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Veronique Brault - One of the best experts on this subject based on the ideXlab platform.
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transmission of turnip yellows virus by myzus persicae is reduced by feeding aphids on double stranded rna targeting the ephrin receptor protein
Frontiers in Microbiology, 2018Co-Authors: Michael Mulot, Baptiste Monsion, Sylvaine Boissinot, Maryam Rastegar, Sophie Meyer, Nicole Bochet, Veronique BraultAbstract:Aphid-transmitted plant viruses are a threat for major crops causing massive economic loss worldwide. Members in the Luteoviridae family are transmitted by aphids in a circulative and non-replicative mode. Virions are acquired by aphids when ingesting sap from infected plants and are transported through the gut and the accessory salivary gland (ASG) cells by a transcytosis mechanism relying on virus-specific receptors largely unknown. Once released into the salivary canal, virions are inoculated to plants, together with saliva, during a subsequent feeding. In this paper, we bring in vivo evidence that the membrane-bound Ephrin receptor (Eph) is a novel aphid protein involved in the transmission of the Turnip yellows virus (TuYV, Polerovirus genus, Luteoviridae family) by Myzus persicae. The minor capsid protein of TuYV, essential for aphid transmission, was able to bind the external domain of Eph in yeast. Feeding M. persicae on in planta- or in vitro-synthesized dsRNA targeting Eph-mRNA (dsRNAEph) did not affect aphid feeding behavior but reduced accumulation of TuYV genomes in the aphid's body. Consequently, TuYV transmission efficiency by the dsRNAEph-treated aphids was reproducibly inhibited and we brought evidence that Eph is likely involved in intestinal uptake of the virion. The inhibition of virus uptake after dsRNAEph acquisition was also observed for two other Poleroviruses transmitted by M. persicae, suggesting a broader role of Eph in Polerovirus transmission. Finally, dsRNAEph acquisition by aphids did not affect nymph production. These results pave the way toward an ecologically safe alternative of insecticide treatments that are used to lower aphid populations and reduce Polerovirus damages.
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Production of a Beet chlorosis virus full-length cDNA clone by means of gibson assembly and analysis of biological properties
Journal of General Virology, 2018Co-Authors: Veronika Wetzel, Veronique Brault, Mark VarrelmannAbstract:Beet chlorosis virus (genus Polerovirus, family Luteoviridae), which is persistently transmitted by the aphid Myzus persicae, is part of virus yellows in sugar beet and causes interveinal yellowing as well as significant yield loss in Beta vulgaris. To allow reverse genetic studies and replace vector transmission, an infectious cDNA clone under cauliflower mosaic virus 35S control in a binary vector for agrobacterium-mediated infection was constructed using Gibson assembly. Following agroinoculation, the BChV full-length clone was able to induce a systemic infection of the cultivated B. vulgaris. The engineered virus was successfully aphid-transmitted when acquired from infected B. vulgaris and displayed the same host plant spectrum as wild-type virus. This new Polerovirus infectious clone is a valuable tool to identify the viral determinants involved in host range and study BChV protein function, and can be used to screen sugar beet for BChV resistance.
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Presentation5.PPTX
2018Co-Authors: Michael Mulot, Baptiste Monsion, Sylvaine Boissinot, Maryam Rastegar, Sophie Meyer, Nicole Bochet, Veronique BraultAbstract:Aphid-transmitted plant viruses are a threat for major crops causing massive economic loss worldwide. Members in the Luteoviridae family are transmitted by aphids in a circulative and non-replicative mode. Virions are acquired by aphids when ingesting sap from infected plants and are transported through the gut and the accessory salivary gland (ASG) cells by a transcytosis mechanism relying on virus-specific receptors largely unknown. Once released into the salivary canal, virions are inoculated to plants, together with saliva, during a subsequent feeding. In this paper, we bring in vivo evidence that the membrane-bound Ephrin receptor (Eph) is a novel aphid protein involved in the transmission of the Turnip yellows virus (TuYV, Polerovirus genus, Luteoviridae family) by Myzus persicae. The minor capsid protein of TuYV, essential for aphid transmission, was able to bind the external domain of Eph in yeast. Feeding M. persicae on in planta- or in vitro-synthesized dsRNA targeting Eph-mRNA (dsRNAEph) did not affect aphid feeding behavior but reduced accumulation of TuYV genomes in the aphid's body. Consequently, TuYV transmission efficiency by the dsRNAEph-treated aphids was reproducibly inhibited and we brought evidence that Eph is likely involved in intestinal uptake of the virion. The inhibition of virus uptake after dsRNAEph acquisition was also observed for two other Poleroviruses transmitted by M. persicae, suggesting a broader role of Eph in Polerovirus transmission. Finally, dsRNAEph acquisition by aphids did not affect nymph production. These results pave the way toward an ecologically safe alternative of insecticide treatments that are used to lower aphid populations and reduce Polerovirus damages.
