Publications by authors named "Etienne Bucher"

29 Publications

  • Page 1 of 1

The plant mobile domain proteins MAIN and MAIL1 interact with the phosphatase PP7L to regulate gene expression and silence transposable elements in Arabidopsis thaliana.

PLoS Genet 2020 04 14;16(4):e1008324. Epub 2020 Apr 14.

LGDP-UMR5096, CNRS, Perpignan, France.

Transposable elements (TEs) are DNA repeats that must remain silenced to ensure cell integrity. Several epigenetic pathways including DNA methylation and histone modifications are involved in the silencing of TEs, and in the regulation of gene expression. In Arabidopsis thaliana, the TE-derived plant mobile domain (PMD) proteins have been involved in TE silencing, genome stability, and control of developmental processes. Using a forward genetic screen, we found that the PMD protein MAINTENANCE OF MERISTEMS (MAIN) acts synergistically and redundantly with DNA methylation to silence TEs. We found that MAIN and its close homolog MAIN-LIKE 1 (MAIL1) interact together, as well as with the phosphoprotein phosphatase (PPP) PP7-like (PP7L). Remarkably, main, mail1, pp7l single and mail1 pp7l double mutants display similar developmental phenotypes, and share common subsets of upregulated TEs and misregulated genes. Finally, phylogenetic analyses of PMD and PP7-type PPP domains among the Eudicot lineage suggest neo-association processes between the two protein domains to potentially generate new protein function. We propose that, through this interaction, the PMD and PPP domains may constitute a functional protein module required for the proper expression of a common set of genes, and for silencing of TEs.
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http://dx.doi.org/10.1371/journal.pgen.1008324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7156037PMC
April 2020

The NRPD1 N-terminus contains a Pol IV-specific motif that is critical for genome surveillance in Arabidopsis.

Nucleic Acids Res 2019 09;47(17):9037-9052

Institut de Biologie Moléculaire des Plantes, CNRS, Université de Strasbourg, F-67084 Strasbourg, France.

RNA-guided surveillance systems constrain the activity of transposable elements (TEs) in host genomes. In plants, RNA polymerase IV (Pol IV) transcribes TEs into primary transcripts from which RDR2 synthesizes double-stranded RNA precursors for small interfering RNAs (siRNAs) that guide TE methylation and silencing. How the core subunits of Pol IV, homologs of RNA polymerase II subunits, diverged to support siRNA biogenesis in a TE-rich, repressive chromatin context is not well understood. Here we studied the N-terminus of Pol IV's largest subunit, NRPD1. Arabidopsis lines harboring missense mutations in this N-terminus produce wild-type (WT) levels of NRPD1, which co-purifies with other Pol IV subunits and RDR2. Our in vitro transcription and genomic analyses reveal that the NRPD1 N-terminus is critical for robust Pol IV-dependent transcription, siRNA production and DNA methylation. However, residual RNA-directed DNA methylation observed in one mutant genotype indicates that Pol IV can operate uncoupled from the high siRNA levels typically observed in WT plants. This mutation disrupts a motif uniquely conserved in Pol IV, crippling the enzyme's ability to inhibit retrotransposon mobilization. We propose that the NRPD1 N-terminus motif evolved to regulate Pol IV function in genome surveillance.
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http://dx.doi.org/10.1093/nar/gkz618DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6753494PMC
September 2019

Apple whole genome sequences: recent advances and new prospects.

Hortic Res 2019 5;6:59. Epub 2019 Apr 5.

1Department of Horticulture, Washington State University, Pullman, WA 99164 USA.

In 2010, a major scientific milestone was achieved for tree fruit crops: publication of the first draft whole genome sequence (WGS) for apple (). This WGS, v1.0, was valuable as the initial reference for sequence information, fine mapping, gene discovery, variant discovery, and tool development. A new, high quality apple WGS, GDDH13 v1.1, was released in 2017 and now serves as the reference genome for apple. Over the past decade, these apple WGSs have had an enormous impact on our understanding of apple biological functioning, trait physiology and inheritance, leading to practical applications for improving this highly valued crop. Causal gene identities for phenotypes of fundamental and practical interest can today be discovered much more rapidly. Genome-wide polymorphisms at high genetic resolution are screened efficiently over hundreds to thousands of individuals with new insights into genetic relationships and pedigrees. High-density genetic maps are constructed efficiently and quantitative trait loci for valuable traits are readily associated with positional candidate genes and/or converted into diagnostic tests for breeders. We understand the species, geographical, and genomic origins of domesticated apple more precisely, as well as its relationship to wild relatives. The WGS has turbo-charged application of these classical research steps to crop improvement and drives innovative methods to achieve more durable, environmentally sound, productive, and consumer-desirable apple production. This review includes examples of basic and practical breakthroughs and challenges in using the apple WGSs. Recommendations for "what's next" focus on necessary upgrades to the genome sequence data pool, as well as for use of the data, to reach new frontiers in genomics-based scientific understanding of apple.
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http://dx.doi.org/10.1038/s41438-019-0141-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6450873PMC
April 2019

Functional and molecular characterization of the conserved Arabidopsis PUMILIO protein, APUM9.

Plant Mol Biol 2019 May 13;100(1-2):199-214. Epub 2019 Mar 13.

Université d'Angers, UMR1345 Institut de Recherche en Horticulture et Semences (IRHS-INRA), 42 rue Georges Morel, 24, 49071, Beaucouzé, France.

