1 GLOBAL ALTERATION OF MICRORNAS AND TRANSPOSON-DERIVED SMALL RNAS IN COTTON (GOSSYPIUM HIRSUTUM) DURING COTTON LEAFROLL DWARF POLEROVIRUS (CLRDV) INFECTION Elisson Romanel 1, Tatiane da Franca Silva 1, Régis L. Corrêa 1, Laurent Farinelli 2, Jennifer Hawkins 3, Carlos E. G. Schrago 1 & Maite F. S. Vaslin 1 1 -UFRJ. Rio de Janeiro. Brazil, 2 -Fasteris Co., Plan-les-Ouates. Switzerland, 3 -West Virginia University. Morgantown. United States Laboratório de Virologia Molecular Vegetal Depto. Virologia, IMPPG - Universidade Federal do Rio de Janeiro Rio de Janeiro, Brasil Profa. Maïté Vaslin F. Silva
2 Cotton: Gossypium hirsutum L. Family: Malvaceae The most widely produced natural fiber in the world; Represents about 40 per cent of the world textile market
3 Cotton Diseases Caused by Viruses: Leaf crumple/leaf curl - Cotton leaf curl virus (CLCuV), Geminivirus Anthocyanosis Blue disease
4 Blue Disease in the World Cotton blue disease in Brazil Associated damages: - Susceptible cvs. planted in cerrado until end of 90s - Losses around 1500 kg.ha-1 in MT at GO - 91% of productivity per plant reduction -Cotton weight (-53,3%) e fiber (-43,0%). -In soft infections: reduction of 36%
5 Symptoms of CBD are leaf rolling, intense green foliage, vein yellowing and a severe to moderate stunting caused by internodal shortening.
6 How CLRDV (casual agent of CBD) can led to the strong developmental alterations observed in infected plants? Each mechanisms are involved? mirna are an important class of gene expression regulation RNA working in several developmental genes downregulation So, how are mirnas profiles during virus infection??
7 ICGI 2012 Expression of Cotton DCL ribonucleases during infection Quantitative RT-PCR analysis. Expression levels of Gh-miR162 (A) and cotton Dcl mrnas (B) in infected and uninfected plants. Silva et al. BMC Molecular Biology 2011, 12:40
12 Profile of all small RNAs from the infected and the uninfected libraries 24-nt 21-nt predominant classes Size distribution of sequenced Gossypium hirsutum small RNAs (srnas) in uninfected and infected leaves. Size distribution of unique (only one read) (A) and redundant (B) cotton srnas from the uninfected (light gray bars) and infected (dark gray bars) libraries. Histograms represent the number srna reads.
13 Is the profile of cellular non-coding RNA altered by virus infection? Identification of distinct non-coding cellular and viral small RNA (srna) sequences in the Gossypium hirsutum uninfected (UIL) and infected (IL) libraries. redundant cotton srnas unique cotton srnas the number of redundant cprna and rrna reads was drastically decreased in the IL compared with the UIL Histograms represent the number of reads within each length class mapping to known cellular and viral RNAs in the uninfected (left) and infected libraries (right) (asterisk)
15 deep sequencing the small RNAs Small RNAs Uninfected cotton Remoção de adaptadores 5 e 3 Small RNAs Infected cotton Elimination of cellular non-coding RNAs rrna, trna, snorna, mtrna e cprna BACs sequences (Cotton DB) Cotton EST (CGI-TIGR) G. raimondii WGS (500 Mb) mirna identification(pmrd) Identification of mirna precursors (mfold) mirnas tagets
16 60 mirna families/ 19 new/novel
17 Differential expression of 21-nt Ghr-miRNAs during virus infection Ghr-miR159 (74,8% of total mirnas) and Ghr-miR3476 (10%) families are overexpressed in UIL libraries
18 Differential expression of 21-nt Ghr-miRNAs during virus infection deep sequencing results validation by RT-qPCR mir162: DCL1 regulation mir159: leaf development mir472: NBS-LRR ptns (Todesco et al., 2010) mir3476: Descritos somente em algodão mir2118, 2910, 2914: Pela primeira vez descritos
19 mir163 (20nt) and mir319, 525 and 3476 (of 22nt) are drastically reduced
20 Target prediction of the mirnas differentially expressed during infection Target prediction: psrnatarget Ghr-miRNAs Sequences 5`- 3' Number of reads Uninfected Infected Gene ID Putative target functions 156ª UUGACAGAAGAGAGUGAGCAC 37,97 110,72 TC Proteína de ligação ao promotor do gene Squamosa C,D,E 157b UUGACAGAAGAUAGAGAGCAC 67,50 690,87 TC Proteína de ligação ao promotor do gene Squamosa B,C,D,E 159ª UUUGGAUUGAAGGGAGCUCUA , ,57 TC ATP sintase F0 C 160ª UGCCUGGCUCCCUGUAUGCCA 978,80 893,39 TC Fator de resposta a Auxina (ARF) B,C,D,E 162ª UCGAUAAACCUCUGCAUCCAG 374,43 500,64 TC237985; Proteína LETM1; Proteína de transporte de TC Auxina 164ª UGGAGAAGCAGGGCACGUGCA 50,62 125,87 CO Fator transcricional NAC C,D,E 165ª UCGGACCAGGCUUCAUCCCCC 23,20 16,08 TC Fator transcricional HD-ZIP C,D,E 166h UCGGACCAGGCUUCAUUCCCG 10751, ,38 ES Fator transcricional HD-ZIP B,C,D,E 167c UGAAGCUGCCAGCAUGAUCUC 24,25 304,74 DW Proteína com domínio LIM 167g GAAGCUGCCAGCAUGAUCUGG 974,58 207,26 ES TC Fator de resposta a Auxina C,D
21 Identification of putative targets Predicted Ghr-miRNA targets for 50 mirna families, which include genes involved in disease resistance, auxin response, transcription factors, metabolism and metal ion transport.
