that the miRNA pathway imposes an important limitation on PTGS efficiency, which could help protect endogenous mRNAs from being routinely targeted by PTGS.

RNA. 2011 Aug ;17 (8):1502-10
Double-stranded RNA binding proteins DRB2 and DRB4 have an antagonistic impact on polymerase IV-dependent siRNA levels in Arabidopsis.
Thierry Pélissier, Marion Clavel, Cristian Chaparro, Marie-Noëlle Pouch-Pélissier, Hervé Vaucheret, Jean-Marc Deragon
Université de Perpignan Via Domitia, CNRS UMR5096 LGDP, 66860 Perpignan Cedex, France.
Biogenesis of the vast majority of plant siRNAs depends on the activity of the plant-specific RNA polymerase IV (PolIV) enzyme. As part of the RNA-dependent DNA methylation (RdDM) process, PolIV-dependent siRNAs (p4-siRNAs) are loaded onto an ARGONAUTE4-containing complex and guide de novo DNA methyltransferases to target loci. Here we show that the double-stranded RNA binding proteins DRB2 and DRB4 are required for proper accumulation of p4-siRNAs. In flowers, loss of DRB2 results in increased accumulation of p4-siRNAs but not ta-siRNAs, inverted repeat (IR)-derived siRNAs, or miRNA. Loss of DRB2 does not impair uniparental expression of p4-dependent siRNAs in developing endosperm, indicating that p4-siRNA increased accumulation is not the result of the activation of the polIV pathway in the male gametophyte. In contrast to drb2, drb4 mutants exhibit reduced p4-siRNA levels, but the extent of this reduction is variable, according to the nature and size of the p4-siRNAs. Loss of DRB4 also leads to a spectacular increase of p4-independent IR-derived 24-nt siRNAs, suggesting a reallocation of factors from p4-dependent to p4-independent siRNA pathways in drb4. Opposite effects of drb2 and drb4 mutations on the accumulation of p4-siRNAs were also observed in vegetative tissues. Moreover, transgenic plants overexpressing DRB2 mimicked drb4 mutants at the morphological and molecular levels, confirming the antagonistic roles of DRB2 and DRB4.

Plant Cell. 2011 Apr ;23 (4):1625-38
The 21-nucleotide, but not 22-nucleotide, viral secondary small interfering RNAs direct potent antiviral defense by two cooperative argonautes in Arabidopsis thaliana.
Xian-Bing Wang, Juan Jovel, Petchthai Udomporn, Ying Wang, Qingfa Wu, Wan-Xiang Li, Virginie Gasciolli, Herve Vaucheret, Shou-Wei Ding
Department of Plant Pathology and Microbiology, University of California, Riverside, California 92521, USA.
Arabidopsis thaliana defense against distinct positive-strand RNA viruses requires production of virus-derived secondary small interfering RNAs (siRNAs) by multiple RNA-dependent RNA polymerases. However, little is known about the biogenesis pathway and effector mechanism of viral secondary siRNAs. Here, we describe a mutant of Cucumber mosaic virus (CMV-Δ2b) that is silenced predominantly by the RNA-DEPENDENT RNA POLYMERASE6 (RDR6)-dependent viral secondary siRNA pathway. We show that production of the viral secondary siRNAs targeting CMV-Δ2b requires SUPPRESSOR OF GENE SILENCING3 and DICER-LIKE4 (DCL4) in addition to RDR6. Examination of 25 single, double, and triple mutants impaired in nine ARGONAUTE (AGO) genes combined with coimmunoprecipitation and deep sequencing identifies an essential function for AGO1 and AGO2 in defense against CMV-Δ2b, which act downstream the biogenesis of viral secondary siRNAs in a nonredundant and cooperative manner. Our findings also illustrate that dicing of the viral RNA precursors of primary and secondary siRNA is insufficient to confer virus resistance. Notably, although DCL2 is able to produce abundant viral secondary siRNAs in the absence of DCL4, the resultant 22-nucleotide viral siRNAs alone do not guide efficient silencing of CMV-Δ2b. Possible mechanisms for the observed qualitative difference in RNA silencing between 21- and 22-nucleotide secondary siRNAs are discussed.

