Small RNA Deep-Sequencing Analyses Reveal a New Regulator of Virulence in Agrobacterium fabrum C58.

Novel ways of regulating Ti plasmid functions were investigated by studying small RNAs (sRNAs) that are known to act as posttranscriptional regulators in plant pathogenic bacteria. sRNA-seq analyses of Agrobacterium fabrum C58 allowed us to identify 1,108 small transcripts expressed in several growth conditions that could be sRNAs. A quarter of them were confirmed by bioinformatics or by biological experiments. Antisense RNAs represent 24% of the candidates and they are over-represented on the pTi (with 62% of pTi sRNAs), suggesting differences in the regulatory mechanisms between the essential and accessory replicons. Moreover, a large number of these pTi antisense RNAs are transcribed opposite to those genes involved in virulence. Others are 5'- and 3'-untranslated region RNAs and trans-encoded RNAs. We have validated, by rapid amplification of cDNA ends polymerase chain reaction, the transcription of 14 trans-encoded RNAs, among which RNA1111 is expressed from the pTiC58. Its deletion decreased the aggressiveness of A. fabrum C58 on tomatoes, tobaccos, and kalanchoe, suggesting that this sRNA activates virulence. The identification of its putative target mRNAs (6b gene, virC2, virD3, and traA) suggests that this sRNA may coordinate two of the major pTi functions, the infection of plants and its dissemination among bacteria.

[1]  J. Belasco,et al.  Messenger RNA degradation in bacterial cells. , 2014, Annual review of genetics.

[2]  Konrad U. Förstner,et al.  Profound Impact of Hfq on Nutrient Acquisition, Metabolism and Motility in the Plant Pathogen Agrobacterium tumefaciens , 2014, PloS one.

[3]  Q. Zeng,et al.  Genome-wide identification of Hfq-regulated small RNAs in the fire blight pathogen Erwinia amylovora discovered small RNAs with virulence regulatory function , 2014, BMC Genomics.

[4]  Patrick R. Wright,et al.  Two separate modules of the conserved regulatory RNA AbcR1 address multiple target mRNAs in and outside of the translation initiation region , 2014, RNA biology.

[5]  L. Paulin,et al.  Phylogeny of the Rhizobium-Allorhizobium-Agrobacterium clade supports the delineation of Neorhizobium gen. nov. , 2014, Systematic and applied microbiology.

[6]  R. Giegerich,et al.  Riboregulation in plant-associated α-proteobacteria , 2014, RNA biology.

[7]  Andreas S. Richter,et al.  Bioinformatics of prokaryotic RNAs , 2014, RNA biology.

[8]  T. Rattei,et al.  Ultra Deep Sequencing of Listeria monocytogenes sRNA Transcriptome Revealed New Antisense RNAs , 2014, PloS one.

[9]  J. Kalinowski,et al.  Comprehensive discovery and characterization of small RNAs in Corynebacterium glutamicum ATCC 13032 , 2013, BMC Genomics.

[10]  Jian-Bing Fan,et al.  A Genome-Wide Survey of Highly Expressed Non-Coding RNAs and Biological Validation of Selected Candidates in Agrobacterium tumefaciens , 2013, PloS one.

[11]  B. Tjaden,et al.  Computational analysis of bacterial RNA-Seq data , 2013, Nucleic acids research.

[12]  J. Coppee,et al.  Genome-Wide Identification of Regulatory RNAs in the Human Pathogen Clostridium difficile , 2013, PLoS genetics.

[13]  R. Giegerich,et al.  Global mapping of transcription start sites and promoter motifs in the symbiotic α-proteobacterium Sinorhizobium meliloti 1021 , 2013, BMC Genomics.

[14]  É. Massé,et al.  New insights into small RNA-dependent translational regulation in prokaryotes. , 2013, Trends in genetics : TIG.

[15]  Stefan Engelen,et al.  MicroScope—an integrated microbial resource for the curation and comparative analysis of genomic and metabolic data , 2012, Nucleic Acids Res..

[16]  Wuju Li,et al.  Predicting sRNAs and Their Targets in Bacteria , 2012, Genom. Proteom. Bioinform..

[17]  S. Molin,et al.  Genome-wide identification of novel small RNAs in Pseudomonas aeruginosa. , 2012, Environmental microbiology.

[18]  F. Narberhaus,et al.  Hfq Influences Multiple Transport Systems and Virulence in the Plant Pathogen Agrobacterium tumefaciens , 2012, Journal of bacteriology.

[19]  M. Nowrousian,et al.  Deep sequencing uncovers numerous small RNAs on all four replicons of the plant pathogen Agrobacterium tumefaciens , 2012, RNA biology.

[20]  P. Stadler,et al.  Genome-wide transcriptome analysis of the plant pathogen Xanthomonas identifies sRNAs with putative virulence functions , 2011, Nucleic acids research.

[21]  G. Storz,et al.  Regulation by small RNAs in bacteria: expanding frontiers. , 2011, Molecular cell.

[22]  F. Hommais,et al.  Genomic Species Are Ecological Species as Revealed by Comparative Genomics in Agrobacterium tumefaciens , 2011, Genome biology and evolution.

[23]  Qian Liu,et al.  sTarPicker: A Method for Efficient Prediction of Bacterial sRNA Targets Based on a Two-Step Model for Hybridization , 2011, PloS one.

[24]  Peter F. Stadler,et al.  RNApredator: fast accessibility-based prediction of sRNA targets , 2011, Nucleic Acids Res..