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Systemic Propagation of a Fluorescent Infectious Clone of a Polerovirus Following Inoculation by Agrobacteria and Aphids
Viruses, 2017Co-Authors: Sylvaine Boissinot, Danièle Scheidecker, Ziegler-graff Véronique, Elodie Pichon, Céline Sorin, Céline Piccini, Veronique BraultAbstract:A fluorescent viral clone of the Polerovirus Turnip yellows virus (TuYV) was engineered by introducing the Enhanced Green Fluorescent Protein (EGFP) sequence into the non-structural domain sequence of the readthrough protein, a minor capsid protein. The resulting recombinant virus, referred to as TuYV-RTGFP, was infectious in several plant species when delivered by agroinoculation and invaded efficiently non-inoculated leaves. As expected for Poleroviruses, which infect only phloem cells, the fluorescence emitted by TuYV-RTGFP was restricted to the vasculature of infected plants. In addition, TuYV-RTGFP was aphid transmissible and enabled the observation of the initial sites of infection in the phloem after aphid probing in epidermal cells. The aphid-transmitted virus moved efficiently to leaves distant from the inoculation sites and importantly retained the EGFP sequence in the viral genome. This work reports on the first engineered member in the Luteoviridae family that can be visualized by fluorescence emission in systemic leaves of different plant species after agroinoculation or aphid transmission
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Insights in luteovirid structural biology guided by chemical cross-linking and high resolution mass spectrometry
2016Co-Authors: Mariko M. Alexander, Veronique Brault, Véronique Ziegler-graff, Jared Jared P. Mohr, Stacy L. Deblasio, Juan D. Chavez, James E. Bruce, Michelle (cilia) HeckAbstract:Interactions among plant pathogenic viruses in the family Luteoviridae and their plant hosts and insect vectors are governed by the topology of the viral capsid, which is the sole vehicle for long distance movement of the viral genome. Previous application of a mass spectrometry-compatible cross-linker to preparations of the luteovirid Potato leafroll virus (PLRV; Luteoviridae: Polerovirus) revealed a detailed network of interactions between viral structural proteins and enabled generation of the first cross-linking guided coat protein models. In this study, we extended application of chemical cross-linking technology to the related Turnip yellows virus (TuYV; Luteoviridae: Polerovirus). Remarkably, all cross-links found between sites in the viral coat protein found for TuYV were also found in PLRV. Guided by these data, we present two models for the TuYV coat protein trimer, the basic structural unit of luteovirid virions. Additional cross-links found between the TuYV coat protein and a site in the viral protease domain suggest a possible role for the luteovirid protease in regulating the structural biology of these viruses.
Sylvaine Boissinot - One of the best experts on this subject based on the ideXlab platform.
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transmission of turnip yellows virus by myzus persicae is reduced by feeding aphids on double stranded rna targeting the ephrin receptor protein
Frontiers in Microbiology, 2018Co-Authors: Michael Mulot, Baptiste Monsion, Sylvaine Boissinot, Maryam Rastegar, Sophie Meyer, Nicole Bochet, Veronique BraultAbstract:Aphid-transmitted plant viruses are a threat for major crops causing massive economic loss worldwide. Members in the Luteoviridae family are transmitted by aphids in a circulative and non-replicative mode. Virions are acquired by aphids when ingesting sap from infected plants and are transported through the gut and the accessory salivary gland (ASG) cells by a transcytosis mechanism relying on virus-specific receptors largely unknown. Once released into the salivary canal, virions are inoculated to plants, together with saliva, during a subsequent feeding. In this paper, we bring in vivo evidence that the membrane-bound Ephrin receptor (Eph) is a novel aphid protein involved in the transmission of the Turnip yellows virus (TuYV, Polerovirus genus, Luteoviridae family) by Myzus persicae. The minor capsid protein of TuYV, essential for aphid transmission, was able to bind the external domain of Eph in yeast. Feeding M. persicae on in planta- or in vitro-synthesized dsRNA targeting Eph-mRNA (dsRNAEph) did not affect aphid feeding behavior but reduced accumulation of TuYV genomes in the aphid's body. Consequently, TuYV transmission efficiency by the dsRNAEph-treated aphids was reproducibly inhibited and we brought evidence that Eph is likely involved in intestinal uptake of the virion. The inhibition of virus uptake after dsRNAEph acquisition was also observed for two other Poleroviruses transmitted by M. persicae, suggesting a broader role of Eph in Polerovirus transmission. Finally, dsRNAEph acquisition by aphids did not affect nymph production. These results pave the way toward an ecologically safe alternative of insecticide treatments that are used to lower aphid populations and reduce Polerovirus damages.
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Presentation5.PPTX
2018Co-Authors: Michael Mulot, Baptiste Monsion, Sylvaine Boissinot, Maryam Rastegar, Sophie Meyer, Nicole Bochet, Veronique BraultAbstract:Aphid-transmitted plant viruses are a threat for major crops causing massive economic loss worldwide. Members in the Luteoviridae family are transmitted by aphids in a circulative and non-replicative mode. Virions are acquired by aphids when ingesting sap from infected plants and are transported through the gut and the accessory salivary gland (ASG) cells by a transcytosis mechanism relying on virus-specific receptors largely unknown. Once released into the salivary canal, virions are inoculated to plants, together with saliva, during a subsequent feeding. In this paper, we bring in vivo evidence that the membrane-bound Ephrin receptor (Eph) is a novel aphid protein involved in the transmission of the Turnip yellows virus (TuYV, Polerovirus genus, Luteoviridae family) by Myzus persicae. The minor capsid protein of TuYV, essential for aphid transmission, was able to bind the external domain of Eph in yeast. Feeding M. persicae on in planta- or in vitro-synthesized dsRNA targeting Eph-mRNA (dsRNAEph) did not affect aphid feeding behavior but reduced accumulation of TuYV genomes in the aphid's body. Consequently, TuYV transmission efficiency by the dsRNAEph-treated aphids was reproducibly inhibited and we brought evidence that Eph is likely involved in intestinal uptake of the virion. The inhibition of virus uptake after dsRNAEph acquisition was also observed for two other Poleroviruses transmitted by M. persicae, suggesting a broader role of Eph in Polerovirus transmission. Finally, dsRNAEph acquisition by aphids did not affect nymph production. These results pave the way toward an ecologically safe alternative of insecticide treatments that are used to lower aphid populations and reduce Polerovirus damages.