Key Message: Here we demonstrate that the APUM9 RNA-binding protein and its co-factors play a role in mRNA destabilization and how this activity might regulate early plant development. APUM9 is a conserved PUF RNA-binding protein (RBP) under complex transcriptional control mediated by a transposable element (TE) that restricts its expression in Arabidopsis. Currently, little is known about the functional and mechanistic details of the plant PUF regulatory system and the biological relevance of the TE-mediated repression of APUM9 in plant development and stress responses. By combining a range of transient assays, we show here, that APUM9 binding to target transcripts can trigger their rapid decay via its conserved C-terminal RNA-binding domain. APUM9 directly interacts with DCP2, the catalytic subunit of the decapping complex and DCP2 overexpression induces rapid decay of APUM9 targeted mRNAs. We show that APUM9 negatively regulates the expression of ABA signaling genes during seed imbibition, and thereby might contribute to the switch from dormant stage to seed germination. By contrast, strong TE-mediated repression of APUM9 is important for normal plant growth in the later developmental stages. Finally, APUM9 overexpression plants show slightly enhanced heat tolerance suggesting that TE-mediated control of APUM9, might have a role not only in embryonic development, but also in plant adaptation to heat stress conditions.
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http://dx.doi.org/10.1007/s11103-019-00853-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6513901PMC
May 2019

A large-scale circular RNA profiling reveals universal molecular mechanisms responsive to drought stress in maize and Arabidopsis.

Plant J 2019 05 5;98(4):697-713. Epub 2019 Mar 5.

National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, 430070, China.

Drought is a major abiotic stress that threatens global food security. Circular RNAs (circRNAs) are endogenous RNAs. How these molecules influence plant stress responses remains elusive. Here, a large-scale circRNA profiling identified 2174 and 1354 high-confidence circRNAs in maize and Arabidopsis, respectively, and most were differentially expressed in response to drought. A substantial number of drought-associated circRNA-hosting genes were involved in conserved or species-specific pathways in drought responses. Comparative analysis revealed that circRNA biogenesis was more complex in maize than in Arabidopsis. In most cases, maize circRNAs were negatively correlated with sRNA accumulation. In 368 maize inbred lines, the circRNA-hosting genes were enriched for single nucleotide polymorphisms (SNPs) associated with circRNA expression and drought tolerance, implying either important roles of circRNAs in maize drought responses or their potential use as biomarkers for breeding drought-tolerant maize. Additionally, the expression levels of circRNAs derived from drought-responsible genes encoding calcium-dependent protein kinase and cytokinin oxidase/dehydrogenase were significantly associated with drought tolerance of maize seedlings. Specifically, Arabidopsis plants overexpressing circGORK (Guard cell outward-rectifying K -channel) were hypersensitive to abscisic acid, but insensitive to drought, suggesting a positive role of circGORK in drought tolerance. We report the transcriptomic profiling and transgenic studies of circRNAs in plant drought responses, and provide evidence highlighting the universal molecular mechanisms involved in plant drought tolerance.
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http://dx.doi.org/10.1111/tpj.14267DOI Listing
May 2019

The SCOOP12 peptide regulates defense response and root elongation in Arabidopsis thaliana.

J Exp Bot 2019 02;70(4):1349-1365

IRHS (Institut de Recherche en Horticulture et Semences), UMR 1345, INRA, Agrocampus-Ouest, Université d'Angers, QuaSaV, Beaucouzé, France.

Small secreted peptides are important players in plant development and stress response. Using a targeted in silico approach, we identified a family of 14 Arabidopsis genes encoding precursors of serine-rich endogenous peptides (PROSCOOP). Transcriptomic analyses revealed that one member of this family, PROSCOOP12, is involved in processes linked to biotic and oxidative stress as well as root growth. Plants defective in this gene were less susceptible to Erwinia amylovora infection and showed an enhanced root growth phenotype. In PROSCOOP12 we identified a conserved motif potentially coding for a small secreted peptide. Exogenous application of synthetic SCOOP12 peptide induces various defense responses in Arabidopsis. Our findings show that SCOOP12 has numerous properties of phytocytokines, activates the phospholipid signaling pathway, regulates reactive oxygen species response, and is perceived in a BAK1 co-receptor-dependent manner.
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http://dx.doi.org/10.1093/jxb/ery454DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6382344PMC
February 2019

The 5'-3' Exoribonuclease XRN4 Regulates Auxin Response via the Degradation of Auxin Receptor Transcripts.

Genes (Basel) 2018 Dec 17;9(12). Epub 2018 Dec 17.

Botanical Institute, University of Basel, Zurich-Basel Plant Science Center, Part of the Swiss Plant Science Web, Schönbeinstrasse 6, 4056 Basel, Switzerland.

Auxin is a major hormone which plays crucial roles in instructing virtually all developmental programs of plants. Its signaling depends primarily on its perception by four partially redundant receptors of the TIR1/AFB2 clade (TAARs), which subsequently mediate the specific degradation of AUX/IAA transcriptional repressors to modulate the expression of primary auxin-responsive genes. Auxin homeostasis depends on complex regulations at the level of synthesis, conjugation, and transport. However, the mechanisms and principles involved in the homeostasis of its signaling are just starting to emerge. We report that mutants exhibit pleiotropic developmental defects and strong auxin hypersensitivity phenotypes. We provide compelling evidences that these phenotypes are directly caused by improper regulation of TAAR transcript degradation. We show that the cytoplasmic 5'-3' exoribonuclease XRN4 is required for auxin response. Thus, our work identifies new targets of XRN4 and a new level of regulation for TAAR transcripts important for auxin response and for plant development.
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http://dx.doi.org/10.3390/genes9120638DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6316084PMC
December 2018

Ecological plant epigenetics: Evidence from model and non-model species, and the way forward.

