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Agrisera Educational Poster 6 "Plant Small RNAs: Biogenesis and Functions". Graphic Design by Dmitry Shevela (SciGrafik, Sweden). Printed copies of the poster are available free of charge from Agrisera's website (https://www.agrisera.com/en/educational-posters).

Epigenomics is the study of molecular signatures associated with discrete regions within genomes, many of which are important for a wide range of nuclear processes. The ability to profile the epigenomic landscape associated with genes, repetitive regions, transposons, transcription, differential expression, cis-regulatory elements, and 3D chromatin interactions has vastly improved our understanding of plant genomes. However, many epigenomic and single-cell genomic assays are challenging to perform in plants, leading to a wide range of data quality issues; thus, the data require rigorous evaluation prior to downstream analyses and interpretation. In this commentary, we provide considerations for the evaluation of plant epigenomics and single-cell genomics data quality with the aim of improving the quality and utility of studies using those data across diverse plant species.

Additional file of Deep sequencing reveals the complex and coordinated transcriptional regulation of genes related to grain quality in rice cultivars

In plants, RNA-directed DNA methylation (RdDM) is a silencing mechanism relying on the production of 24-nt small interfering RNAs (siRNAs) by RNA POLYMERASE IV (Pol IV) to trigger methylation and inactivation of transposable elements (TEs). We present the construction and characterization of osnrpd1, a knock-down RNA interference line of OsNRPD1 gene that encodes the largest subunit of Pol IV in rice (Oryza sativa ssp japonica cv Nipponbare). We show that osnrpd1 displays a lower accumulation of OsNRPD1 transcripts, associated with an overall reduction of 24-nt siRNAs and DNA methylation level in all three contexts, CG, CHG and CHH. We uncovered new insertions of known active TEs, the LTR retrotransposons Tos17 and Lullaby and the long interspersed nuclear element-type retrotransposon Karma. However, we did not observe any clear developmental phenotype, contrary to what was expected for a mutant severely affected in RdDM. In addition, despite the presence of many putatively functional TEs in the rice genome, we found no evidence of in planta global reactivation of transposition. This knock-down of OsNRPD1 likely led to a weakly affected line, with no effect on development and a limited effect on transposition. We discuss the possibility that a knock-out mutation of OsNRPD1 would cause sterility in rice.This article is part of the discussion meeting issue ‘Crossroads between transposons and gene regulation’.

Additional file 1:Filter results of the five MPSS and SBS libraries. A) A total of 39,288 distinct 17-base expressed signatures from the five MPSS libraries were processed according to three filters: significance, reliability, and genomic match. B) Similarly, 397,543 signatures from the five SBS libraries were also processed using these same filters as previously described by Meyers et al. [45]. (PPT 134 KB)

Additional file 2:Classification of the MPSS and SBS signatures from the five libraries based on their location on the annotated gene (hits = 1) (See Meyers et al. 2004[45]for details).(DOC 60 KB)

Authors’ original file for figure 2

In plants, RNA-directed DNA methylation (RdDM) is a silencing mechanism relying on the production of 24-nt small interfering RNAs (siRNAs) by RNA POLYMERASE IV (Pol IV) to trigger methylation and inactivation of transposable elements (TEs). We present the construction and characterization of osnrpd1, a knock-down RNA interference line of OsNRPD1 gene that encodes the largest subunit of Pol IV in rice (Oryza sativa ssp japonica cv Nipponbare). We show that osnrpd1 displays a lower accumulation of OsNRPD1 transcripts, associated with an overall reduction of 24-nt siRNAs and DNA methylation level in all three contexts, CG, CHG and CHH. We uncovered new insertions of known active TEs, the LTR retrotransposons Tos17 and Lullaby and the long interspersed nuclear element-type retrotransposon Karma. However, we did not observe any clear developmental phenotype, contrary to what was expected for a mutant severely affected in RdDM. In addition, despite the presence of many putatively functional TEs in the rice genome, we found no evidence of in planta global reactivation of transposition. This knock-down of OsNRPD1 likely led to a weakly affected line, with no effect on development and a limited effect on transposition. We discuss the possibility that a knock-out mutation of OsNRPD1 would cause sterility in rice.This article is part of the discussion meeting issue ‘Crossroads between transposons and gene regulation’.