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Systemic Propagation of a Fluorescent Infectious Clone of a Polerovirus Following Inoculation by Agrobacteria and Aphids
Viruses, 2017Co-Authors: Sylvaine Boissinot, Danièle Scheidecker, Ziegler-graff Véronique, Elodie Pichon, Céline Sorin, Céline Piccini, Veronique BraultAbstract:A fluorescent viral clone of the Polerovirus Turnip yellows virus (TuYV) was engineered by introducing the Enhanced Green Fluorescent Protein (EGFP) sequence into the non-structural domain sequence of the readthrough protein, a minor capsid protein. The resulting recombinant virus, referred to as TuYV-RTGFP, was infectious in several plant species when delivered by agroinoculation and invaded efficiently non-inoculated leaves. As expected for Poleroviruses, which infect only phloem cells, the fluorescence emitted by TuYV-RTGFP was restricted to the vasculature of infected plants. In addition, TuYV-RTGFP was aphid transmissible and enabled the observation of the initial sites of infection in the phloem after aphid probing in epidermal cells. The aphid-transmitted virus moved efficiently to leaves distant from the inoculation sites and importantly retained the EGFP sequence in the viral genome. This work reports on the first engineered member in the Luteoviridae family that can be visualized by fluorescence emission in systemic leaves of different plant species after agroinoculation or aphid transmission
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both structural and non structural forms of the readthrough protein of cucurbit aphid borne yellows virus are essential for efficient systemic infection of plants
PLOS ONE, 2014Co-Authors: Veronique Brault, Veronique Zieglergraff, Baptiste Monsion, Sylvaine Boissinot, Monique ErdingerAbstract:Cucurbit aphid-borne yellows virus (CABYV) is a Polerovirus (Luteoviridae family) with a capsid composed of the major coat protein and a minor component referred to as the readthrough protein (RT). Two forms of the RT were reported: a full-length protein of 74 kDa detected in infected plants and a truncated form of 55 kDa (RT*) incorporated into virions. Both forms were detected in CABYV-infected plants. To clarify the specific roles of each protein in the viral cycle, we generated by deletion a Polerovirus mutant able to synthesize only the RT* which is incorporated into the particle. This mutant was unable to move systemically from inoculated leaves inferring that the C-terminal half of the RT is required for efficient long-distance transport of CABYV. Among a collection of CABYV mutants bearing point mutations in the central domain of the RT, we obtained a mutant impaired in the correct processing of the RT which does not produce the RT*. This mutant accumulated very poorly in upper non-inoculated leaves, suggesting that the RT* has a functional role in long-distance movement of CABYV. Taken together, these results infer that both RT proteins are required for an efficient CABYV movement.
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Rack-1, GAPDH3, and actin: proteins of Myzus persicae potentially involved in the transcytosis of beet western yellows virus particles in the aphid
Virology, 2004Co-Authors: Pascale Seddas, Sylvaine Boissinot, Jean-marc Strub, Alain Van Dorsselaer, Marc H. V. Van Regenmortel, Franc PattusAbstract:Beet western yellows virus (BWYV) is a Polerovirus that relies on the aphid Myzus persicae for its transmission, in a persistent-circulative mode. To be transmitted, the virus must cross the midgut and the accessory salivary glands (ASG) epithelial barriers in a transcytosis mechanism where vector receptors interact with virions. In this paper, we report in vitro interaction experiments between BWYV and aphid components. Using the M. persicae clone from Colmar, we showed that a set of aphid polypeptides, separated by SDS-PAGE or 2D electrophoresis (2DE), can bind in vitro to purified wild type or mutant particles. Using subcellular fractionation, we showed that the 65-kDa polypeptide identified as symbionin is a soluble protein whereas the other polypeptides seem to be associated more or less strongly to the membrane. We hypothesize that three polypeptides, identified by mass spectrometry as Rack-1, GAPDH3, and actin, may be involved in the epithelial transcytosis of virus particles in the aphid vector.
Boissinot Sylvaine - One of the best experts on this subject based on the ideXlab platform.
