In Vitro Acquisition of Specific Small Interfering RNAs Inhibits the Expression of Some Target Genes in the Plant Ectoparasite Xiphinema index

Xiphinema index is an important plant parasitic nematode that induces direct damages and specifically transmits the Grapevine fanleaf virus, which is particularly harmful for grapevines. Genomic resources of this nematode species are still limited and no functional gene validation technology is available. RNA interference (RNAi) is a powerful technology to study gene function and here we describe the application of RNAi on several genes in X. index. Soaking the nematodes for 48 h in a suspension containing specific small interfering RNAs resulted in a partial inhibition of the accumulation of some targeted mRNA. However, low reproducible silencing efficiency was observed which could arise from X. index silencing pathway deficiencies. Indeed, essential accustomed proteins for these pathways were not found in the X. index proteome predicted from transcriptomic data. The most reproducible silencing effect was obtained when targeting the piccolo gene potentially involved in endo-exocytosis of synaptic molecules. This represents the first report of gene silencing in a nematode belonging to the Longidoridae family

The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics

The Carbohydrate-Active Enzyme (CAZy) database is a knowledge-based resource specialized in the enzymes that build and breakdown complex carbohydrates and glycoconjugates. As of September 2008, the database describes the present knowledge on 113 glycoside hydrolase, 91 glycosyltransferase, 19 polysaccharide lyase, 15 carbohydrate esterase and 52 carbohydrate-binding module families. These families are created based on experimentally characterized proteins and are populated by sequences from public databases with significant similarity. Protein biochemical information is continuously curated based on the available literature and structural information. Over 6400 proteins have assigned EC numbers and 700 proteins have a PDB structure. The classification (i) reflects the structural features of these enzymes better than their sole substrate specificity, (ii) helps to reveal the evolutionary relationships between these enzymes and (iii) provides a convenient framework to understand mechanistic properties. This resource has been available for over 10 years to the scientific community, contributing to information dissemination and providing a transversal nomenclature to glycobiologists. More recently, this resource has been used to improve the quality of functional predictions of a number genome projects by providing expert annotation. The CAZy resource resides at URL: http://www.cazy.org/

Intraspecific Variation in Vertical Habitat Use by Tiger Sharks (Galeocerdo cuvier) in the Western North Atlantic

Tiger sharks (Galeocerdo cuvier) are a wide ranging, potentially keystone predator species that display a variety of horizontal movement patterns, making use of coastal and pelagic waters. Far less, however, is known about their vertical movements and use of the water column. We used pop-up satellite archival tags with two data sampling rates (high rate and standard rate tags) to investigate the vertical habitat use and diving behavior of tiger sharks tagged on the Puerto Rico–Virgin Islands platform and off Bermuda between 2008 and 2009. Useable data were received from nine of 14 sharks tagged, tracked over a total of 529 days. Sharks spent the majority of their time making yo-yo dives within the upper 50 m of the water column and considerable time within the upper 5 m of the water column. As a result, sharks typically occupied a narrow daily temperature range (~2°C). Dives to greater than 200 m were common, and all sharks made dives to at least 250 m, with one shark reaching a depth of 828 m. Despite some similarities among individuals, a great deal of intraspecific variability in vertical habit use was observed. Four distinct depth distributions that were not related to tagging location, horizontal movements, sex, or size were detected. In addition, similar depth distributions did not necessitate similar dive patterns among sharks. Recognition of intraspecific variability in habitat use of top predators can be crucial for effective management of these species and for understanding their influence on ecosystem dynamics

Evidence for a novel coding sequence overlapping the 5'-terminal ~90 codons of the Gill-associated and Yellow head okavirus envelope glycoprotein gene

The genus Okavirus (order Nidovirales) includes a number of viruses that infect crustaceans, causing major losses in the shrimp industry. These viruses have a linear positive-sense ssRNA genome of ~26-27 kb, encoding a large replicase polyprotein that is expressed from the genomic RNA, and several additional proteins that are expressed from a nested set of 3'-coterminal subgenomic RNAs. In this brief report, we describe the bioinformatic discovery of a new, apparently coding, ORF that overlaps the 5' end of the envelope glycoprotein encoding sequence, ORF3, in the +2 reading frame. The new ORF has a strong coding signature and, in fact, is more conserved at the amino acid level than the overlapping region of ORF3. We propose that translation of the new ORF initiates at a conserved AUG codon separated by just 2 nt from the ORF3 AUG initiation codon, resulting in a novel 86 amino acid protein