In plants, RNA-directed DNA methylation (RdDM) is a silencing mechanism relying on the production of 24-nt small interfering RNAs (siRNAs) by RNA POLYMERASE IV (Pol IV) to trigger methylation and inactivation of transposable elements (TEs). We present the construction and characterization of osnrpd1, a knock-down RNA interference line of OsNRPD1 gene that encodes the largest subunit of Pol IV in rice (Oryza sativa ssp japonica cv Nipponbare). We show that osnrpd1 displays a lower accumulation of OsNRPD1 transcripts, associated with an overall reduction of 24-nt siRNAs and DNA methylation level in all three contexts, CG, CHG and CHH. We uncovered new insertions of known active TEs, the LTR retrotransposons Tos17 and Lullaby and the long interspersed nuclear element-type retrotransposon Karma. However, we did not observe any clear developmental phenotype, contrary to what was expected for a mutant severely affected in RdDM. In addition, despite the presence of many putatively functional TEs in the rice genome, we found no evidence of in planta global reactivation of transposition. This knock-down of OsNRPD1 likely led to a weakly affected line, with no effect on development and a limited effect on transposition. We discuss the possibility that a knock-out mutation of OsNRPD1 would cause sterility in rice.This article is part of the discussion meeting issue ‘Crossroads between transposons and gene regulation’.

Additional file 7:Conserved cis elements in the promoter region of the highly induced genes (≥50 fold) in Cypress (compared to LaGrue and Nipponbare) and Ilpumbyeo (compared to YR15965 and Nipponbare) that are involved in seed development. (DOC 46 KB)

Authors’ original file for figure 1

Additional figures

In plants, RNA-directed DNA methylation (RdDM) is a silencing mechanism relying on the production of 24-nt small interfering RNAs (siRNAs) by RNA POLYMERASE IV (Pol IV) to trigger methylation and inactivation of transposable elements (TEs). We present the construction and characterization of osnrpd1, a knock-down RNA interference line of OsNRPD1 gene that encodes the largest subunit of Pol IV in rice (Oryza sativa ssp japonica cv Nipponbare). We show that osnrpd1 displays a lower accumulation of OsNRPD1 transcripts, associated with an overall reduction of 24-nt siRNAs and DNA methylation level in all three contexts, CG, CHG and CHH. We uncovered new insertions of known active TEs, the LTR retrotransposons Tos17 and Lullaby and the long interspersed nuclear element-type retrotransposon Karma. However, we did not observe any clear developmental phenotype, contrary to what was expected for a mutant severely affected in RdDM. In addition, despite the presence of many putatively functional TEs in the rice genome, we found no evidence of in planta global reactivation of transposition. This knock-down of OsNRPD1 likely led to a weakly affected line, with no effect on development and a limited effect on transposition. We discuss the possibility that a knock-out mutation of OsNRPD1 would cause sterility in rice.This article is part of the discussion meeting issue ‘Crossroads between transposons and gene regulation’.

Many evolutionary conserved microRNAs (miRNAs) in plants regulate transcription factors with key functions in development. Hence, mutations in the core components of the miRNA biogenesis machinery cause strong growth defects. An essential aspect of miRNA biogenesis is the precise excision of the small RNA from its precursor. In plants, miRNA precursors are largely variable in size and shape and can be processed by different modes. Here, we optimized an approach to detect intermediates during miRNA biogenesis. We characterized a miRNA whose processing is triggered by a terminal branched loop. Plant miRNA processing can be initiated by internal bubbles, small terminal loops or branched loops followed by dsRNA segments of 15-17 bp. Interestingly, precision and efficiency vary with the processing modes. Despite the various potential structural determinants present in a single a miRNA precursor, DCL1 is mostly guided by a predominant structural region in each precursor in wild-type plants. However, our studies in fiery1, hyl1 and se mutants revealed the existence of cleavage signatures consistent with the recognition of alternative processing determinants. The results provide a general view of the mechanisms underlying the specificity of miRNA biogenesis in plants.