Ecol Lett 2017 Dec 12;20(12):1576-1590. Epub 2017 Oct 12.

Netherlands Institute of Ecology (NIOO-KNAW), Wageningen, The Netherlands.

Growing evidence shows that epigenetic mechanisms contribute to complex traits, with implications across many fields of biology. In plant ecology, recent studies have attempted to merge ecological experiments with epigenetic analyses to elucidate the contribution of epigenetics to plant phenotypes, stress responses, adaptation to habitat, and range distributions. While there has been some progress in revealing the role of epigenetics in ecological processes, studies with non-model species have so far been limited to describing broad patterns based on anonymous markers of DNA methylation. In contrast, studies with model species have benefited from powerful genomic resources, which contribute to a more mechanistic understanding but have limited ecological realism. Understanding the significance of epigenetics for plant ecology requires increased transfer of knowledge and methods from model species research to genomes of evolutionarily divergent species, and examination of responses to complex natural environments at a more mechanistic level. This requires transforming genomics tools specifically for studying non-model species, which is challenging given the large and often polyploid genomes of plants. Collaboration among molecular geneticists, ecologists and bioinformaticians promises to enhance our understanding of the mutual links between genome function and ecological processes.
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http://dx.doi.org/10.1111/ele.12858DOI Listing
December 2017

Inhibition of RNA polymerase II allows controlled mobilisation of retrotransposons for plant breeding.

Genome Biol 2017 07 7;18(1):134. Epub 2017 Jul 7.

IRHS, Université d'Angers, INRA, AGROCAMPUS-Ouest, SFR4207 QUASAV, Université Bretagne Loire, 49045, Angers, France.

Background: Retrotransposons play a central role in plant evolution and could be a powerful endogenous source of genetic and epigenetic variability for crop breeding. To ensure genome integrity several silencing mechanisms have evolved to repress retrotransposon mobility. Even though retrotransposons fully depend on transcriptional activity of the host RNA polymerase II (Pol II) for their mobility, it was so far unclear whether Pol II is directly involved in repressing their activity.

Results: Here we show that plants defective in Pol II activity lose DNA methylation at repeat sequences and produce more extrachromosomal retrotransposon DNA upon stress in Arabidopsis and rice. We demonstrate that combined inhibition of both DNA methylation and Pol II activity leads to a strong stress-dependent mobilization of the heat responsive ONSEN retrotransposon in Arabidopsis seedlings. The progenies of these treated plants contain up to 75 new ONSEN insertions in their genome which are stably inherited over three generations of selfing. Repeated application of heat stress in progeny plants containing increased numbers of ONSEN copies does not result in increased activation of this transposon compared to control lines. Progenies with additional ONSEN copies show a broad panel of environment-dependent phenotypic diversity.

Conclusions: We demonstrate that Pol II acts at the root of transposon silencing. This is important because it suggests that Pol II can regulate the speed of plant evolution by fine-tuning the amplitude of transposon mobility. Our findings show that it is now possible to study induced transposon bursts in plants and unlock their use to induce epigenetic and genetic diversity for crop breeding.
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http://dx.doi.org/10.1186/s13059-017-1265-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5501947PMC
July 2017

High-quality de novo assembly of the apple genome and methylome dynamics of early fruit development.

Nat Genet 2017 Jul 5;49(7):1099-1106. Epub 2017 Jun 5.

Institut de Recherche en Horticulture et Semences (IRHS), Université d'Angers, INRA, AGROCAMPUS-Ouest, SFR4207 QUASAV, Université Bretagne Loire, Angers, France.

Using the latest sequencing and optical mapping technologies, we have produced a high-quality de novo assembly of the apple (Malus domestica Borkh.) genome. Repeat sequences, which represented over half of the assembly, provided an unprecedented opportunity to investigate the uncharacterized regions of a tree genome; we identified a new hyper-repetitive retrotransposon sequence that was over-represented in heterochromatic regions and estimated that a major burst of different transposable elements (TEs) occurred 21 million years ago. Notably, the timing of this TE burst coincided with the uplift of the Tian Shan mountains, which is thought to be the center of the location where the apple originated, suggesting that TEs and associated processes may have contributed to the diversification of the apple ancestor and possibly to its divergence from pear. Finally, genome-wide DNA methylation data suggest that epigenetic marks may contribute to agronomically relevant aspects, such as apple fruit development.
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http://dx.doi.org/10.1038/ng.3886DOI Listing
July 2017

Recurrent evolution of heat-responsiveness in Brassicaceae COPIA elements.

Genome Biol 2016 10 11;17(1):209. Epub 2016 Oct 11.

Department of Plant Breeding and Genetics, Max Planck Institute for Plant Breeding Research, Cologne, 50829, Germany.

Background: The mobilization of transposable elements (TEs) is suppressed by host genome defense mechanisms. Recent studies showed that the cis-regulatory region of Arabidopsis thaliana COPIA78/ONSEN retrotransposons contains heat-responsive elements (HREs), which cause their activation during heat stress. However, it remains unknown whether this is a common and potentially conserved trait and how it has evolved.