PLoS One. 2011 ;6 (2):e16724
A novel fry1 allele reveals the existence of a mutant phenotype unrelated to 5'->3' exoribonuclease (XRN) activities in Arabidopsis thaliana roots.
Judith Hirsch, Julie Misson, Peter A Crisp, Pascale David, Vincent Bayle, Gonzalo M Estavillo, Hélène Javot, Serge Chiarenza, Allison C Mallory, Alexis Maizel, Marie Declerck, Barry J Pogson, Hervé Vaucheret, Martin Crespi, Thierry Desnos, Marie-Christine Thibaud, Laurent Nussaume, Elena Marin
CEA, DSV IBEB, Laboratoire de Biologie du Développement des Plantes, UMR 6191 CNRS, CEA, Aix-Marseille II, Saint-Paul-lez-Durance, France.
Mutations in the FRY1/SAL1 Arabidopsis locus are highly pleiotropic, affecting drought tolerance, leaf shape and root growth. FRY1 encodes a nucleotide phosphatase that in vitro has inositol polyphosphate 1-phosphatase and 3',(2'),5'-bisphosphate nucleotide phosphatase activities. It is not clear which activity mediates each of the diverse biological functions of FRY1 in planta. A fry1 mutant was identified in a genetic screen for Arabidopsis mutants deregulated in the expression of Pi High affinity Transporter 1;4 (PHT1;4). Histological analysis revealed that, in roots, FRY1 expression was restricted to the stele and meristems. The fry1 mutant displayed an altered root architecture phenotype and an increased drought tolerance. All of the phenotypes analyzed were complemented with the AHL gene encoding a protein that converts 3'-polyadenosine 5'-phosphate (PAP) into AMP and Pi. PAP is known to inhibit exoribonucleases (XRN) in vitro. Accordingly, an xrn triple mutant with mutations in all three XRNs shared the fry1 drought tolerance and root architecture phenotypes. Interestingly these two traits were also complemented by grafting, revealing that drought tolerance was primarily conferred by the rosette and that the root architecture can be complemented by long-distance regulation derived from leaves. By contrast, PHT1 expression was not altered in xrn mutants or in grafting experiments. Thus, PHT1 up-regulation probably resulted from a local depletion of Pi in the fry1 stele. This hypothesis is supported by the identification of other genes modulated by Pi deficiency in the stele, which are found induced in a fry1 background. Our results indicate that the 3',(2'),5'-bisphosphate nucleotide phosphatase activity of FRY1 is involved in long-distance as well as local regulatory activities in roots. The local up-regulation of PHT1 genes transcription in roots likely results from local depletion of Pi and is independent of the XRNs.

Plant Cell. 2010 Dec ;22 (12):3879-89
Form, function, and regulation of ARGONAUTE proteins.
Allison Mallory, Hervé Vaucheret
Institut Jean-Pierre Bourgin, Unité Mixte de Recherche 1318, Institut National de la Recherche Agronomique, 78026 Versailles Cedex, France. allison.mallory@versailles.inra.fr
Both transcriptional (TGS) and posttranscriptional gene silencing (PTGS) are conserved eukaryotic gene regulatory mechanisms, integral for taming exogenous (viruses and bacteria) or endogenous (repetitive elements and transposons) invasive nucleic acids to minimize their impact on genome integrity and function. TGS and PTGS also are essential for controlling the expression of protein coding genes throughout development or in response to environmental stimuli. In plants and animals, at least one member of the conserved ARGONAUTE (AGO) protein family comprises the catalytic engine of the silencing complex, which is guided by sequence-specific small RNA to cognate RNA. In this review, we present general features of plant and animal AGO proteins and detail our knowledge on the 10 Arabidopsis thaliana AGOs.

Plant Cell. 2010 Aug ;22 (8):2697-709
The conserved RNA trafficking proteins HPR1 and TEX1 are involved in the production of endogenous and exogenous small interfering RNA in Arabidopsis.
Vincent Jauvion, Taline Elmayan, Hervé Vaucheret
Institut Jean-Pierre Bourgin, Institut National de la Recherche Agronomique, 78026 Versailles Cedex, France.
We previously identified Arabidopsis thaliana mutants defective in sense transgene posttranscriptional gene silencing (S-PTGS) that defined six loci; here, we describe mutants that define nine additional loci, including HYPER RECOMBINATION1 (HPR1), SILENCING DEFECTIVE3 (SDE3), and SDE5. Our analyses extend previous findings by showing that the requirement for the putative RNA helicase SDE3 is inversely proportional to the strength of the PTGS inducer and that the putative RNA trafficking protein SDE5 is an essential component of the trans-acting small interfering RNA (tasiRNA) pathway and is required for S-PTGS but not inverted repeat transgene-mediated PTGS (IR-PTGS). Our screen also identified HPR1 as a PTGS actor. We show that hpr1 mutations negatively impact S-PTGS, IR-PTGS, and tasiRNA pathways, resulting in increased accumulation of siRNA precursors and decreased accumulation of mature siRNA. In animals, HPR1/THO1 is a member of the conserved RNA trafficking THO/TREX complex, which also includes TEX1/THO3. We show that tex1 mutants, like hpr1 mutants, impact TAS precursor and mature tasiRNA levels, suggesting that a THO/TREX complex exists in plants and that this complex is important for the integrity of the tasiRNA pathway. We propose that both HPR1 and TEX1 participate in the trafficking of siRNA precursors to the ARGONAUTE catalytic center.

