1,721,065 research outputs found
Next generation sequencing for studying viruses and RNA silencing-based antiviral defense in crop plants
Full text linkThe main objectives of this work have been to use next generation sequencing (NGS) and develop bioinformatics tools for plant virus diagnostics and genome reconstruction as well as for investigation of RNA silencing-based antiviral defense. In virus-infected plants, the host Dicer-like (DCL) enzymes process viral double-stranded RNAs into 21-24 nucleotide (nt) short interfering RNAs (siRNAs) which can potentially associate with Argonaute (AGO) proteins and guide the resulting RNA-induce silencing complexes (RISCs) to target complementary viral RNA for post-transcriptional silencing and, in the case of DNA viruses, complementary viral DNA for transcriptional silencing. In the pioneering work, Kreuze et al. (2009) have demonstrated that an RNA virus genome can be reconstructed from multiple siRNA contigs generated by the short sequencing read assembler Velvet.
In this PhD study, we developed a bioinformatics pipeline to analyze viral siRNA populations in various model and crop plants experimentally infected with known viruses and naturally infected with unknown viruses. First, we developed a bioinformatics tool (MISIS) to view and analyze maps of small RNAs derived from viruses and genomic loci that generate multiple small RNAs (Seguin et al. 2014b). Using MISIS, we discovered that viral siRNAs cover the entire genomes of RNA and DNA viruses as well as viroids in both sense and antisense orientation without gaps (Aregger et al. 2012; Seguin et al. 2014a; Rajeswaran et al. 2014a, 2014b), thus allowing for de novo reconstruction of any plant virus or viroid from siRNAs. Then, we developed a de novo assembly pipeline to reconstruct complete viral genomes as single contigs of viral siRNAs, in which Velvet was used in combination with other assemblers: Metavelvet or Oases to generate contigs from viral redundant or non-redundant siRNA reads and Seqman to merge the contigs. Furthermore, we employed the mapping tool BWA and the map viewing tool IGV to verify the reconstructed genomes and identify a consensus master genome and its variants present in the virus quasispecies. The approach combining deep siRNA sequencing with the bioinformatics tools and algorithms, which enabled us to reconstruct consensus master genomes of RNA and DNA viruses, was named siRNA omics (siRomics) (Seguin et al. 2014a).
We utilized siRomics to reconstruct a DNA virus and two viroids associated with an emerging grapevine red leaf disease and generate an infectious wild type genome clone of oilseed rape mosaic virus (Seguin et al. 2014a). Furthermore, siRomics was used to investigate siRNA-based antiviral defense in banana plants persistently infected with six distinct banana streak pararetroviruses (Rajeswaran et al. 2014a) and rice plants infected with rice tungro bacilliform pararetrovirus (Rajeswaran et al. 2014b). Our results revealed that multiple host DCLs generate abundant and diverse populations of 21-, 22- and 24-nt viral siRNAs that can potentially associate with multiple AGO proteins to target viral genes for post-transcriptional and transcriptional silencing. However, pararetroviruses appear to have evolved silencing evasion mechanisms such as overexpression of decoy dsRNA from a short non-coding region of the virus genome to engage the silencing machinery in massive siRNA production and thereby protect other regions of the virus genome from repressive action of viral siRNAs (Rajeswaran et al. 2014b). Furthermore, despite massive production of 24-nt siRNAs, the circular viral DNA remains unmethylated and therefore transcriptionally active, while the host genome is extensively methylated (Rajeswaran et al. 2014b). These findings shed new light at the siRNA generating machinery of economically-important crop plants. Our analysis of plant small RNAs in banana and rice revealed a novel class of highly abundant 20-nt small RNAs with 5'-terminal guanidine (5'G), which has not been identified in dicot plants. Interestingly, the 20-nt 5'G-RNA-generating pathway does not target the pararetroviruses, which correlates with silencing evasion (Rajeswaran et al. 2014a, 2014b).