Results: We show that ONSEN, COPIA37, TERESTRA, and ROMANIAT5 are the major families of heat-responsive TEs in A. lyrata and A. thaliana. Heat-responsiveness of COPIA families is correlated with the presence of putative high affinity heat shock factor binding HREs within their long terminal repeats in seven Brassicaceae species. The strong HRE of ONSEN is conserved over millions of years and has evolved by duplication of a proto-HRE sequence, which was already present early in the evolution of the Brassicaceae. However, HREs of most families are species-specific, and in Boechera stricta, the ONSEN HRE accumulated mutations and lost heat-responsiveness.

Conclusions: Gain of HREs does not always provide an ultimate selective advantage for TEs, but may increase the probability of their long-term survival during the co-evolution of hosts and genomic parasites.
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http://dx.doi.org/10.1186/s13059-016-1072-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5059998PMC
October 2016

HISTONE DEACETYLASE6 Controls Gene Expression Patterning and DNA Methylation-Independent Euchromatic Silencing.

Plant Physiol 2015 Aug 27;168(4):1298-308. Epub 2015 Apr 27.

Botanical Institute, Zürich-Basel Plant Science Center, University of Basel, CH-4056 Basel, Switzerland (E.H., K.F., L.K., E.B.); andUniversité d'Angers, UMR1345 Institut de Recherche en Horticulture et Semences, 49071 Beaucouzé, France (D.W., E.B.)

To investigate the role of chromatin regulators in patterning gene expression, we employed a unique epigenetically controlled and highly tissue-specific green fluorescent protein reporter line in Arabidopsis (Arabidopsis thaliana). Using a combination of forward and reverse genetic approaches on this line, we show here that distinct epigenetic regulators are involved in silencing the transgene in different tissues. The forward genetic screen led to the identification of a novel HISTONE DEACETYLASE6 (HDA6) mutant allele (epigenetic control1, hda6-8). This allele differs from the previously reported alleles, as it did not affect DNA methylation and only had a very modest effect on the release of transposable elements and other heterochromatic transcripts. Overall, our data shows that HDA6 has at least two clearly separable activities in different genomic regions. In addition, we present an unexpected role for HDA6 in the control of DNA methylation at CG dinucleotides.
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http://dx.doi.org/10.1104/pp.15.00177DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4528735PMC
August 2015

Heterosis and inbreeding depression of epigenetic Arabidopsis hybrids.

Nat Plants 2015 Jul 6;1:15092. Epub 2015 Jul 6.

Department of Plant Biology, University of Geneva, Sciences III, Geneva 4 CH-1211, Switzerland.

We have addressed the possible epigenetic contribution to heterosis using epigenetic inbred lines (epiRILs) with varying levels and distributions of DNA methylation. One line consistently displayed parent-of-origin heterosis for growth-related traits. Genome-wide transcription profiling followed by a candidate gene approach revealed 33 genes with altered regulation in crosses of this line that could contribute to the observed heterosis. Although none of the candidate genes could explain hybrid vigour, we detected intriguing, hybrid-specific transcriptional regulation of the RPP5 gene, encoding a growth suppressor. RPP5 displayed intermediate transcript levels in heterotic hybrids; surprisingly however, with global loss of fitness of their F2 progeny, we observed striking under-representation of the hybrid-like intermediate levels. Thus, in addition to genetic factors contributing to heterosis, our results strongly suggest that epigenetic diversity and epigenetic regulation of transcription play a role in hybrid vigour and inbreeding depression, and also in the absence of parental genetic diversity.
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http://dx.doi.org/10.1038/nplants.2015.92DOI Listing
July 2015

Epigenetic control of transposon transcription and mobility in Arabidopsis.

Curr Opin Plant Biol 2012 Nov 31;15(5):503-10. Epub 2012 Aug 31.

Botanical Institute, University of Basel, Hebelstrasse 1, 4056 Basel, Switzerland.

The mobility of genetic elements called transposable elements (TEs) was discovered half a century ago by Barbara McClintock. Although she had recognized them as chromosomal controlling elements, for much of the consequent time TEs were primarily considered as parasites of the host genome. However the recent explosion of discoveries in the fields of genomics and epigenetics have unambiguously shown the importance of TEs in genome function and evolution. Bursts of endogenous TEs have been reported in plants with epigenetic misregulation, revealing the molecular mechanisms underlying their control. We review here the different steps in TE invasion of the host genome involving epigenetic control and environmental stress responses. As TEs propagate in plant genomes and attract epigenetic marks, their neo-insertions can lead to the formation of new, heritable epigenetic variants (epialleles) of genes in their vicinity and impact on host gene regulatory networks. The epigenetic interplay between TE and genes thus plays a crucial role in the TE-host co-evolution.
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http://dx.doi.org/10.1016/j.pbi.2012.08.006DOI Listing
November 2012

Loss of DNA methylation affects the recombination landscape in Arabidopsis.

Proc Natl Acad Sci U S A 2012 Apr 26;109(15):5880-5. Epub 2012 Mar 26.

Department of Plant Biology, Sciences III, University of Geneva, CH-1211 Geneva 4, Switzerland.