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The Ephrin receptor: a potential actor in Polerovirus transmission by aphids
HAL CCSD, 2017Co-Authors: Mulot Michaël, Boissinot Sylvaine, Monsion Baptiste, Meyer Sophie, Bochet Nicole, Brault VeroniqueAbstract:BGPI : équipe 2Poleroviruses are phloem-limited RNA viruses strictly transmitted by aphids in a circulative and non-propagative mode. Virus particles, acquired by aphids on infected plants, successively cross intestinal and accessory salivary gland cells by transcytosis before being released, together with saliva, into a plant during aphid feeding. Virus transport through the epithelia relies on the presence of specific virus receptors. Identification of these receptors could eventually result in the development of innovative strategies to block virus acquisition by aphids. The Ephrin receptor (Ephr) from Myzus persicae has been reviously identified by yeast two hybrid as a potential partner of the structural proteins of Turnip yellows virus (TuYV, genus Polerovirus, family Luteoviridae). Ephr is a membrane protein involved in cell communication and endocytosis in mammalian cells. In order to address its function in TuYV transmission by M. persicae, we developed several strategies (1), all based on the RNA interference mechanism, to inhibit expression of Ephr in M. persicae. The oral acquisition of in vitro synthetized dsRNA targeting Ephr and the acquisition of dsRNA, or siRNA, from transgenic plants expressing an RNA hairpin, resulted in an inhibition of the accumulation of Ephr-mRNA in M. persicae. When fed on a viral source, these Ephr-silenced aphids showed a lower internalization of viral genomes into their body. Although the feeding behaviour and the fecundity of the Ephr-silenced aphids were not affected by the reduction of Ephr expression, TuYV transmission was reproducibly reduced. Preliminary experiments showed that transmission inhibition of another related Polerovirus, the Beet mild yellowing virus, was also obtained when using Ephr-silenced aphids. Taken together, these experiments strongly suggest implication of Ephr in Polerovirus transmission. Whether Ephr encodes a true virus receptor or a co-receptor still needs to be elucidated
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The Ephrin receptor: a potential actor in Polerovirus transmission by aphids
2017Co-Authors: Mulot Michaël, Boissinot Sylvaine, Monsion Baptiste, Meyer Sophie, Bochet Nicole, Brault VeroniqueAbstract:Poleroviruses are phloem-limited RNA viruses strictly transmitted by aphids in a circulative and non-propagative mode. Virus particles, acquired by aphids on infected plants, successively cross intestinal and accessory salivary gland cells by transcytosis before being released, together with saliva, into a plant during aphid feeding. Virus transport through the epithelia relies on the presence of specific virus receptors. Identification of these receptors could eventually result in the development of innovative strategies to block virus acquisition by aphids. The Ephrin receptor (Ephr) from Myzus persicae has been reviously identified by yeast two hybrid as a potential partner of the structural proteins of Turnip yellows virus (TuYV, genus Polerovirus, family Luteoviridae). Ephr is a membrane protein involved in cell communication and endocytosis in mammalian cells. In order to address its function in TuYV transmission by M. persicae, we developed several strategies (1), all based on the RNA interference mechanism, to inhibit expression of Ephr in M. persicae. The oral acquisition of in vitro synthetized dsRNA targeting Ephr and the acquisition of dsRNA, or siRNA, from transgenic plants expressing an RNA hairpin, resulted in an inhibition of the accumulation of Ephr-mRNA in M. persicae. When fed on a viral source, these Ephr-silenced aphids showed a lower internalization of viral genomes into their body. Although the feeding behaviour and the fecundity of the Ephr-silenced aphids were not affected by the reduction of Ephr expression, TuYV transmission was reproducibly reduced. Preliminary experiments showed that transmission inhibition of another related Polerovirus, the Beet mild yellowing virus, was also obtained when using Ephr-silenced aphids. Taken together, these experiments strongly suggest implication of Ephr in Polerovirus transmission. Whether Ephr encodes a true virus receptor or a co-receptor still needs to be elucidated
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Systemic Propagation of a Fluorescent Infectious Clone of a Polerovirus Following Inoculation by Agrobacteria and Aphids
'MDPI AG', 2017Co-Authors: Boissinot Sylvaine, Véronique Ziegler-graff, Scheidecker Danièle, Pichon Elodie, Sorin Céline, Piccini Céline, Brault VeroniqueAbstract:BGPI : équipe 2A fluorescent viral clone of the Polerovirus Turnip yellows virus (TuYV) was engineered by introducing the Enhanced Green Fluorescent Protein (EGFP) sequence into the non-structural domain sequence of the readthrough protein, a minor capsid protein. The resulting recombinant virus, referred to as TuYV-RTGFP, was infectious in several plant species when delivered by agroinoculation and invaded efficiently non-inoculated leaves. As expected for Poleroviruses, which infect only phloem cells, the fluorescence emitted by TuYV-RTGFP was restricted to the vasculature of infected plants. In addition, TuYV-RTGFP was aphid transmissible and enabled the observation of the initial sites of infection in the phloem after aphid probing in epidermal cells. The aphid-transmitted virus moved efficiently to leaves distant from the inoculation sites and importantly retained the EGFP sequence in the viral genome. This work reports on the first engineered member in the Luteoviridae family that can be visualized by fluorescence emission in systemic leaves of different plant species after agroinoculation or aphid transmissio