This thesis work is a part of the European Cooperation in Science and Technology (COST) action that aims develop an RNA-based vaccine to immunize crop plants against viral infection. Our analysis of viral siRNA profiles in various virus-infected plants allowed to identify the regions in the viral genomes that generate low-abundance siRNAs, which are the candidate regions to be targeted by RNA interference (RNAi). Our analysis of RNAi transgenic tomato plants confirmed that targeting of the low-abundance siRNA region of Tomato yellow leaf curl virus (TYLCV) by transgene-derived siRNAs renders immunity to TYLCV disease, one of the major constraints for tomato cultivation worldwide
Revisiting the roles of replicase complex proteins in tobamovirus replication and suppression of RNA silencing
Full text linkTobamoviral replicase possesses an RNA-dependent RNA polymerase (RDR) domain and is translated from genomic (g)RNA via a stop codon readthrough mechanism at a one-to-ten ratio relative to a shorter protein lacking the RDR domain. The two proteins share methyltransferase and helicase domains and form a heterodimer implicated in gRNA replication. The shorter protein is also implicated in suppressing RNA silencing based antiviral defenses. Using a stop codon mutant of Oilseed rape mosaic tobamovirus (ORMV), we demonstrate that the readthrough replicase (p182) is sufficient for gRNA replication and for subgenomic RNA transcription during systemic infection in Nicotiana benthamiana and Arabidopsis thaliana. However, the mutant virus displays milder symptoms and does not interfere with HEN1-mediated methylation of viral short interfering (si)RNAs or plant small (s)RNAs. The mutant virus tends to revert the stop codon, thereby restoring expression of the shorter protein (p125), even in the absence of plant Dicer-like activities that generate viral siRNAs. Plant RDR activities that generate endogenous siRNA precursors do not prevent replication or movement of the mutant virus, and double-stranded precursors of viral siRNAs representing the entire virus genome are likely synthesized by p182. Transgenic expression of p125 partially recapitulates the ORMV disease symptoms associated with overaccumulation of plant sRNAs. Taken together, the readthrough replicase p182 is sufficient for viral replication and transcription but not for silencing suppression. By contrast, the shorter p125 protein suppresses silencing, provokes severe disease symptoms, causes overaccumulation of unmethylated viral and plant sRNAs but it is not an essential component of the viral replicase complex
The role of viral effector proteins in suppression of plant antiviral defenses based on RNA silencing and innate immunity
Full text linkPlant viruses are widespread and economically important pathogens. Currently, there are more than one thousand viruses that are known to be potentially capable of infecting plants and new viruses are being discovered every day. Many of them could cause important diseases of various cultivated plants that humans grow for food, fiber, feed, construction material and biofuel. Therefore understanding the biology of plant viruses is important for development and improvement of cultivated plant resistance to viral pathogens.
A major role in plant resistance against viruses belongs to the process called RNA silencing, that targets both RNA and DNA viruses through the small RNA-directed RNA degradation and DNA methylation pathways. In addition, plants respond to virus infection using an innate immune system that recognizes microbe-associated molecular patterns (MAMPs) of potential pathogens and elicits both local and systemic defense responses. However, in order to be succesfull and break the host resistance, plant viruses have evolved a variety of counter-defense mechanisms such as expressing effector proteins, which are used to downregulate plant antiviral responses. Here, we performed comparative investigation of viral effector proteins from two distanly-related pararetroviruses, Cauliflower mosaic virus (CaMV) and Rice tungro bacilliform virus (RTBV), to understand their role in the suppression of plant antiviral defenses based on RNA silencing and innate immunity. The CaMV P6 protein has previously been shown to serve as a silencing suppressor, while the function of RTBV P4 protein was unknown. Through the use of a classical transient assay in leaves of the N. benthamiana transgenic line 16c we show that RTBV P4 can suppress cell-to-cell spread of transgene silencing, but enhance cell autonomous transgene silencing, which correlates with reduced accumulation of 21-nt siRNAs and increased accumulation of 22-nt siRNAs, respectively. Furthermore, we demonstrate that CaMV P6 from strain CM1841 and RTBV P4 proteins are able to suppress the early plant innate immunity responses, such as oxidative burst. In contrast, CaMV P6 from strain D4 failed to suppress innate immunity, but was capable of suppressing RNA silencing as P6 protein from strain CM1841.
We also elucidated the role of P4 F-box-like motif and N-terminal domain that are required for RTBV P4 effector functions and protein stability, respectively.