[25]  É. Massé,et al.  New insights into riboswitch regulation mechanisms , 2011, Molecular microbiology.

[26]  B. Voß,et al.  Small RNA‐mediated control of the Agrobacterium tumefaciens GABA binding protein , 2011, Molecular microbiology.

[27]  J. Vogel,et al.  An experimentally anchored map of transcriptional start sites in the model cyanobacterium Synechocystis sp. PCC6803 , 2011, Proceedings of the National Academy of Sciences.

[28]  R. Giegerich,et al.  A genome-wide survey of sRNAs in the symbiotic nitrogen-fixing alpha-proteobacterium Sinorhizobium meliloti , 2010, BMC Genomics.

[29]  H. Hirt,et al.  New insights into an old story: Agrobacterium‐induced tumour formation in plants by plant transformation , 2010, The EMBO journal.

[30]  C. Buchrieser,et al.  A trans-Acting Riboswitch Controls Expression of the Virulence Regulator PrfA in Listeria monocytogenes , 2009, Cell.

[31]  Samuel A. Assefa,et al.  A Strand-Specific RNA–Seq Analysis of the Transcriptome of the Typhoid Bacillus Salmonella Typhi , 2009, PLoS genetics.

[32]  J. Glover,et al.  Agrobacterium tumefaciens VirC2 enhances T-DNA transfer and virulence through its C-terminal ribbon–helix–helix DNA-binding fold , 2009, Proceedings of the National Academy of Sciences.

[33]  D. Faure,et al.  Different regulation and roles of lactonases AiiB and AttM in Agrobacterium tumefaciens C58. , 2009, Molecular plant-microbe interactions : MPMI.

[34]  M. Lawrence,et al.  Experimental discovery of sRNAs in Vibrio cholerae by direct cloning, 5S/tRNA depletion and parallel sequencing , 2009, Nucleic acids research.

[35]  Rolf Backofen,et al.  IntaRNA: efficient prediction of bacterial sRNA targets incorporating target site accessibility and seed regions , 2008, Bioinform..

[36]  J. Livny,et al.  Prediction of Sinorhizobium meliloti sRNA genes and experimental detection in strain 2011 , 2008, BMC Genomics.

[37]  F. Hommais,et al.  PecS Is a Global Regulator of the Symptomatic Phase in the Phytopathogenic Bacterium Erwinia chrysanthemi 3937 , 2008, Journal of bacteriology.

[38]  F. Barloy-Hubler,et al.  Identification of chromosomal alpha-proteobacterial small RNAs by comparative genome analysis and detection in Sinorhizobium meliloti strain 1021 , 2007, BMC Genomics.

[39]  E. Rivas,et al.  Identification of differentially expressed small non-coding RNAs in the legume endosymbiont Sinorhizobium meliloti by comparative genomics , 2007, Molecular microbiology.

[40]  Hironaka Tsukagoshi,et al.  An Oncoprotein from the Plant Pathogen Agrobacterium Has Histone Chaperone–Like Activity[W] , 2007, The Plant Cell Online.

[41]  J. Zoń,et al.  Abnormal accumulation of sugars and phenolics in tobacco roots expressing the Agrobacterium T-6b oncogene and the role of these compounds in 6b-induced growth. , 2007, Molecular plant-microbe interactions : MPMI.

[42]  Robert G. Martin,et al.  Detection of low-level promoter activity within open reading frame sequences of Escherichia coli , 2005, Nucleic acids research.

[43]  S. C. Winans,et al.  VirA and VirG activate the Ti plasmid repABC operon, elevating plasmid copy number in response to wound-released chemical signals. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[44]  S. C. Winans,et al.  A small antisense RNA downregulates expression of an essential replicase protein of an Agrobacterium tumefaciens Ti plasmid , 2005, Molecular microbiology.

[45]  Jean YH Yang,et al.  Bioconductor: open software development for computational biology and bioinformatics , 2004, Genome Biology.

[46]  T. Fujita,et al.  The Protein Encoded by Oncogene 6b from Agrobacterium tumefaciens Interacts with a Nuclear Protein of Tobacco Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.010360. , 2002, The Plant Cell Online.

[47]  J A Eisen,et al.  The Genome of the Natural Genetic Engineer Agrobacterium tumefaciens C58 , 2001, Science.

[48]  J. Mullikin,et al.  SSAHA: a fast search method for large DNA databases. , 2001, Genome research.

[49]  Kim Rutherford,et al.  Artemis: sequence visualization and annotation , 2000, Bioinform..

[50]  A. Das,et al.  The Agrobacterium tumefaciens virD3 gene is not essential for tumorigenicity on plants , 1992, Journal of bacteriology.

[51]  C. Kado,et al.  Vir box sequences in Agrobacterium tumefaciens pTiC58 and A6. , 1988, Nucleic acids research.

[52]  M. Montagu,et al.  Substrate induction of conjugative activity of Agrobacterium tumefaciens Ti plasmids , 1978, Nature.

[53]  N. Chua,et al.  Molecular insights into plant cell proliferation disturbance by Agrobacterium protein 6b. , 2011, Genes & development.

[54]  I. Galis,et al.  Reduction of polar auxin transport in tobacco by the tumorigenic Agrobacterium tumefaciens AK-6b gene , 2005, Planta.

[55]  B. Goldman,et al.  Genome Sequence of the Plant Pathogen and Biotechnology Agent Agrobacterium tumefaciens C58 , 2001, Science.