During sexual reproduction, one-half of the genetic material is deposited in gametes, and a complete set of chromosomes is restored upon fertilization. Reduction of the genetic information before gametogenesis occurs in meiosis, when cross-overs (COs) between homologous chromosomes secure an exchange of their genetic information. COs are not evenly distributed along chromosomes and are suppressed in chromosomal regions encompassing compact, hypermethylated centromeric and pericentromeric DNA. Therefore, it was postulated that DNA hypermethylation is inhibitory to COs. Here, when analyzing meiotic recombination in mutant plants with hypomethylated DNA, we observed unexpected and counterintuitive effects of DNA methylation losses on CO distribution. Recombination was further promoted in the hypomethylated chromosome arms while it was inhibited in heterochromatic regions encompassing pericentromeric DNA. Importantly, the total number of COs was not affected, implying that loss of DNA methylation led to a global redistribution of COs along chromosomes. To determine by which mechanisms altered levels of DNA methylation influence recombination--whether directly in cis or indirectly in trans by changing expression of genes encoding recombination components--we analyzed CO distribution in wild-type lines with randomly scattered and well-mapped hypomethylated chromosomal segments. The results of these experiments, supported by expression profiling data, suggest that DNA methylation affects meiotic recombination in cis. Because DNA methylation exhibits significant variation even within a single species, our results imply that it may influence the evolution of plant genomes through the control of meiotic recombination.
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http://dx.doi.org/10.1073/pnas.1120841109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3326504PMC
April 2012

An siRNA pathway prevents transgenerational retrotransposition in plants subjected to stress.

Nature 2011 Apr 13;472(7341):115-9. Epub 2011 Mar 13.

Department of Plant Biology, University of Geneva, Sciences III, 30 Quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland.

Eukaryotic genomes consist to a significant extent of retrotransposons that are suppressed by host epigenetic mechanisms, preventing their uncontrolled propagation. However, it is not clear how this is achieved. Here we show that in Arabidopsis seedlings subjected to heat stress, a copia-type retrotransposon named ONSEN (Japanese 'hot spring') not only became transcriptionally active but also synthesized extrachromosomal DNA copies. Heat-induced ONSEN accumulation was stimulated in mutants impaired in the biogenesis of small interfering RNAs (siRNAs); however, there was no evidence of transposition occurring in vegetative tissues. After stress, both ONSEN transcripts and extrachromosomal DNA gradually decayed and were no longer detected after 20-30 days. Surprisingly, a high frequency of new ONSEN insertions was observed in the progeny of stressed plants deficient in siRNAs. Insertion patterns revealed that this transgenerational retrotransposition occurred during flower development and before gametogenesis. Therefore in plants with compromised siRNA biogenesis, memory of stress was maintained throughout development, priming ONSEN to transpose during differentiation of generative organs. Retrotransposition was not observed in the progeny of wild-type plants subjected to stress or in non-stressed mutant controls, pointing to a crucial role of the siRNA pathway in restricting retrotransposition triggered by environmental stress. Finally, we found that natural and experimentally induced variants in ONSEN insertions confer heat responsiveness to nearby genes, and therefore mobility bursts may generate novel, stress-responsive regulatory gene networks.
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http://dx.doi.org/10.1038/nature09861DOI Listing
April 2011

Stress-induced activation of heterochromatic transcription.

PLoS Genet 2010 Oct 28;6(10):e1001175. Epub 2010 Oct 28.

Department of Plant Biology, University of Geneva, Geneva, Switzerland.

Constitutive heterochromatin comprising the centromeric and telomeric parts of chromosomes includes DNA marked by high levels of methylation associated with histones modified by repressive marks. These epigenetic modifications silence transcription and ensure stable inheritance of this inert state. Although environmental cues can alter epigenetic marks and lead to modulation of the transcription of genes located in euchromatic parts of the chromosomes, there is no evidence that external stimuli can globally destabilize silencing of constitutive heterochromatin. We have found that heterochromatin-associated silencing in Arabidopsis plants subjected to a particular temperature regime is released in a genome-wide manner. This occurs without alteration of repressive epigenetic modifications and does not involve common epigenetic mechanisms. Such induced release of silencing is mostly transient, and rapid restoration of the silent state occurs without the involvement of factors known to be required for silencing initiation. Thus, our results reveal new regulatory aspects of transcriptional repression in constitutive heterochromatin and open up possibilities to identify the molecular mechanisms involved.
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http://dx.doi.org/10.1371/journal.pgen.1001175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2965753PMC
October 2010

RNA-directed DNA methylation and plant development require an IWR1-type transcription factor.

EMBO Rep 2010 Jan 11;11(1):65-71. Epub 2009 Dec 11.

Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Dr Bohr-Gasse 3, 1030 Vienna, Austria.

RNA-directed DNA methylation (RdDM) in plants requires two RNA polymerase (Pol) II-related RNA polymerases, namely Pol IV and Pol V. A genetic screen designed to reveal factors that are important for RdDM in a developmental context in Arabidopsis identified DEFECTIVE IN MERISTEM SILENCING 4 (DMS4). Unlike other mutants defective in RdDM, dms4 mutants have a pleiotropic developmental phenotype. The DMS4 protein is similar to yeast IWR1 (interacts with RNA polymerase II), a conserved putative transcription factor that interacts with Pol II subunits. The DMS4 complementary DNA partly complements the K1 killer toxin hypersensitivity of a yeast iwr1 mutant, suggesting some functional conservation. In the transgenic system studied, mutations in DMS4 directly or indirectly affect Pol IV-dependent secondary short interfering RNAs, Pol V-mediated RdDM, Pol V-dependent synthesis of intergenic non-coding RNA and expression of many Pol II-driven genes. These data suggest that DMS4 might be a regulatory factor for several RNA polymerases, thus explaining its diverse roles in the plant.
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http://dx.doi.org/10.1038/embor.2009.246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2816620PMC
January 2010

MOM1 and Pol-IV/V interactions regulate the intensity and specificity of transcriptional gene silencing.