An exploration of ambigrammatic sequences in narnaviruses

Narnaviruses have been described as positive-sense RNA viruses with a remarkably simple genome of ~3 kb, encoding only a highly conserved RNA-dependent RNA polymerase (RdRp). Many narnaviruses, however, are 'ambigrammatic' and harbour an additional uninterrupted open reading frame (ORF) covering almost the entire length of the reverse complement strand. No function has been described for this ORF, yet the absence of stops is conserved across diverse narnaviruses, and in every case the codons in the reverse ORF and the RdRp are aligned. The >3 kb ORF overlap on opposite strands, unprecedented among RNA viruses, motivates an exploration of the constraints imposed or alleviated by the codon alignment. Here, we show that only when the codon frames are aligned can all stop codons be eliminated from the reverse strand by synonymous single-nucleotide substitutions in the RdRp gene, suggesting a mechanism for de novo gene creation within a strongly conserved amino-acid sequence. It will be fascinating to explore what implications this coding strategy has for other aspects of narnavirus biology. Beyond narnaviruses, our rapidly expanding catalogue of viral diversity may yet reveal additional examples of this broadly-extensible principle for ambigrammatic-sequence development

Candidates in Astroviruses, Seadornaviruses, Cytorhabdoviruses and Coronaviruses for +1 frame overlapping genes accessed by leaky scanning

<p>Abstract</p> <p>Background</p> <p>Overlapping genes are common in RNA viruses where they serve as a mechanism to optimize the coding potential of compact genomes. However, annotation of overlapping genes can be difficult using conventional gene-finding software. Recently we have been using a number of complementary approaches to systematically identify previously undetected overlapping genes in RNA virus genomes. In this article we gather together a number of promising candidate new overlapping genes that may be of interest to the community.</p> <p>Results</p> <p>Overlapping gene predictions are presented for the astroviruses, seadornaviruses, cytorhabdoviruses and coronaviruses (families <it>Astroviridae</it>, <it>Reoviridae</it>, <it>Rhabdoviridae </it>and <it>Coronaviridae</it>, respectively).</p

Intron retention in the Drosophila melanogaster Rieske iron sulphur protein gene generated a new protein

Genomes can encode a variety of proteins with unrelated architectures and activities. It is known that protein-coding genes of de novo origin have significantly contributed to this diversity. However, the molecular mechanisms and evolutionary processes behind these originations are still poorly understood. Here we show that the last 102 codons of a novel gene, Noble, assembled directly from non-coding DNA following an intronic deletion that induced alternative intron retention at the Drosophila melanogaster Rieske Iron Sulphur Protein (RFeSP) locus. A systematic analysis of the evolutionary processes behind the origin of Noble showed that its emergence was strongly biased by natural selection on and around the RFeSP locus. Noble mRNA is shown to encode a bona fide protein that lacks an iron sulphur domain and localizes to mitochondria. Together, these results demonstrate the generation of a novel protein at a naturally selected site

The Hexamer Structure of the Rift Valley Fever Virus Nucleoprotein Suggests a Mechanism for its Assembly into Ribonucleoprotein Complexes

Rift Valley fever virus (RVFV), a Phlebovirus with a genome consisting of three single-stranded RNA segments, is spread by infected mosquitoes and causes large viral outbreaks in Africa. RVFV encodes a nucleoprotein (N) that encapsidates the viral RNA. The N protein is the major component of the ribonucleoprotein complex and is also required for genomic RNA replication and transcription by the viral polymerase. Here we present the 1.6 Å crystal structure of the RVFV N protein in hexameric form. The ring-shaped hexamers form a functional RNA binding site, as assessed by mutagenesis experiments. Electron microscopy (EM) demonstrates that N in complex with RNA also forms rings in solution, and a single-particle EM reconstruction of a hexameric N-RNA complex is consistent with the crystallographic N hexamers. The ring-like organization of the hexamers in the crystal is stabilized by circular interactions of the N terminus of RVFV N, which forms an extended arm that binds to a hydrophobic pocket in the core domain of an adjacent subunit. The conformation of the N-terminal arm differs from that seen in a previous crystal structure of RVFV, in which it was bound to the hydrophobic pocket in its own core domain. The switch from an intra- to an inter-molecular interaction mode of the N-terminal arm may be a general principle that underlies multimerization and RNA encapsidation by N proteins from Bunyaviridae. Furthermore, slight structural adjustments of the N-terminal arm would allow RVFV N to form smaller or larger ring-shaped oligomers and potentially even a multimer with a super-helical subunit arrangement. Thus, the interaction mode between subunits seen in the crystal structure would allow the formation of filamentous ribonucleocapsids in vivo. Both the RNA binding cleft and the multimerization site of the N protein are promising targets for the development of antiviral drugs

The genome of the yellow potato cyst nematode, Globodera rostochiensis, reveals insights into the basis of parasitism and virulence