Principal investigators John P. Vogel1, David F. Garvin2, Todd C. Mockler3, Jeremy Schmutz4, Dan Rokhsar5,6, Michael W. Bevan7; DNA sequencing and assembly Kerrie Barry5, Susan Lucas5, Miranda Harmon-Smith5, Kathleen Lail5, Hope Tice5, Jeremy Schmutz4 (Leader), Jane Grimwood4, Neil McKenzie7, Michael W. Bevan7; Pseudomolecule assembly and BACend sequencing Naxin Huo1, Yong Q.Gu1,Gerard R. Lazo1, Olin D.Anderson1, John P. Vogel1 (Leader), Frank M. You8,Ming-Cheng Luo8, Jan Dvorak8, Jonathan Wright7, Melanie Febrer7, Michael W. Bevan7, Dominika Idziak9, Robert Hasterok9, David F. Garvin2; Transcriptome sequencing and analysis Erika Lindquist5, Mei Wang5, Samuel E. Fox3, Henry D. Priest3, Sergei A. Filichkin3, Scott A. Givan3, Douglas W. Bryant3, Jeff H.Chang3, Todd C.Mockler3 (Leader), Haiyan Wu10,24, Wei Wu10, An-Ping Hsia10, Patrick S. Schnable10,24, Anantharaman Kalyanaraman11, Brad Barbazuk12, Todd P.Michael13, Samuel P.Hazen14, Jennifer N. Bragg1, Debbie Laudencia-Chingcuanco1, John P. Vogel1, David F. Garvin2, Yiqun Weng15, Neil McKenzie7, Michael W. Bevan7; Gene analysis and annotation Georg Haberer16, Manuel Spannagl16, Klaus Mayer16 (Leader), Thomas Rattei17, Therese Mitros6, Dan Rokhsar6, Sang-Jik Lee18, Jocelyn K.C. Rose18, Lukas A. Mueller19, Thomas L. York19; Repeats analysis Thomas Wicker20 (Leader), Jan P. Buchmann20, Jaakko Tanskanen21, Alan H. Schulman21 (Leader), Heidrun Gundlach16, Jonathan Wright7, Michael Bevan7, Antonio Costa de Oliveira22, Luciano da C. Maia22, William Belknap1, Yong Q. Gu1, Ning Jiang23, Jinsheng Lai24, Liucun Zhu25, JianxinMa25, Cheng Sun26, Ellen Pritham26; Comparative genomics Jerome Salse27 (Leader), Florent Murat27, Michael Abrouk27, Georg Haberer16, Manuel Spannagl16, Klaus Mayer16, Remy Bruggmann13, Joachim Messing13, Frank M. You8, Ming-Cheng Luo8, Jan Dvorak8; Small RNA analysis Noah Fahlgren3, Samuel E. Fox3, Christopher M. Sullivan3, Todd C. Mockler3, James C. Carrington3, Elisabeth J. Chapman3,28, Greg D.May29, Jixian Zhai30, Matthias Ganssmann30, Sai Guna Ranjan Gurazada30, Marcelo German30, Blake C. Meyers30, Pamela J. Green30 (Leader); Manual annotation and gene family analysis Jennifer N. Bragg1, Ludmila Tyler1,6, Jiajie Wu1,8, Yong Q. Gu1, Gerard R. Lazo1, Debbie Laudencia-Chingcuanco1, James Thomson1, John P. Vogel1 (Leader), Samuel P. Hazen14, Shan Chen14, Henrik V. Scheller31, Jesper Harholt32, Peter Ulvskov32, Samuel E. Fox3, Sergei A. Filichkin3, Noah Fahlgren3, Jeffrey A. Kimbrel3, Jeff H. Chang3, Christopher M. Sullivan3, Elisabeth J. Chapman3,27, James C. Carrington3, Todd C. Mockler3, Laura E. Bartley8,31, Peijian Cao8,31, Ki-Hong Jung8,31, Manoj K Sharma8,31, Miguel Vega-Sanchez8,31, Pamela Ronald8,31, Christopher D.Dardick33, Stefanie De Bodt34,Wim Verelst34, Dirk Inze34, Maren Heese35, Arp Schnittger35, Xiaohan Yang36, Udaya C. Kalluri36, Gerald A. Tuskan36, Zhihua Hua37, Richard D. Vierstra37, David F. Garvin3, Yu Cui24, Shuhong Ouyang24, Qixin Sun24, Zhiyong Liu24, Alper Yilmaz38, Erich Grotewold38, Richard Sibout39, Kian Hematy39, Gregory Mouille39, Herman Höfte39, Todd Michael13, Jérome Pelloux40, Devin O'Connor41, James Schnable41, Scott Rowe41, Frank Harmon41, Cynthia L. Cass42, John C. Sedbrook42, Mary E. Byrne7, Sean Walsh7, Janet Higgins7, Michael Bevan7, Pinghua Li19, Thomas Brutnell19, Turgay Unver43,Hikmet Budak43, Harry Belcram44, Mathieu Charles44, Boulos Chalhoub44, Ivan Baxter45 1 USDA-ARS Western Regional Research Center, Albany, California 94710, USA. 2 USDA-ARS Plant Science Research Unit and University of Minnesota, St Paul, Minnesota 55108, USA. 3 Oregon State University, Corvallis, Oregon 97331-4501, USA. 4 HudsonAlpha Institute, Huntsville, Alabama 35806, USA. 5 US DOE Joint Genome Institute, Walnut Creek, California 94598, USA. 6 University of California Berkeley, Berkeley, California 94720, USA. 7 John Innes Centre, Norwich NR4 7UJ, UK. 8 University of California Davis, Davis, California 95616, USA. 9 University of Silesia, 40-032 Katowice, Poland. 