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Systemic Propagation of a Fluorescent Infectious Clone of a Polerovirus Following Inoculation by Agrobacteria and Aphids
'MDPI AG', 2017Co-Authors: Boissinot Sylvaine, Véronique Ziegler-graff, Scheidecker Danièle, Pichon Elodie, Sorin Céline, Piccini Céline, Brault VeroniqueAbstract:BGPI : équipe 2International audienceA fluorescent viral clone of the Polerovirus Turnip yellows virus (TuYV) was engineered by introducing the Enhanced Green Fluorescent Protein (EGFP) sequence into the non-structural domain sequence of the readthrough protein, a minor capsid protein. The resulting recombinant virus, referred to as TuYV-RTGFP, was infectious in several plant species when delivered by agroinoculation and invaded efficiently non-inoculated leaves. As expected for Poleroviruses, which infect only phloem cells, the fluorescence emitted by TuYV-RTGFP was restricted to the vasculature of infected plants. In addition, TuYV-RTGFP was aphid transmissible and enabled the observation of the initial sites of infection in the phloem after aphid probing in epidermal cells. The aphid-transmitted virus moved efficiently to leaves distant from the inoculation sites and importantly retained the EGFP sequence in the viral genome. This work reports on the first engineered member in the Luteoviridae family that can be visualized by fluorescence emission in systemic leaves of different plant species after agroinoculation or aphid transmissio
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Partners and role in viral cycle of the different forms of Cucurbit aphid-borne yellows virus RT protein
2013Co-Authors: Boissinot SylvaineAbstract:Les polérovirus infectent de nombreuses plantes d’intérêt économique telles que la pomme de terre, la betterave à sucre et les cucurbitacées. Ces virus icosaédriques renferment un ARN simple brin et leur capside est constituée d’une protéine majeure (CP) et d’un composant mineur (RT*) localisé à la surface des virions. Ces virus sont restreints aux cellules du phloème dans lesquelles ils se multiplient et se déplacent. Les protéines CP et RT sont essentielles à la dissémination du virus par le puceron vecteur et à son mouvement dans la plante. L’objectif de cette étude a consisté à identifier dans les cellules du phloème, les protéines associées aux virions susceptibles d’intervenir dans le cycle viral en criblant une banque d’ADNc de cellules compagnes (CC) d’A. thaliana avec les protéines de structure ou des domaines protéiques du CABYV. Quatre gènes codant pour une protéine Heat Shock (HSP), la profiline 3 (PRF3) une glysosyl hydrolase ; et la protéine « Response to low sulfur 3 » ont été identifiés. Tous ces gènes candidats interagissent avec le domaine RTC-ter du CABYV et avec la protéine RT* pour la protéine HSP. En plus de ces gènes candidats, je me suis intéressée à la protéine ALY, identifiée au laboratoire, au cours du criblage d’une banque d’ADNc de puceron entier avec les deux protéines de structure du Turnip yellows virus (un autre polérovirus). Cette protéine possède quatre orthologues chez Arabidopsis susceptibles d’être impliquées dans le mécanisme de gene silencing mis en place contre le Tomato Bushy Stunt Virus. Les protéines ALY sont donc des candidats intéressants et j’ai montré une interaction entre les protéines de structure du CABYV et du TuYV et les quatre orthologues d’Arabidopsis. L’implication de ces gènes candidats n’a pas pu être confirmée à ce jour dans des mutants knock-out d’arabidopsis. Les résultats complexes obtenus pour le candidat PRF3 au cours des analyses de validation fonctionnelle, m’a conduit à étudier l’interaction entre ce candidat et le domaine RTC-ter du CABYV in planta par FLIM mais aucune interaction n’a pu être confirmée à ce jour. Tous les candidats isolés lors du criblage de la banque d’ADNc de CC interagissant avec le domaine RTC-ter du CABYV, ce travail m’a conduit à analyser le rôle dans le cycle viral de ce domaine et de la protéine RT (sous sa forme complète ou dépourvue du domaine RTC-ter), en étudiant l’accumulation de ces mutants dans les plantes et le clivage de la protéine RT. Tout d’abord, afin de localiser précisément le site de clivage de la protéine RT, des mutants ponctuels dans la zone de clivage ont été réalisés ce qui a permis de montrer que la structure secondaire de la protéine est importante pour son clivage. Puis, afin d’analyser le rôle du domaine RTC-ter dans le cycle viral, j’ai obtenu par délétion, un mutant n’exprimant plus ce domaine. Ce mutant synthétise uniquement la protéine RT tronquée, forme des particules virales semblables au virus sauvage et est transmissible par puceron. Par contre, de façon surprenante, ce mutant est incapable d’envahir les feuilles non-inoculées d’une plante. Ce résultat suggère que les deux formes de la protéine RT (complète et tronquée) sont indispensables au mouvement à longue distance du virus et nous proposons un modèle dans lequel le domaine C-terminal de la protéine RT agit en trans sur la particule virale pour promouvoir le mouvement du CABYV à longue distance.Poleroviruses infect a wide range of cultivated plants such as potatoes, sugar beet and plants of Cucurbitaceae family. These viruses are restricted to phloem tissue where they replicate in nucleated cells and translocate over long distances through sieve elements. Polerovirus capsid is composed of the major coat protein (CP) and of a minor component referred to as the readthrough (RT*) protein and exposed at the outside of the particles. CP and RT proteins are essential for virus movement and transmission by aphids. The aim of this study is to identify phloem proteins interacting with viral proteins and potentially involved in viral cycle, by screening an A. thaliana companion cell (CC) cDNA library with structural proteins or protein domains of CABYV. Four genes encoding for a heat shock protein (HSP), a profilin (PRF3), a glycosyl hydrolase and the protein ”Response to low sulfur ” (LSU3) were identified