Nucleic Acids Res. 2010 Sep 1;38 (17):5844-50
siRNAs compete with miRNAs for methylation by HEN1 in Arabidopsis.
Bin Yu, Liu Bi, Jixian Zhai, Manu Agarwal, Shengben Li, Qingfa Wu, Shou-Wei Ding, Blake C Meyers, Hervé Vaucheret, Xuemei Chen
Department of Botany and Plant Sciences, Institute of Integrative Genome Biology, University of California, Riverside, CA 92521, USA.
Plant microRNAs (miRNAs) and small interfering RNAs (siRNAs) bear a 2'-O-methyl group on the 3'-terminal nucleotide. This methyl group is post-synthetically added by the methyltransferase protein HEN1 and protects small RNAs from enzymatic activities that target the 3'-OH. A mutagenesis screen for suppressors of the partial loss-of-function hen1-2 allele in Arabidopsis identified second-site mutations that restore miRNA methylation. These mutations affect two subunits of the DNA-dependent RNA polymerase IV (Pol IV), which is essential for the biogenesis of 24 nt endogenous siRNAs. A mutation in RNA-dependent RNA polymerase 2, another essential gene for the biogenesis of endogenous 24-nt siRNAs, also rescued the defects in miRNA methylation of hen1-2, revealing a previously unsuspected, negative influence of siRNAs on HEN1-mediated miRNA methylation. In addition, our findings imply the existence of a negative modifier of HEN1 activity in the Columbia genetic background.

Science 5 August 2005
Luck of the Draw
Lisa D. Chong
Genetically identical organisms that have been raised in identical environments age at different rates, suggesting that in addition to genes and environment, chance physiological phenomena can influence life span. Rea et al. report that the stress response system of Caenorhabditis elegans is subject to an underlying physiological randomness that affects how it copes with environmental insults. They placed the gene encoding green fluorescent protein (GFP) under the control of the regulatory region from the gene encoding a heat shock protein, creating an easily scored biomarker. Upon exposure to heat, isogenic worms exhibited considerable variation in fluorescence, and those expressing the highest amount of GFP tolerated heat the best and lived the longest. The physiological state indexed by GFP expression level was not heritable, and the authors suggest that stochastic variation in molecular and biochemical reactions could account for the variation in individual robustness and longevity.

Nature Genetics 37(8): 894-898 (2005)
A stress-sensitive reporter predicts longevity in isogenic populations of Caenorhabditis elegans.
Shane L Rea, Deqing Wu, James R Cypser, James W Vaupel & Thomas E Johnson
When both genotype and environment are held constant, 'chance' variation in the lifespan of individuals in a population is still quite large. Using isogenic populations of the nematode Caenorhabditis elegans, we show that, on the first day of adult life, chance variation in the level of induction of a green fluorescent protein (GFP) reporter coupled to a promoter from the gene hsp-16.2 predicts as much as a fourfold variation in subsequent survival. The same reporter is also a predictor of ability to withstand a subsequent lethal thermal stress. The level of induction of GFP is not heritable, and GFP expression levels in other reporter constructs are not associated with differences in longevity. HSP-16.2 itself is probably not responsible for the observed differences in survival but instead probably reflects a hidden, heterogeneous, but now quantifiable, physiological state that dictates the ability of an organism to deal with the rigors of living.

Genetics. 2005 Aug;170(4):1633-52. Epub 2005 Jun 8.
Imprinting capacity of gamete lineages in Caenorhabditis elegans.
Sha K, Fire A.
We have observed a gamete-of-origin imprinting effect in C. elegans using a set of GFP reporter transgenes. From a single progenitor line carrying an extrachromosomal unc-54::gfp transgene array, we generated three independent autosomal integrations of the unc-54::gfp transgene. The progenitor line, two of its three integrated derivatives, and a nonrelated unc-119:gfp transgene exhibit an imprinting effect: single-generation transmission of these transgenes through the male germline results in approximately ~1.5- to 2.0-fold greater expression than transmission through the female germline. There is a detectable resetting of the imprint after passage through the opposite germline for a single generation, indicating that the imprinted status of the transgenes is reversible. In cases where the transgene is maintained in either the oocyte lineage or sperm lineage for multiple, consecutive generations, a full reset requires passage through the opposite germline for several generations. Taken together, our results indicate that C. elegans has the ability to imprint chromosomes and that differences in the cell and/or molecular biology of oogenesis and spermatogenesis are manifest in an imprint that can persist in both somatic and germline gene expression for multiple generations.