Inmaculada Ferriol

Genetic variation and evolutionary analysis of broad bean wilt virus 2

The genetic variation and evolutionary mechanisms of broad bean wilt virus 2 (BBWV-2) were studied by nucleotide sequence analysis of four genomic regions of 30 isolates from different countries. Nucleotide diversity was high (0.198) for a plant virus. Phylogenetic and genetic structure analyses showed low population subdivision, suggesting a significant gene flow between distant geographic areas. Analysis of synonymous and nonsynonymous substitutions showed different negative selection pressures for different parts of the coding regions, but no positive selection was found. Several recombination detection methods showed that some BBWV-2 genomes might have originated from recombination or reassortment.

Detection and absolute quantitation of Broad bean wilt virus 1 (BBWV-1) and BBWV-2 by real time RT-PCR

Broad bean wilt virus 1 (BBWV-1) and BBWV-2 are the two most significant viruses in the genus Fabavirus, causing damage to many economically important agricultural crops worldwide. A quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR) procedure using two TaqMan(®)MGB probes was developed for sensitive and specific detection and quantitation of BBWV-1 and BBWV-2. Primers and probes were designed from conserved sequence stretches to detect all isolates of each virus. Standard curves using RNA transcripts identical to both TaqMan(®)MGB probes enabled absolute quantitation, with a wide dynamic range and high sensitivity (10(3)-10(10) RNA molecules). RT-qPCR was assayed with genetically divergent BBWV-1 and BBWV-2 isolates from different plant hosts and countries, and was used to evaluate the temporal accumulation of BBWV-1 RNA in two plant hosts.

Comparison of the Oilseed rape mosaic virus and Tobacco mosaic virus movement proteins (MP) reveals common and dissimilar MP functions for tobamovirus spread

Tobacco mosaic virus (TMV) is a longstanding model for studying virus movement and macromolecular transport through plasmodesmata (PD). Its movement protein (MP) interacts with cortical microtubule (MT)-associated ER sites (C-MERs) to facilitate the formation and transport of ER-associated viral replication complexes (VRCs) along the ER-actin network towards PD. To investigate whether this movement mechanism might be conserved between tobamoviruses, we compared the functions of Oilseed rape mosaic virus (ORMV) MP with those of MP(TMV). We show that MP(ORMV) supports TMV movement more efficiently than MP(TMV). Moreover, MP(ORMV) localizes to C-MERs like MP(TMV) but accumulates to lower levels and does not localize to larger inclusions/VRCs or along MTs, patterns regularly seen for MP(TMV). Our findings extend the role of C-MERs in viral cell-to-cell transport to a virus commonly used for functional genomics in Arabidopsis. Moreover, accumulation of tobamoviral MP in inclusions or along MTs is not required for virus movement.

Detection and identification of Fabavirus species by one-step RT-PCR and multiplex RT-PCR

The genus Fabavirus of the family Secoviridae comprises a group of poorly characterized viruses. To date, only five species have been described: Broad bean wilt virus 1 (BBWV-1), Broad bean wilt virus 2 (BBWV-2), Lamium mild mosaic virus (LMMV), Gentian mosaic virus (GeMV) and Cucurbit mild mosaic virus (CuMMV). The development is described of two RT-PCR procedures for the detection and identification of Fabavirus species: a one-step RT-PCR using a single pair of conserved primers for the detection of all fabaviruses, and a one-step multiplex RT-PCR using species-specific primers for the simultaneous detection and identification of the above-mentioned species of the genus Fabavirus. These methods were applied successfully to field samples and the results were compared with those obtained by molecular hybridization and ELISA. The combination of the two techniques enables rapid, sensitive and reliable identification of the five known fabavirus species, as well as the possibility of discovering new species of this genus.

Experimental virus evolution reveals a role of plant microtubule dynamics and TORTIFOLIA1/SPIRAL2 in RNA trafficking.

The cytoskeleton is a dynamic network composed of filamentous polymers and regulatory proteins that provide a flexible structural scaffold to the cell and plays a fundamental role in developmental processes. Mutations that alter the spatial orientation of the cortical microtubule (MT) array of plants are known to cause important changes in the pattern of cell wall synthesis and developmental phenotypes; however, the consequences of such alterations on other MT-network-associated functions in the cytoplasm are not known. In vivo observations suggested a role of cortical MTs in the formation and movement of Tobacco mosaic virus (TMV) RNA complexes along the endoplasmic reticulum (ER). Thus, to probe the significance of dynamic MT behavior in the coordination of MT-network-associated functions related to TMV infection and, thus, in the formation and transport of RNA complexes in the cytoplasm, we performed an evolution experiment with TMV in Arabidopsis thaliana tor1/spr2 and tor2 mutants with specific defects in MT dynamics and asked whether TMV is sensitive to these changes. We show that the altered cytoskeleton induced genetic changes in TMV that were correlated with efficient spread of infection in the mutant hosts. These observations demonstrate a role of dynamic MT rearrangements and of the MT-associated protein TORTIFOLIA1/SPIRAL2 in cellular functions related to virus spread and indicate that MT dynamics and MT-associated proteins represent constraints for virus evolution and adaptation. The results highlight the importance of the dynamic plasticity of the MT network in directing cytoplasmic functions in macromolecular assembly and trafficking and illustrate the value of experimental virus evolution for addressing the cellular functions of dynamic, long-range order systems in multicellular organisms.