EMBO J 2010 Jan 12;29(2):340-51. Epub 2009 Nov 12.

Department of Plant Biology, University of Geneva, Sciences III, Geneva, Switzerland.

It is commonly observed that onset or release of transcriptional gene silencing (TGS) correlates with alteration of repressive epigenetic marks. The TGS regulator MOM1 in Arabidopsis is exceptional since it regulates transcription in intermediate heterochromatin with only minor changes in epigenetic marks. We have isolated an enhancer of the mom1 mutation that points towards regulatory interplay between MOM1 and RNA polymerase-V (Pol-V). Pol-V transcribes heterochromatic loci, which seems to be required for maintenance of their silencing; however, it is still not clear how Pol-V is targeted to heterochromatin. We now provide evidence that Pol-V is required for MOM1-mediated suppression of transcription at a subset of its chromosomal targets. Thus, Pol-V genetically interacts with MOM1 in the control of gene silencing. Interestingly, functional cooperation of MOM1 and Pol-V not only broadens the range of the controlled loci in comparison to each individual factor, but also determines the degree of TGS.
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http://dx.doi.org/10.1038/emboj.2009.328DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2824458PMC
January 2010

Selective epigenetic control of retrotransposition in Arabidopsis.

Nature 2009 Sep 6;461(7262):427-30. Epub 2009 Sep 6.

Department of Plant Biology, University of Geneva, Sciences III, 30 Quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland.

Retrotransposons are mobile genetic elements that populate chromosomes, where the host largely controls their activities. In plants and mammals, retrotransposons are transcriptionally silenced by DNA methylation, which in Arabidopsis is propagated at CG dinucleotides by METHYLTRANSFERASE 1 (MET1). In met1 mutants, however, mobilization of retrotransposons is not observed, despite their transcriptional activation. A post-transcriptional mechanism therefore seems to be preventing retrotransposition. Here we show that a copia-type retrotransposon (Evadé, French for 'fugitive') evaded suppression of its movement during inbreeding of hybrid epigenomes consisting of met1- and wild-type-derived chromosomes. Evadé (EVD) reinsertions caused a series of developmental mutations that allowed its identification. Genetic testing of host control of the EVD life cycle showed that transcriptional suppression occurred by CG methylation supported by RNA-directed DNA methylation. On transcriptional reactivation, subsequent steps of the EVD cycle were inhibited by plant-specific RNA polymerase IV/V and the histone methyltransferase KRYPTONITE (KYP). Moreover, genome resequencing demonstrated retrotransposition of EVD but no other potentially active retroelements when this combination of epigenetic mechanisms was compromised. Our results demonstrate that epigenetic control of retrotransposons extends beyond transcriptional suppression and can be individualized for particular elements.
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http://dx.doi.org/10.1038/nature08328DOI Listing
September 2009

Compromised stability of DNA methylation and transposon immobilization in mosaic Arabidopsis epigenomes.

Genes Dev 2009 Apr;23(8):939-50

Department of Plant Biology, University of Geneva, Sciences III, Geneva, Switzerland.

Transgenerational epigenetic inheritance has been defined by the study of relatively few loci. We examined a population of recombinant inbred lines with epigenetically mosaic chromosomes consisting of wild-type and CG methylation-depleted segments (epiRILs). Surprisingly, transposons that were immobile in the parental lines displayed stochastic movement in 28% of the epiRILs. Although analysis after eight generations of inbreeding, supported by genome-wide DNA methylation profiling, identified recombined parental chromosomal segments, these were interspersed with unexpectedly high frequencies of nonparental methylation polymorphism. Hence, epigenetic inheritance in hybrids derived from parents with divergent epigenomes permits long-lasting epi-allelic interactions that violate Mendelian expectations. Such persistently "unstable" epigenetic states complicate linkage-based epigenomic mapping. Thus, future epigenomic analyses should consider possible genetic instabilities and alternative mapping strategies.
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http://dx.doi.org/10.1101/gad.524609DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2675864PMC
April 2009

A stepwise pathway for biogenesis of 24-nt secondary siRNAs and spreading of DNA methylation.

EMBO J 2009 Jan 11;28(1):48-57. Epub 2008 Dec 11.

Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Vienna, Austria.