10 Iowa State University, Ames, Iowa 50011, USA. 11 Washington State University, Pullman, Washington 99163, USA. 12 University of Florida, Gainsville, Florida 32611, USA. 13 Rutgers University, Piscataway, New Jersey 08855-0759, USA. 14 University of Massachusetts, Amherst, Massachusetts 01003-9292, USA. 15 USDA-ARS Vegetable Crops Research Unit, Horticulture Department, University of Wisconsin, Madison, Wisconsin 53706, USA. 16 Helmholtz Zentrum München, D-85764 Neuherberg, Germany. 17 Technical University München, 80333 München, Germany. 18 Cornell University, Ithaca, New York 14853, USA. 19 Boyce Thompson Institute for Plant Research, Ithaca, New York 14853-1801, USA. 20 University of Zurich, 8008 Zurich, Switzerland. 21 MTT Agrifood Research and University of Helsinki, FIN-00014 Helsinki, Finland. 22 Federal University of Pelotas, Pelotas, 96001-970, RS, Brazil. 23 Michigan State University, East Lansing, Michigan 48824, USA. 24 China Agricultural University, Beijing 10094, China. 25 Purdue University, West Lafayette, Indiana 47907, USA. 26 The University of Texas, Arlington, Arlington, Texas 76019, USA. 27 Institut National de la Recherche Agronomique UMR 1095, 63100 Clermont-Ferrand, France. 28 University of California San Diego, La Jolla, California 92093, USA. 29 National Centre for Genome Resources, Santa Fe, New Mexico 87505, USA. 30 University of Delaware, Newark, Delaware 19716, USA. 31 Joint Bioenergy Institute, Emeryville, California 94720, USA. 32 University of Copenhagen, Frederiksberg DK-1871, Denmark. 33 USDA-ARS Appalachian Fruit Research Station, Kearneysville, West Virginia 25430, USA. 34 VIB Department of Plant Systems Biology, VIB and Department of Plant Biotechnology and Genetics, Ghent University, Technologiepark 927, 9052 Gent, Belgium. 35 Institut de Biologie Moléculaire des Plantes du CNRS, Strasbourg 67084, France. 36 BioEnergy Science Center and Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-6422, USA. 37 University of Wisconsin-Madison, Madison, Wisconsin 53706, USA. 38 The Ohio State University, Columbus, Ohio 43210, USA. 39 Institut Jean-Pierre Bourgin, UMR1318, Institut National de la Recherche Agronomique, 78026 Versailles cedex, France. 40 Université de Picardie, Amiens 80039, France. 41 Plant Gene Expression Center, University of California Berkeley, Albany, California 94710, USA. 42 Illinois State University and DOE Great Lakes Bioenergy Research Center, Normal, Illinois 61790, USA. 43 Sabanci University, Istanbul 34956, Turkey. 44 Unité de Recherche en Génomique Végétale: URGV (INRA-CNRS-UEVE), Evry 91057, France. 45 USDA-ARS/Donald Danforth Plant Science Center, St Louis, Missouri 63130, USA.; Three subfamilies of grasses, the Ehrhartoideae, Panicoideae and Pooideae, provide the bulk of human nutrition and are poised to become major sources of renewable energy. Here we describe the genome sequence of the wild grass Brachypodium distachyon (Brachypodium), which is, to our knowledge, the first member of the Pooideae subfamily to be sequenced. Comparison of the Brachypodium, rice and sorghum genomes shows a precise history of genome evolution across a broad diversity of the grasses, and establishes a template for analysis of the large genomes of economically important pooid grasses such as wheat. The high-quality genome sequence, coupled with ease of cultivation and transformation, small size and rapid life cycle, will help Brachypodium reach its potential as an important model system for developing new energy and food crops.