Finally, through the use of agroinoculation of Oryza sativa plants with RTBV infectious clone we tested if the P4 F-box motif is required for infectivity and our preliminary results show that the F-box mutant virus exhibts drastically reduced infectivity. Furthermore, we found that RTBV circular double-stranded DNA evades siRNA-directed cytosine methylation in infected rice plants and that rice plants overexpressing an OsAGO18 protein are resistant to RTBV infection
siRomics for universal diagnostics of plant viral disease and virus diversity studies
Full text linkTraditional methods of viral diagnostics using specific antibodies and PCR often fail to
identify a viral pathogen. In our EU Marie-Curie IDP bridges project, we used an alternative
novel approach called siRomics which allows not only to detect the virus but also to de novo
reconstruct a complete consensus master genome in the viral quasispecies population.
The main plant antiviral defense system is based on RNA silencing mediated by small RNAs.
In plants infected with DNA and RNA viruses, host Dicer enzymes generate 21-24 nucleotide
(nt) viral small interfering RNAs (siRNAs) that restrict virus replication and systemic spread.
Growing evidence indicates that viral siRNAs are derived from the entire genome sequence
of RNA and DNA viruses and accumulate at high levels. Hence it appears feasible to
reconstruct a complete viral genome simply from viral siRNA species. Current
bioinformatics algorithms enable de novo assembly of genomes and transcriptomes from
short sequencing reads. In the past years, the siRomics pipeline, developed by Seguin et al.
(2014b) in model plants, was further applied in crop plants (Seguin et al. 2014b, 2016,
Rajeswaran et al. 2014a, 2014b, Fuentes et al. 2016). Thus, our siRomics approach has the
potential for universal diagnostics of plant virus disease and de novo reconstruction of viral
genomes in mixed infections.
In this study we applied siRomics for virus detection and virome reconstruction in several
case studies of economically-important viral diseases in Switzerland. In naturally-infected
Solanum tuberosum (potato), one case study revealed a virome comprising Potato virus Y
(genus Potyvirus) and Potato virus X (genus Potexvirus), which was reconstructed by de
novo assembling separate genome-size sRNA contigs. Another case study revealed a virome
comprising NTN and O strains of Potato virus Y, whose sRNAs assembled in chimeric
contigs which could be disentangled on the basis of reference genome sequences.
Both viromes were stable in vegetative potato progeny. In a cross-protection trial of Solanum
lycopersicum (tomato), the supposedly protective mild strain CH2 of Pepino mosaic virus
(Potexvirus) was tested for protection against the strain LP of the same virus. Reciprocal
mechanical inoculations eventually resulted in co-infection of all individual plants with CH2
and LP strains, reconstructed as separate sRNA contigs. LP invasions into CH2-preinfected
plants and vice versa were accompanied by alterations of consensus genome sequences in
viral quasispecies, indicating a potential risk of cross-protection measures. Additionally, the
study also revealed, by reconstruction from sRNAs, the presence of the mechanically non-
transmissible Southern tomato virus (Amalgavirus) in some plants. Our in-depth analysis of
sRNA sizes, 5'-nucleotide frequencies and hotspot maps revealed similarities in sRNA-
generating mechanisms in potato and tomato, differential silencing responses to virome
components and potential for sRNA-directed cross-targeting between viral strains which
could not, however, prevent the formation of stable viromes. Furthermore, by siRomics we
characterized the virome present in cultivated and non-cultivated perennial plants including
grapevine, cherry, fig, privet and larch. As expected, grapevine samples showed a complex
virome, including viroids, in particular Grapevine Fanleaf virus, Grapevine virus A,
Grapevine leafroll associated virus, Yellow speckle viroid 1, Yellow speckle viroid 2, Hopstunt viroid and Australian grapevine viroid. In cherry trees affected by little cherry disease,
we confirmed that the presence of two Little cherry virus (1 and 2, respectively) in one of the
samples, induces more severe symptoms compared with the sample where only Little cherry
virus 1 was present. In a fig tree exhibiting virus-like symptoms coming from a private
garden, new isolates of Fig mosaic virus and Fig Badnavirus-1 were identified and
reconstructed. In the forest bush plant privet (Ligustrum vulgare) showing yellow mosaic
disease, a novel virus distantly related to Barley yellow strip virus and Lychnis ringspot virus
was identified, fully reconstructed and named Ligustrum mosaic virus. Our work combined
multi-disciplinary approaches ranging from advanced molecular methods of next generation