We used a transgene system to study spreading of RNA-directed DNA methylation (RdDM) during transcriptional gene silencing in Arabidopsis thaliana. Forward and reverse genetics approaches using this system delineated a stepwise pathway for the biogenesis of secondary siRNAs and unidirectional spreading of methylation from an upstream enhancer element into downstream sequences. Trans-acting, hairpin-derived primary siRNAs induce primary RdDM, independently of an enhancer-associated 'nascent' RNA, at the target enhancer region. Primary RdDM is a key step in the pathway because it attracts the secondary siRNA-generating machinery, including RNA polymerase IV, RNA-dependent RNA polymerase2 and Dicer-like3 (DCL3). These factors act in a turnover pathway involving a nascent RNA, which normally accumulates stably in non-silenced plants, to produce cis-acting secondary siRNAs that induce methylation in the downstream region. The identification of DCL3 in a forward genetic screen for silencing-defective mutants demonstrated a strict requirement for 24-nt siRNAs to direct methylation. A similar stepwise process for spreading of DNA methylation may occur in mammalian genomes, which are extensively transcribed in upstream regulatory regions.
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http://dx.doi.org/10.1038/emboj.2008.260DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2633084PMC
January 2009

A structural-maintenance-of-chromosomes hinge domain-containing protein is required for RNA-directed DNA methylation.

Nat Genet 2008 May 20;40(5):670-5. Epub 2008 Apr 20.

Gregor Mendel Institute for Molecular Plant Biology, Austrian Academy of Sciences, Dr. Bohrgasse 3, A-1030 Vienna, Austria.

RNA-directed DNA methylation (RdDM) is a process in which dicer-generated small RNAs guide de novo cytosine methylation at the homologous DNA region. To identify components of the RdDM machinery important for Arabidopsis thaliana development, we targeted an enhancer active in meristems for methylation, which resulted in silencing of a downstream GFP reporter gene. This silencing system also features secondary siRNAs, which trigger methylation that spreads beyond the targeted enhancer region. A screen for mutants defective in meristem silencing and enhancer methylation retrieved six dms complementation groups, which included the known factors DRD1 (ref. 3; a SNF2-like chromatin-remodeling protein) and Pol IVb subunits. Additionally, we identified a previously unknown gene DMS3 (At3g49250), encoding a protein similar to the hinge-domain region of structural maintenance of chromosomes (SMC) proteins. This finding implicates a putative chromosome architectural protein that can potentially link nucleic acids in facilitating an RNAi-mediated epigenetic modification involving secondary siRNAs and spreading of DNA methylation.
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http://dx.doi.org/10.1038/ng.119DOI Listing
May 2008

RNA-directed DNA methylation mediated by DRD1 and Pol IVb: a versatile pathway for transcriptional gene silencing in plants.

Biochim Biophys Acta 2007 May-Jun;1769(5-6):358-74. Epub 2007 Mar 12.

Gregor Mendel Institute for Molecular Plant Biology, Austrian Academy of Sciences, Dr. Bohr-Gasse 3, A-1030 Vienna, Austria.

RNA-directed DNA methylation, which is one of several RNAi-mediated pathways in the nucleus, has been highly elaborated in the plant kingdom. RNA-directed DNA methylation requires for the most part conventional DNA methyltransferases, histone modifying enzymes and RNAi proteins; however, several novel, plant-specific proteins that are essential for this process have been identified recently. DRD1 (defective in RNA-directed DNA methylation) is a putative SWI2/SNF2-like chromatin remodelling protein; DRD2 and DRD3 (renamed NRPD2a and NRPD1b, respectively) are subunits of Pol IVb, a putative RNA polymerase found only in plants. Interestingly, DRD1 and Pol IVb appear to be required not only for RNA-directed de novo methylation, but also for full erasure of methylation when the RNA trigger is withdrawn. These proteins thus have the potential to facilitate dynamic regulation of DNA methylation. Prominent targets of RNA-directed DNA methylation in the Arabidopsis thaliana genome include retrotransposon long terminal repeats (LTRs), which have bidirectional promoter/enhancer activities, and other types of intergenic transposons and repeats. Intergenic solitary LTRs that are targeted for reversible methylation by the DRD1/Pol IVb pathway can potentially act as switches or rheostats for neighboring plant genes. The resulting alterations in gene expression patterns may promote physiological flexibility and adaptation to the environment.
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http://dx.doi.org/10.1016/j.bbaexp.2007.03.001DOI Listing
July 2007

Multiple virus resistance at a high frequency using a single transgene construct.

J Gen Virol 2006 Dec;87(Pt 12):3697-3701

Laboratory of Virology, Wageningen University, Binnenhaven 11, 6709 PD Wageningen, The Netherlands.

RNA silencing is a natural antiviral defence in plants, which can be exploited in transgenic plants for preprogramming virus recognition and ensuring enhanced resistance. By arranging viral transgenes as inverted repeats it is thus possible to obtain strong repression of incoming viruses. Due to the high sequence specificity of RNA silencing, this technology has hitherto been limited to the targeting of single viruses. Here it is shown that efficient simultaneous targeting of four different tospoviruses can be achieved by using a single small transgene based on the production of minimal sized chimaeric cassettes. Due to simultaneous RNA silencing, as demonstrated by specific siRNA accumulation, the transgenic expression of these cassettes rendered up to 82 % of the transformed plant lines heritably resistant against all four viruses. Thus RNA silencing can be further improved for high frequency multiple virus resistance by combining small RNA fragments from a series of target viruses.
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http://dx.doi.org/10.1099/vir.0.82276-0DOI Listing
December 2006

The influenza A virus NS1 protein binds small interfering RNAs and suppresses RNA silencing in plants.

J Gen Virol 2004 Apr;85(Pt 4):983-991

Laboratory of Virology, Wageningen University, Binnenhaven 11, 6709 PD Wageningen, The Netherlands.