Genetic variability and evolution of broad bean wilt virus 1: role of recombination, selection and gene flow

Analysis of four genomic regions from 37 geographically diverse isolates of broad bean wilt virus 1 (BBWV-1) showed high genetic diversity in comparison to most plant viruses. Comparison of synonymous and nonsynonymous substitutions of the small coat protein gene (SCP) revealed negative selection for most amino acid positions. Phylogenetic analysis of SCP showed that some BBWV-1 isolates from distant geographical areas were genetically close, suggesting long-distance migration. Analysis of genetic differentiation revealed high gene flow between Spanish and Near Eastern subpopulations, which were separated from North-Central and South-Eastern European subpopulations. Finally, putative recombinant and reassortant genomes were also identified.

Construction of Agrobacterium tumefaciens-mediated tomato black ring virus infectious cDNA clones

Tomato black ring virus (TBRV, genus Nepovirus) infects a wide range of economically important plants such as tomato, potato, tobacco and cucumber. Here, a successful construction of infectious full-length cDNA clones of the TBRV genomic RNAs (RNA1 and RNA2) is reported for the first time. The engineered constructs consisting of PCR-amplified DNAs were cloned into binary vector pJL89 immediately downstream of a double cauliflower mosaic virus (CaMV) 35S promoter, and upstream of the hepatitis delta virus (HDV) ribozyme and nopaline synthase terminator (NOS). The symptoms induced on plants agroinoculated with both constructs were indistinguishable from those caused by the wild-type virus. The infectivity of obtained clones was verified by reinoculation to Nicotiana tabacum cv. Xanthi, Chenopodium quinoa and Cucumis sativus. The presence of viral particles and RNA was confirmed by electron microscopy and reverse transcription polymerase chain reaction, respectively. Constructed full-length infectious cDNA clones will serve as an excellent tool to study virus-host-vector interactions.

The complete genome sequence of Lamium mild mosaic virus , a member of the genus Fabavirus

Lamium mild mosaic virus (LMMV) is the only one of the five members of the genus Fabavirus for which there are no nucleotide sequence data. In this study, the complete genome sequence of LMMV was determined and compared with the available complete genome sequences of other members of the genus Fabavirus. The genome was the largest of the genus but maintained the typical organization, with RNA 1 of 6080 nucleotides (nt), RNA 2 of 4065 nt, and an unusually long 3′ untranslated region in RNA 2 of 603 nt. Phylogenetic analysis of the amino acid sequences of the protease-polymerase (Pro-Pol) region and the two coat proteins confirmed that LMMV belongs to a distinct species within the genus Fabavirus.

Identification of the cleavage sites of the RNA2-encoded polyproteins for two members of the genus Torradovirus by N-terminal sequencing of the virion capsid proteins.

Torradoviruses, family Secoviridae, are emergent bipartite RNA plant viruses. RNA1 is ca. 7kb and has one open reading frame (ORF) encoding for the protease, helicase and RNA-dependent RNA polymerase (RdRp). RNA2 is ca. 5kb and has two ORFs. RNA2-ORF1 encodes for a putative protein with unknown function(s). RNA2-ORF2 encodes for a putative movement protein and three capsid proteins. Little is known about the replication and polyprotein processing strategies of torradoviruses. Here, the cleavage sites in the RNA2-ORF2-encoded polyproteins of two torradoviruses, Tomato marchitez virus isolate M (ToMarV-M) and tomato chocolate spot virus, were determined by N-terminal sequencing, revealing that the amino acid (aa) at the -1 position of the cleavage sites is a glutamine. Multiple aa sequence comparison confirmed that this glutamine is conserved among other torradoviruses. Finally, site-directed mutagenesis of conserved aas in the ToMarV-M RdRp and protease prevented substantial accumulation of viral coat proteins or RNAs.

Detection and absolute quantitation of Tomato torrado virus (ToTV) by real time RT-PCR.

Tomato torrado virus (ToTV) causes serious damage to the tomato industry and significant economic losses. A quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR) method using primers and a specific TaqMan(®) MGB probe for ToTV was developed for sensitive detection and quantitation of different ToTV isolates. A standard curve using RNA transcripts enabled absolute quantitation, with a dynamic range from 10(4) to 10(10) ToTV RNA copies/ng of total RNA. The specificity of the RT-qPCR was tested with twenty-three ToTV isolates from tomato (Solanum lycopersicum L.), and black nightshade (Solanum nigrum L.) collected in Spain, Australia, Hungary and France, which covered the genetic variation range of this virus. This new RT-qPCR assay enables a reproducible, sensitive and specific detection and quantitation of ToTV, which can be a valuable tool in disease management programs and epidemiological studies.