sequencing to sophisticated bioinformatics algorithms for virus genome reconstruction. The
results of our study are informative for further understanding the mechanisms of RNA
silencing-based antiviral defense, which would contribute to basic research in the field of
plant-pathogen interaction, and for developing novel strategies of virus control, which could
potentially be implemented in the future in Swiss agriculture though our recommendations to
the policy makers. In modern agriculture, horticulture and (bio-) farming, it becomes critical
to assess the risk of emerging plant infections and to control the spread of plant viral
diseases
Molecular interactions of banana bunchy top virus and its alphasatellite with host plants and aphid vectors
No full textLe banana bunchy top virus (BBTV) est le virus le plus dommageable de la culture de la banane, une monocotylédone considérée comme la 4ème plus grande culture mondiale. Appartenant à la famille des Nanoviridae et au genre Babuvirus, le BBTV est un virus à ADN circulaire simple brin (ssDNA) multipartite composé de 6 ADN viraux différents. Transmis de plante à plante par le puceron vecteur Pentalonia nigronervosa, ce virus peux également être retrouvé associé avec un alphasatellite. Un alphasatellite est une molécule d'ADN circulaire simple brin appartenant à la famille des Alphasatellitidae, qui code pour une protéine nommée Rep-like lui permettant de s'autorépliquer. Toutefois, il ne peut pas trans-répliquer son virus assistant et nécessite ce dernier afin de se déplacer dans la plante, être encapsider et être transmis de plante à plante. Très peu d'étude ont été menées sur les alphasatellites et leur rôle dans l'infection virale n'est pas défini. Dans ces travaux de thèse, nous montrons l'émergence d'un nouvel alphasatellite associé au BBTV provenant de République Démocratique du Congo. C'est le premier alphasatellite découvert associé au BBTV en Afrique. Sa caractérisation a montré qu'il impactait son virus assistant au travers de la transmission, de la réplication et la transcription virale, et de la production des petits ARNs viraux. De plus, cet alphasatellite est plus proche phylogénétiquement des alphasatellites découverts chez les dicotylédones que ceux découverts associés au BBTV et forme le genre des Banaphisatellite que nous décrivons. De plus, notre étude d'échantillons de BBTV provenant d'Asie du sud-est montre la forte prévalence d'alphasatellite dans cette région du monde et la découverte des alphasatellites formant deux autres espèces différentes du genre Banaphisatellite. Enfin, notre caractérisation des plantes dicotylédones montre qu'elles sont des hôtes alternatifs du BBTV et leur alphasatellites. Possédant également des alphasatellites du genre Banaphisatellite, elles appuient l'hypothèse d'un passage des dicotylédones à monocotylédone de ce genre d'alphasatellite.Banana bunchy top virus (BBTV) is the most damaging virus of monocot bananas and plantains, considered the 4th largest crop plants in the world. Belonging to the family Nanoviridae and the genus Babuvirus, BBTV is a multipartite circular single-stranded DNA (ssDNA) virus composed of six viral DNA components. Transmitted from plant to plant by the aphid vector Pentalonia nigronervosa, this virus can also be associated with one or more alphasatellites. Alphasatellites are circular ssDNA molecules belonging to the family Alphasatellitidae which encode a Rep-like protein that allows alphasatellites to self-replicate. However, alphasatellites cannot trans-replicate helper virus components and require the helper virus for encapsidation, movement and transmission by insect vectors. Very few studies have been conducted on alphasatellites, and their role in viral infection and disease transmission is not defined. In this thesis, we report the emergence of a new alphasatellite associated with BBTV in Democratic Republic of the Congo. This is the first BBTV alphasatellite discovered in Africa. We demonstrate that this alphasatellite affects its helper virus transmission, replication, gene expression and evasion of antiviral defences generating viral small interfering RNAs. Furthermore, this alphasatellite forms a new genus Banaphisatellite and is more phylogenetically related to alphasatellites infecting dicots than those previously found to be associated with monocot-infecting BBTV in South-East Asia. In addition, our surveys of BBTV-infected plants from South-East Asia showed the high prevalence of alphasatellites in this region of the world and among other alphasatellites we discovered two additional novel species representing the genus Banaphisatellite. Finally, we demonstrate that dicot plants can serve as alternative hosts of BBTV and its alphasatellites including the banaphisatellites, thus supporting the hypothesis of a dicot to monocot passage of this alphasatellite genus
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
- …