RNA silencing comprises a set of sequence-specific RNA degradation pathways that occur in a wide range of eukaryotes, including animals, fungi and plants. A hallmark of RNA silencing is the presence of small interfering RNA molecules (siRNAs). The siRNAs are generated by cleavage of larger double-stranded RNAs (dsRNAs) and provide the sequence specificity for degradation of cognate RNA molecules. In plants, RNA silencing plays a key role in developmental processes and in control of virus replication. It has been shown that many plant viruses encode proteins, denoted RNA silencing suppressors, that interfere with this antiviral response. Although RNA silencing has been shown to occur in vertebrates, no relationship with inhibition of virus replication has been demonstrated to date. Here we show that the NS1 protein of human influenza A virus has an RNA silencing suppression activity in plants, similar to established RNA silencing suppressor proteins of plant viruses. In addition, NS1 was shown to be capable of binding siRNAs. The data presented here fit with a potential role for NS1 in counteracting innate antiviral responses in vertebrates by sequestering siRNAs.
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http://dx.doi.org/10.1099/vir.0.19734-0DOI Listing
April 2004

Antiviral immune responses in gene-targeted mice expressing the immunoglobulin heavy chain of virus-neutralizing antibodies.

Proc Natl Acad Sci U S A 2003 Oct 20;100(22):12883-8. Epub 2003 Oct 20.

Institute of Experimental Immunology, Department of Pathology, University Hospital, CH-8091 Zurich, Switzerland.

Two gene-targeted immunoglobulin heavy chain transgenic mouse strains, TgH(KL25) and TgH(VI10), expressing neutralizing specificities for lymphocytic choriomeningitis virus and vesicular stomatitis virus, respectively, have been generated. Three days after lymphocytic choriomeningitis virus infection, TgH(KL25) mice showed a thymus-independent neutralizing IgM response followed by thymus-dependent (TD) IgG. In contrast, WT mice mounted only a TD IgG response around day 80. These observations indicated that not only structural properties of the virus but also immunological parameters such as the frequency of B cells were indicative for the induction of thymus-independent versus TD Ig responses. Naïve vesicular stomatitis virusspecific Ig heavy chain transgenic mice displayed greatly elevated natural antibody titers. However, despite these high naïve titers, de novo activation of naïve CD4+ T and B cells was not blocked. Therefore, B cells giving rise to natural antibodies do not participate in virus-induced antibody responses.
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http://dx.doi.org/10.1073/pnas.2135542100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC240713PMC
October 2003

Resistance mechanisms to plant viruses: an overview.

Virus Res 2003 Apr;92(2):207-12

Laboratory of Virology, Wageningen University, Binnenhaven 11, PD-6709 Wageningen, The Netherlands.

To obtain virus-resistant host plants, a range of operational strategies can be followed nowadays. While for decades plant breeders have been able to introduce natural resistance genes in susceptible genotypes without knowing precisely what these resistance traits were, currently a growing number of (mostly) dominant resistance genes have been cloned and analyzed. This has led not only to a better understanding of the plant's natural defence systems, but also opened the way to use these genes beyond species borders. Besides using natural resistance traits, also several novel, "engineered" forms of virus resistance have been developed over the past 15 years. The first successes were obtained embarking from the principle of pathogen-derived resistance (PDR) by transforming host plants with viral genes or sequences with the purpose to block a specific step during virus multiplication in the plant. As an unforeseen spin-off of these investments, the phenomenon of post-translational gene silencing (PTGS) was discovered, which to date is by far the most successful way to engineer resistance. It is generally believed that PTGS reflects a natural defence system of the plant, and part of the hypothesized components required for PTGS have been identified. As counteracting strategy, and confirming PTGS to be a natural phenomenon, a considerable number of viruses have acquired gene functions by which they can suppress PTGS. In addition to PDR and PTGS, further strategies for engineered virus resistance have been explored, including the use of pokeweed antiviral protein (PAP), 2',5'-oligoadenylate synthetase and "plantibodies". This paper will give a brief overview of the major strategies that have become operational during the past 10 years.
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http://dx.doi.org/10.1016/s0168-1702(02)00353-2DOI Listing
April 2003

Negative-strand tospoviruses and tenuiviruses carry a gene for a suppressor of gene silencing at analogous genomic positions.

J Virol 2003 Jan;77(2):1329-36

Laboratory of Virology, Wageningen University, The Netherlands.

Posttranscriptional silencing of a green fluorescent protein (GFP) transgene in Nicotiana benthamiana plants was suppressed when these plants were infected with Tomato spotted wilt virus (TSWV), a plant-infecting member of the BUNYAVIRIDAE: Infection with TSWV resulted in complete reactivation of GFP expression, similar to the case for Potato virus Y, but distinct from that for Cucumber mosaic virus, two viruses known to carry genes encoding silencing suppressor proteins. Agrobacterium-based leaf injections with individual TSWV genes identified the NS(S) gene to be responsible for the RNA silencing-suppressing activity displayed by this virus. The absence of short interfering RNAs in NS(S)-expressing leaf sectors suggests that the tospoviral NS(S) protein interferes with the intrinsic RNA silencing present in plants. Suppression of RNA silencing was also observed when the NS3 protein of the Rice hoja blanca tenuivirus, a nonenveloped negative-strand virus, was expressed. These results indicate that plant-infecting negative-strand RNA viruses carry a gene for a suppressor of RNA silencing.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC140852PMC
http://dx.doi.org/10.1128/jvi.77.2.1329-1336.2003DOI Listing
January 2003