and interact with the C-terminal part of the RT protein (RTC‑ter) and with the RT* protein for the HSP. An additional gene encoding for the protein ALY, identified in the laboratory, by screening an aphid cDNA library with structural proteins of the Turnip yellows virus (another Polerovirus) was studied. This protein has four orthologues in Arabidopsis, involved in the gene silencing mechanism against Tomato Bushy Stunt Virus. Here we show that CABYV and TuYV structural proteins interact with the four orthologues of Arabidopsis. Involvement of these candidate genes was not confirmed in Arabidopsis knock-out mutants. In functional experiments, ambiguous results were obtained with PRF3 arabidopsis mutants, and this lead me to study the interaction between PRF3 protein CABYV RT c-ter domain by FLIM, but no interaction was found so far. As all candidat interact with the RTC-ter domain, we studied more precisely the role of this domain in the viral cycle and the role of the complete RT protein. We studied the in vivo RT protein processing and its consequences on systemic movement of CABYV mutants. Using a collection of point mutations introduced in the central domain of the CABYV RT protein, we approached the site of the RT processing and proposed that this process is affected by the secondary structure around the cleavage site. We also reported for the first time the generation of a Polerovirus mutant able to synthesize only the RT* protein and to incorporate it into the particle. This mutant was unable to move systemically. Conversely another mutant producing a full-length RT protein impaired in correct processing and incorporating a shorter version of the RT* protein showed very weak systemic infection. These data are strongly in favor of a role of both RT proteins in efficient CABYV movement. An inefficient virus transport was still maintained in the absence of RT proteins suggesting an RT-independent movement pathway. Based on these results, we propose a model for CABYV long-distance transport in which the complete RT protein, or its C-terminal part, acts in trans on wild-type virions to promote their efficient long-distance transport
Brault Veronique - One of the best experts on this subject based on the ideXlab platform.
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The Ephrin receptor: a potential actor in Polerovirus transmission by aphids
HAL CCSD, 2017Co-Authors: Mulot Michaël, Boissinot Sylvaine, Monsion Baptiste, Meyer Sophie, Bochet Nicole, Brault VeroniqueAbstract:BGPI : équipe 2Poleroviruses are phloem-limited RNA viruses strictly transmitted by aphids in a circulative and non-propagative mode. Virus particles, acquired by aphids on infected plants, successively cross intestinal and accessory salivary gland cells by transcytosis before being released, together with saliva, into a plant during aphid feeding. Virus transport through the epithelia relies on the presence of specific virus receptors. Identification of these receptors could eventually result in the development of innovative strategies to block virus acquisition by aphids. The Ephrin receptor (Ephr) from Myzus persicae has been reviously identified by yeast two hybrid as a potential partner of the structural proteins of Turnip yellows virus (TuYV, genus Polerovirus, family Luteoviridae). Ephr is a membrane protein involved in cell communication and endocytosis in mammalian cells. In order to address its function in TuYV transmission by M. persicae, we developed several strategies (1), all based on the RNA interference mechanism, to inhibit expression of Ephr in M. persicae. The oral acquisition of in vitro synthetized dsRNA targeting Ephr and the acquisition of dsRNA, or siRNA, from transgenic plants expressing an RNA hairpin, resulted in an inhibition of the accumulation of Ephr-mRNA in M. persicae. When fed on a viral source, these Ephr-silenced aphids showed a lower internalization of viral genomes into their body. Although the feeding behaviour and the fecundity of the Ephr-silenced aphids were not affected by the reduction of Ephr expression, TuYV transmission was reproducibly reduced. Preliminary experiments showed that transmission inhibition of another related Polerovirus, the Beet mild yellowing virus, was also obtained when using Ephr-silenced aphids. Taken together, these experiments strongly suggest implication of Ephr in Polerovirus transmission. Whether Ephr encodes a true virus receptor or a co-receptor still needs to be elucidated
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The Ephrin receptor: a potential actor in Polerovirus transmission by aphids
2017Co-Authors: Mulot Michaël, Boissinot Sylvaine, Monsion Baptiste, Meyer Sophie, Bochet Nicole, Brault VeroniqueAbstract:Poleroviruses are phloem-limited RNA viruses strictly transmitted by aphids in a circulative and non-propagative mode. Virus particles, acquired by aphids on infected plants, successively cross intestinal and accessory salivary gland cells by transcytosis before being released, together with saliva, into a plant during aphid feeding. Virus transport through the epithelia relies on the presence of specific virus receptors. Identification of these receptors could eventually result in the development of innovative strategies to block virus acquisition by aphids. The Ephrin receptor (Ephr) from Myzus persicae has been reviously identified by yeast two hybrid as a potential partner of the structural proteins of Turnip yellows virus (TuYV, genus Polerovirus, family Luteoviridae). Ephr is a membrane protein involved in cell communication and endocytosis in mammalian cells. In order to address its function in TuYV transmission by M. persicae, we developed several strategies (1), all based on the RNA interference mechanism, to inhibit expression of Ephr in M. persicae. The oral acquisition of in vitro synthetized dsRNA targeting Ephr and the acquisition of dsRNA, or siRNA, from transgenic plants expressing an RNA hairpin, resulted in an inhibition of the accumulation of Ephr-mRNA in M. persicae. When fed on a viral source, these Ephr-silenced aphids showed a lower internalization of viral genomes into their body. Although the feeding behaviour and the fecundity of the Ephr-silenced aphids were not affected by the reduction of Ephr expression, TuYV transmission was reproducibly reduced. Preliminary experiments showed that transmission inhibition of another related Polerovirus, the Beet mild yellowing virus, was also obtained when using Ephr-silenced aphids. Taken together, these experiments strongly suggest implication of Ephr in Polerovirus transmission. Whether Ephr encodes a true virus receptor or a co-receptor still needs to be elucidated