The genus Phytophthora consists of many notorious pathogens of crops and forestry trees. At present, battling Phytophthora diseases is challenging due to a lack of understanding of their pathogenesis. We investigated the role of small RNAs in regulating soybean defense in response to infection by Phytophthora sojae, the second most destructive pathogen of soybean. Small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), are universal regulators that repress target gene expression in eukaryotes. We identified known and novel small RNAs that differentially accumulated during P.sojae infection in soybean roots. Among them, miR393 and miR166 were induced by heat-inactivated P.sojae hyphae, indicating that they may be involved in soybean basal defense. Indeed, knocking down the level of mature miR393 led to enhanced susceptibility of soybean to P.sojae; furthermore, the expression of isoflavonoid biosynthetic genes was drastically reduced in miR393 knockdown roots. These data suggest that miR393 promotes soybean defense against P.sojae. In addition to miRNAs, P.sojae infection also resulted in increased accumulation of phased siRNAs (phasiRNAs) that are predominantly generated from canonical resistance genes encoding nucleotide binding-leucine rich repeat proteins and genes encoding pentatricopeptide repeat-containing proteins. This work identifies specific miRNAs and phasiRNAs that regulate defense-associated genes in soybean during Phytophthora infection.

Additional file 5:Network of lysine and aspartate family amino acid biosynthesis and degradation. http://www.gramene.org . Only the genes with 5-fold up- or down-regulation in Cypress (PSC) or Ilpumbyeo (PSI) compared with that in LaGrue or YR15965 are shown. The positive number in parenthesis indicates up-regulation and the negative number in parenthesis indicates down-regulation. The first value in parenthesis shows the fold change in expression either in LaGrue or YR15965, and the second value shows the fold change in expression in Nipponbare. The italicized and underlined bold number before the parenthesis shows the MPSS/SBS signature class [45]. Green indicates that the gene was identified by SBS only. Red indicates that the gene was identified by MPSS only. Blue indicates that the gene was identified by both MPSS and SBS. (PPT 261 KB)

Additional file 5:Network of lysine and aspartate family amino acid biosynthesis and degradation. http://www.gramene.org . Only the genes with 5-fold up- or down-regulation in Cypress (PSC) or Ilpumbyeo (PSI) compared with that in LaGrue or YR15965 are shown. The positive number in parenthesis indicates up-regulation and the negative number in parenthesis indicates down-regulation. The first value in parenthesis shows the fold change in expression either in LaGrue or YR15965, and the second value shows the fold change in expression in Nipponbare. The italicized and underlined bold number before the parenthesis shows the MPSS/SBS signature class [45]. Green indicates that the gene was identified by SBS only. Red indicates that the gene was identified by MPSS only. Blue indicates that the gene was identified by both MPSS and SBS. (PPT 261 KB)