BACKGROUND: The yellow potato cyst nematode, Globodera rostochiensis, is a devastating plant pathogen of global economic importance. This biotrophic parasite secretes effectors from pharyngeal glands, some of which were acquired by horizontal gene transfer, to manipulate host processes and promote parasitism. G. rostochiensis is classified into pathotypes with different plant resistance-breaking phenotypes. RESULTS: We generate a high quality genome assembly for G. rostochiensis pathotype Ro1, identify putative effectors and horizontal gene transfer events, map gene expression through the life cycle focusing on key parasitic transitions and sequence the genomes of eight populations including four additional pathotypes to identify variation. Horizontal gene transfer contributes 3.5 % of the predicted genes, of which approximately 8.5 % are deployed as effectors. Over one-third of all effector genes are clustered in 21 putative ‘effector islands’ in the genome. We identify a dorsal gland promoter element motif (termed DOG Box) present upstream in representatives from 26 out of 28 dorsal gland effector families, and predict a putative effector superset associated with this motif. We validate gland cell expression in two novel genes by in situ hybridisation and catalogue dorsal gland promoter element-containing effectors from available cyst nematode genomes. Comparison of effector diversity between pathotypes highlights correlation with plant resistance-breaking. CONCLUSIONS: These G. rostochiensis genome resources will facilitate major advances in understanding nematode plant-parasitism. Dorsal gland promoter element-containing effectors are at the front line of the evolutionary arms race between plant and parasite and the ability to predict gland cell expression a priori promises rapid advances in understanding their roles and mechanisms of action.SE-vdA is supported by BBSRC grant BB/M014207/1. Sequencing was funded by BBSRC grant BB/F000642/1 to the University of Leeds and grant BB/F00334X/1 to the Wellcome Trust Sanger Institute). DRL was supported by a fellowship from The James Hutton Institute and the School of Biological Sciences, University of Edinburgh. GK was supported by a BBSRC PhD studentship. The James Hutton Institute receives funding from the Scottish Government. JAC and NEH are supported by the Wellcome Trust through its core funding of the Wellcome Trust Sanger Institute (grant 098051). This work was also supported by funding from the Canadian Safety and Security Program, project number CRTI09_462RD

Evolutionary origins of Brassicaceae specific genes in Arabidopsis thaliana

<p>Abstract</p> <p>Background</p> <p>All sequenced genomes contain a proportion of lineage-specific genes, which exhibit no sequence similarity to any genes outside the lineage. Despite their prevalence, the origins and functions of most lineage-specific genes remain largely unknown. As more genomes are sequenced opportunities for understanding evolutionary origins and functions of lineage-specific genes are increasing.</p> <p>Results</p> <p>This study provides a comprehensive analysis of the origins of lineage-specific genes (LSGs) in <it>Arabidopsis thaliana </it>that are restricted to the Brassicaceae family. In this study, lineage-specific genes within the nuclear (1761 genes) and mitochondrial (28 genes) genomes are identified. The evolutionary origins of two thirds of the lineage-specific genes within the <it>Arabidopsis thaliana </it>genome are also identified. Almost a quarter of lineage-specific genes originate from non-lineage-specific paralogs, while the origins of ~10% of lineage-specific genes are partly derived from DNA exapted from transposable elements (twice the proportion observed for non-lineage-specific genes). Lineage-specific genes are also enriched in genes that have overlapping CDS, which is consistent with such novel genes arising from overprinting. Over half of the subset of the 958 lineage-specific genes found only in <it>Arabidopsis thaliana </it>have alignments to intergenic regions in <it>Arabidopsis lyrata</it>, consistent with either <it>de novo </it>origination or differential gene loss and retention, with both evolutionary scenarios explaining the lineage-specific status of these genes. A smaller number of lineage-specific genes with an incomplete open reading frame across different <it>Arabidopsis thaliana </it>accessions are further identified as accession-specific genes, most likely of recent origin in <it>Arabidopsis thaliana</it>. Putative <it>de novo </it>origination for two of the <it>Arabidopsis thaliana</it>-only genes is identified via additional sequencing across accessions of <it>Arabidopsis thaliana </it>and closely related sister species lineages. We demonstrate that lineage-specific genes have high tissue specificity and low expression levels across multiple tissues and developmental stages. Finally, stress responsiveness is identified as a distinct feature of Brassicaceae-specific genes; where these LSGs are enriched for genes responsive to a wide range of abiotic stresses.</p> <p>Conclusion</p> <p>Improving our understanding of the origins of lineage-specific genes is key to gaining insights regarding how novel genes can arise and acquire functionality in different lineages. This study comprehensively identifies all of the Brassicaceae-specific genes in <it>Arabidopsis thaliana </it>and identifies how the majority of such lineage-specific genes have arisen. The analysis allows the relative importance (and prevalence) of different evolutionary routes to the genesis of novel ORFs within lineages to be assessed. Insights regarding the functional roles of lineage-specific genes are further advanced through identification of enrichment for stress responsiveness in lineage-specific genes, highlighting their likely importance for environmental adaptation strategies.</p