22 IDENTIFICATION OF PRE-MiCRO RNAs Predicted stem-loop hairpin secondary structures of three new cotton mirnas identified Predição da estrutura secundária mfold: 21 Pré-miRNAs
23 Predicted stem-loop hairpin secondary structures of four novel cotton mirnas
24 reduced frequency of 24-nt srnas in the IL GSS TE libraries from Dr. Jennifer Hawkins, West Virginia University, USA
25 Analysis of cotton 24-nt srnas matching TEs RNAi is necessary to specifically silence TEs through DNA methylation 24-nt srna - methylation A deep-sequencing: 24-nt srnas mapping retrotransposable elements in the uninfected and CLRDV-infected srna libraries. Reads are expressed as reads per 10 million. B Detection of three TEs (Gypsy 2, Copia 1 and Copia 2) transcripts in 5 dpi CLRDV-infected and uninfected cotton leaves by RT-qPCR Copia 2 element was up-regulated in the infected leaf, which correlates well with the observed reduction in the total number of sirnas mapping to this retrotransposon
26 Gypsy-like 2 Copia 1 Fig. 6 The spatial distribution and frequency of cotton 24- nt small RNAs (srnas) along gypsy-like and copia-like retrotransposons in CLRDV-infected and uninfected libraries The distribution of perfectly matching 24-nt srnas along the Gossypium hirsutum Gypsy 2 fulllength sequence in the uninfected (A) and CLRDV-infected (B) libraries. Copia 2
27 Lab. de Virologia Molecular Vegetal Depto. Genética/I.Biologia Prof. Régis Lopes Corrêa Dr. Tatiane da Franca Silva Dr. Jennifer Hawkins, Wesr Virginia University, USA Laurent Farinelli Cécile Deluen Magne Osteras Dr. Elisson Romannel Prof. Carlos Guerra Schrago
UNIT.3 Real-Time PCR Dean Fraga, 1 Tea Meulia, 2 and Steven Fenster 3 1 College of Wooster, Wooster, Ohio 2 Ohio Agricultural Research and Development Center, Wooster, Ohio 3 Ashland University, Ashland,
Insights & Perspectives Introns in UTRs: Why we should stop ignoring them Alicia A. Bicknell 1)y, Can Cenik 1)y, Hon N. Chua 2), Frederick P. Roth 2) and Melissa J. Moore 1)3) * Although introns in 5 0
UNIT.3 Dean Fraga, 1 Tea Meulia, 2 and Steven Fenster 3 1 College of Wooster, Wooster, Ohio 2 Ohio Agricultural Research and Development Center, Wooster, Ohio 3 Ashland University, Ashland, Ohio OVERVIEW
Tan et al. BMC Plant Biology (2015) 15:62 DOI 10.1186/s12870-015-0448-y RESEARCH ARTICLE Open Access Powerful regulatory systems and posttranscriptional gene silencing resist increases in cellulose content
BLOCK-iT RNAi Products BLOCK-iT Products: Powerful tools to advance RNAi analysis With BLOCK-iT RNAi Technology you can: Get powerful blocking and easily generate functional RNAi data Effectively prepare
U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES National Institutes of Health National Institute of General Medical Sciences The New Genetics WHAT IS NIGMS? The National Institute of General Medical Sciences
VIROLOGY 245, 33 46 (1998) ARTICLE NO. VY989146 Internal\Structures Containing Transcriptase-Related Proteins in Top Component Particles of Mammalian Orthoreovirus Kelly A. Dryden,*,,1,2 Diane L. Farsetta,,1
Methodology for Copy Number Variant Detection from High Throughput DNA Exome Sequencing and Application to the Genetic Mapping of Rare Genetic Disorders Case Presentation 3 Word Count - 4996 Michael Epstein
ROTEIN IMORT INTO CHLOROLASTS Jürgen Soll and Enrico Schleiff Chloroplasts are organelles of endosymbiotic origin, and they transferred most of their genetic information to the host nucleus during this
Overexpression of the activated form of the AtAREB1 gene (AtAREB1 QT) improves soybean responses to water deficit J.P. Leite 1, E.G.G. Barbosa 2, S.R.R. Marin 2, J.P. Marinho 3, J.F.C. Carvalho 5, R.F.