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Systemic Propagation of a Fluorescent Infectious Clone of a Polerovirus Following Inoculation by Agrobacteria and Aphids
'MDPI AG', 2017Co-Authors: Boissinot Sylvaine, Véronique Ziegler-graff, Scheidecker Danièle, Pichon Elodie, Sorin Céline, Piccini Céline, Brault VeroniqueAbstract:BGPI : équipe 2A fluorescent viral clone of the Polerovirus Turnip yellows virus (TuYV) was engineered by introducing the Enhanced Green Fluorescent Protein (EGFP) sequence into the non-structural domain sequence of the readthrough protein, a minor capsid protein. The resulting recombinant virus, referred to as TuYV-RTGFP, was infectious in several plant species when delivered by agroinoculation and invaded efficiently non-inoculated leaves. As expected for Poleroviruses, which infect only phloem cells, the fluorescence emitted by TuYV-RTGFP was restricted to the vasculature of infected plants. In addition, TuYV-RTGFP was aphid transmissible and enabled the observation of the initial sites of infection in the phloem after aphid probing in epidermal cells. The aphid-transmitted virus moved efficiently to leaves distant from the inoculation sites and importantly retained the EGFP sequence in the viral genome. This work reports on the first engineered member in the Luteoviridae family that can be visualized by fluorescence emission in systemic leaves of different plant species after agroinoculation or aphid transmissio
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Systemic Propagation of a Fluorescent Infectious Clone of a Polerovirus Following Inoculation by Agrobacteria and Aphids
'MDPI AG', 2017Co-Authors: Boissinot Sylvaine, Véronique Ziegler-graff, Scheidecker Danièle, Pichon Elodie, Sorin Céline, Piccini Céline, Brault VeroniqueAbstract:BGPI : équipe 2International audienceA fluorescent viral clone of the Polerovirus Turnip yellows virus (TuYV) was engineered by introducing the Enhanced Green Fluorescent Protein (EGFP) sequence into the non-structural domain sequence of the readthrough protein, a minor capsid protein. The resulting recombinant virus, referred to as TuYV-RTGFP, was infectious in several plant species when delivered by agroinoculation and invaded efficiently non-inoculated leaves. As expected for Poleroviruses, which infect only phloem cells, the fluorescence emitted by TuYV-RTGFP was restricted to the vasculature of infected plants. In addition, TuYV-RTGFP was aphid transmissible and enabled the observation of the initial sites of infection in the phloem after aphid probing in epidermal cells. The aphid-transmitted virus moved efficiently to leaves distant from the inoculation sites and importantly retained the EGFP sequence in the viral genome. This work reports on the first engineered member in the Luteoviridae family that can be visualized by fluorescence emission in systemic leaves of different plant species after agroinoculation or aphid transmissio
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Discovery of a Small Non-AUG-Initiated ORF in Poleroviruses and Luteoviruses That Is Required for Long-Distance Movement.
PLoS Pathog, 2015Co-Authors: Smirnova Ekaterina, Brault Veronique, Rakotondrafara A. M., Firth Andrew, Miller W Allen, Scheidecker Danièle, Reinbold Catherine, Chung Betty, Ziegler-graff VéroniqueAbstract:Viruses in the family Luteoviridae have positive-sense RNA genomes of around 5.2 to 6.3 kb, and they are limited to the phloem in infected plants. The Luteovirus and Polerovirus genera include all but one virus in the Luteoviridae. They share a common gene block, which encodes the coat protein (ORF3), a movement protein (ORF4), and a carboxy-terminal extension to the coat protein (ORF5). These three proteins all have been reported to participate in the phloem-specific movement of the virus in plants. All three are translated from one subgenomic RNA, sgRNA1. Here, we report the discovery of a novel short ORF, termed ORF3a, encoded near the 5' end of sgRNA1. Initially, this ORF was predicted by statistical analysis of sequence variation in large sets of aligned viral sequences. ORF3a is positioned upstream of ORF3 and its translation initiates at a non-AUG codon. Functional analysis of the ORF3a protein, P3a, was conducted with Turnip yellows virus (TuYV), a Polerovirus, for which translation of ORF3a begins at an ACG codon. ORF3a was translated from a transcript corresponding to sgRNA1 in vitro, and immunodetection assays confirmed expression of P3a in infected protoplasts and in agroinoculated plants. Mutations that prevent expression of P3a, or which overexpress P3a, did not affect TuYV replication in protoplasts or inoculated Arabidopsis thaliana leaves, but prevented virus systemic infection (long-distance movement) in plants. Expression of P3a from a separate viral or plasmid vector complemented movement of a TuYV mutant lacking ORF3a. Subcellular localization studies with fluorescent protein fusions revealed that P3a is targeted to the Golgi apparatus and plasmodesmata, supporting an essential role for P3a in viral movement.ES and WAM were financed through a Gutenberg Chair (Région Alsace) grant awarded to WAM. Work in the AEF lab was funded by grants from the Wellcome Trust (088789) and the UK Biotechnology and Biological Research Council (BBSRC) (BB/J007072/1 and BB/J015652/1). WAM was also funded by a Fulbright Foundation Research Scholarship and grant number 5R01GM067104-09 from the NIH Institute of General Medical Sciences.This is the final version. It was first published by PLOS at http://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1004868#ack
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transmission of turnip yellows virus by myzus persicae is reduced by feeding aphids on double stranded rna targeting the ephrin receptor protein
Frontiers in Microbiology, 2018Co-Authors: Michael Mulot, Baptiste Monsion, Sylvaine Boissinot, Maryam Rastegar, Sophie Meyer, Nicole Bochet, Veronique BraultAbstract:Aphid-transmitted plant viruses are a threat for major crops causing massive economic loss worldwide. Members in the Luteoviridae family are transmitted by aphids in a circulative and non-replicative mode. Virions are acquired by aphids when ingesting sap from infected plants and are transported through the gut and the accessory salivary gland (ASG) cells by a transcytosis mechanism relying on virus-specific receptors largely unknown. Once released into the salivary canal, virions are inoculated to plants, together with saliva, during a subsequent feeding. In this paper, we bring in vivo evidence that the membrane-bound Ephrin receptor (Eph) is a novel aphid protein involved in the transmission of the Turnip yellows virus (TuYV, Polerovirus genus, Luteoviridae family) by Myzus persicae. The minor capsid protein of TuYV, essential for aphid transmission, was able to bind the external domain of Eph in yeast. Feeding M. persicae on in planta- or in vitro-synthesized dsRNA targeting Eph-mRNA (dsRNAEph) did not affect aphid feeding behavior but reduced accumulation of TuYV genomes in the aphid's body. Consequently, TuYV transmission efficiency by the dsRNAEph-treated aphids was reproducibly inhibited and we brought evidence that Eph is likely involved in intestinal uptake of the virion. The inhibition of virus uptake after dsRNAEph acquisition was also observed for two other Poleroviruses transmitted by M. persicae, suggesting a broader role of Eph in Polerovirus transmission. Finally, dsRNAEph acquisition by aphids did not affect nymph production. These results pave the way toward an ecologically safe alternative of insecticide treatments that are used to lower aphid populations and reduce Polerovirus damages.
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Presentation5.PPTX
2018Co-Authors: Michael Mulot, Baptiste Monsion, Sylvaine Boissinot, Maryam Rastegar, Sophie Meyer, Nicole Bochet, Veronique BraultAbstract:Aphid-transmitted plant viruses are a threat for major crops causing massive economic loss worldwide. Members in the Luteoviridae family are transmitted by aphids in a circulative and non-replicative mode. Virions are acquired by aphids when ingesting sap from infected plants and are transported through the gut and the accessory salivary gland (ASG) cells by a transcytosis mechanism relying on virus-specific receptors largely unknown. Once released into the salivary canal, virions are inoculated to plants, together with saliva, during a subsequent feeding. In this paper, we bring in vivo evidence that the membrane-bound Ephrin receptor (Eph) is a novel aphid protein involved in the transmission of the Turnip yellows virus (TuYV, Polerovirus genus, Luteoviridae family) by Myzus persicae. The minor capsid protein of TuYV, essential for aphid transmission, was able to bind the external domain of Eph in yeast. Feeding M. persicae on in planta- or in vitro-synthesized dsRNA targeting Eph-mRNA (dsRNAEph) did not affect aphid feeding behavior but reduced accumulation of TuYV genomes in the aphid's body. Consequently, TuYV transmission efficiency by the dsRNAEph-treated aphids was reproducibly inhibited and we brought evidence that Eph is likely involved in intestinal uptake of the virion. The inhibition of virus uptake after dsRNAEph acquisition was also observed for two other Poleroviruses transmitted by M. persicae, suggesting a broader role of Eph in Polerovirus transmission. Finally, dsRNAEph acquisition by aphids did not affect nymph production. These results pave the way toward an ecologically safe alternative of insecticide treatments that are used to lower aphid populations and reduce Polerovirus damages.
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both structural and non structural forms of the readthrough protein of cucurbit aphid borne yellows virus are essential for efficient systemic infection of plants
PLOS ONE, 2014Co-Authors: Veronique Brault, Veronique Zieglergraff, Baptiste Monsion, Sylvaine Boissinot, Monique ErdingerAbstract:Cucurbit aphid-borne yellows virus (CABYV) is a Polerovirus (Luteoviridae family) with a capsid composed of the major coat protein and a minor component referred to as the readthrough protein (RT). Two forms of the RT were reported: a full-length protein of 74 kDa detected in infected plants and a truncated form of 55 kDa (RT*) incorporated into virions. Both forms were detected in CABYV-infected plants. To clarify the specific roles of each protein in the viral cycle, we generated by deletion a Polerovirus mutant able to synthesize only the RT* which is incorporated into the particle. This mutant was unable to move systemically from inoculated leaves inferring that the C-terminal half of the RT is required for efficient long-distance transport of CABYV. Among a collection of CABYV mutants bearing point mutations in the central domain of the RT, we obtained a mutant impaired in the correct processing of the RT which does not produce the RT*. This mutant accumulated very poorly in upper non-inoculated leaves, suggesting that the RT* has a functional role in long-distance movement of CABYV. Taken together, these results infer that both RT proteins are required for an efficient CABYV movement.