Heading/title Health and Safety Executive The SACGM Compendium of guidance Part 4: Genetic modification work that involves plants (including plant-associated genetically modified microorganisms) HSE Books
Gene Selection for Cancer Classification using Support Vector Machines Isabelle Guyon+, Jason Weston+, Stephen Barnhill, M.D.+ and Vladimir Vapnik* +Barnhill Bioinformatics, Savannah, Georgia, USA * AT&T
Chemistry Guide DNA Sequencing by Capillary Electrophoresis Applied Biosystems Chemistry Guide Second Edition Chemistry Guide DNA Sequencing by Capillary Electrophoresis Applied Biosystems Chemistry Guide
The role of pre-mrn secondary structure in gene splicing in Saccharomyces cerevisiae by Sanja Rogic B.Sc., The Faculty of Mathematics, The niversity of Belgrade, 1994 M.Sc., The niversity of British Columbia,
Real-time PCR for mrna quantitation Marisa L. Wong and Juan F. Medrano BioTechniques 39:75-85 (July 2005) Real-time PCR has become one of the most widely used methods of gene quantitation because it has
Baculovirus Expression Vector System Instruction Manual 6th Edition, May 1999 Baculovirus Expression Vector System Manual 6th Edition May 1999 Instruction Manual General Methods 6xHis and GST Purification
PERSPECTIVE doi:10.1038/nature09764 Charting a course for genomic medicine from base pairs to bedside Eric D. Green 1, Mark S. Guyer 1 & National Human Genome Research Institute* There has been much progress
A-Z of quantitative PCR (Editor: SA Bustin) Chapter3. Quantification strategies in real-time PCR 87 Quantification strategies in real-time PCR Michael W. Pfaffl Chaper 3 pages 87-112 in: A-Z of quantitative
N.M. Luscombe, D. Greenbaum, M. Gerstein Department of Molecular Biophysics and Biochemistry Yale University New Haven, USA Review What is bioinformatics? An introduction and overview Abstract: A flood
PYF12 3/21/05 8:04 PM Page 191 Chapter 12 Gene expression and regulation Bacterial genomes usually contain several thousand different genes. Some of the gene products are required by the cell under all
The Automatic Generation of Templates for Automatic Abstracting Michael P. Oakes Computing Department, Lancaster University Lancaster, England Chris. D. Paice Computing Department, Lancaster University
Biologics and biosimilars An overview Contents An introduction to biotechnology... 3 A brief history of medicine development... 4 What are biologic medicines?... 5 How are biologic medicines developed?...
Structural and functional studies on Nod Like Receptors: insights into NAIP/ inflammasome formation Els F. Halff 2013 Structural and functional studies on Nod Like Receptors: insights into NAIP/ inflammasome
Experimental Cell Research 304 (2005) 391 406 www.elsevier.com/locate/yexcr The cytoplasmic C-terminus of polycystin-1 increases cell proliferation in kidney epithelial cells through serum-activated and
6 Immune function and exercise Position Statement Part one: Immune function and exercise Neil P. Walsh 1, Michael Gleeson 2, Roy J. Shephard 3, Maree Gleeson 4 Jeffrey A. Woods 5, Nicolette C. Bishop 2,
Detecting DNA SMRT Analysis of Microbial Methylomes Background Microbial genomes contain a variety of base modifications, most commonly occurring as methylation at adenine or cytosine residues. These modifications
Progress in Neurobiology 66 (2002) 1 18 Neural stem cells and regulation of cell number Lukas Sommer a, Mahendra Rao b, a Institute of Cell Biology, Swiss Federal Institute of Technology, ETH-Hoenggerberg