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1. Synergy between an emerging monopartite begomovirus and a DNA-B component

Author

Alassane Ouattara, Fidèle Tiendrébéogo, Nathalie Becker, Cica Urbino, Gaël Thébaud, Murielle Hoareau, Agathe Allibert, Frédéric Chiroleu, Marie-Stéphanie Vernerey, Edgar Valentin Traoré, Nicolas Barro, Oumar Traoré, Pierre Lefeuvre, and Jean-Michel Lett

Subjects
Medicine, Science
Abstract

Abstract In recent decades, a legion of monopartite begomoviruses transmitted by the whitefly Bemisia tabaci has emerged as serious threats to vegetable crops in Africa. Recent studies in Burkina Faso (West Africa) reported the predominance of pepper yellow vein Mali virus (PepYVMLV) and its frequent association with a previously unknown DNA-B component. To understand the role of this DNA-B component in the emergence of PepYVMLV, we assessed biological traits related to virulence, virus accumulation, location in the tissue and transmission. We demonstrate that the DNA-B component is not required for systemic movement and symptom development of PepYVMLV (non-strict association), but that its association produces more severe symptoms including growth arrest and plant death. The increased virulence is associated with a higher viral DNA accumulation in plant tissues, an increase in the number of contaminated nuclei of the phloem parenchyma and in the transmission rate by B. tabaci. Our results suggest that the association of a DNA-B component with the otherwise monopartite PepYVMLV is a key factor of its emergence.

Published
2022
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2. Structure-guided mutagenesis of the capsid protein indicates that a nanovirus requires assembled viral particles for systemic infection.

Author

Stefano Trapani, Eijaz Ahmed Bhat, Michel Yvon, Joséphine Lai-Kee-Him, François Hoh, Marie-Stéphanie Vernerey, Elodie Pirolles, Mélia Bonnamy, Guy Schoehn, Jean-Louis Zeddam, Stéphane Blanc, and Patrick Bron

Subjects
Immunologic diseases. Allergy, RC581-607, Biology (General), QH301-705.5
Abstract

Nanoviruses are plant multipartite viruses with a genome composed of six to eight circular single-stranded DNA segments. The distinct genome segments are encapsidated individually in icosahedral particles that measure ≈18 nm in diameter. Recent studies on the model species Faba bean necrotic stunt virus (FBNSV) revealed that complete sets of genomic segments rarely occur in infected plant cells and that the function encoded by a given viral segment can complement the others across neighbouring cells, presumably by translocation of the gene products through unknown molecular processes. This allows the viral genome to replicate, assemble into viral particles and infect anew, even with the distinct genome segments scattered in different cells. Here, we question the form under which the FBNSV genetic material propagates long distance within the vasculature of host plants and, in particular, whether viral particle assembly is required. Using structure-guided mutagenesis based on a 3.2 Å resolution cryogenic-electron-microscopy reconstruction of the FBNSV particles, we demonstrate that specific site-directed mutations preventing capsid formation systematically suppress FBNSV long-distance movement, and thus systemic infection of host plants, despite positive detection of the mutated coat protein when the corresponding segment is agroinfiltrated into plant leaves. These results strongly suggest that the viral genome does not propagate within the plant vascular system under the form of uncoated DNA molecules or DNA:coat-protein complexes, but rather moves long distance as assembled viral particles.

Published
2023
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3. A multicellular way of life for a multipartite virus

Author

Anne Sicard, Elodie Pirolles, Romain Gallet, Marie-Stéphanie Vernerey, Michel Yvon, Cica Urbino, Michel Peterschmitt, Serafin Gutierrez, Yannis Michalakis, and Stéphane Blanc

Subjects
multipartite virus, virus-host interaction, evolution, genome, plant, population genetics, Medicine, Science, Biology (General), QH301-705.5
Abstract

A founding paradigm in virology is that the spatial unit of the viral replication cycle is an individual cell. Multipartite viruses have a segmented genome where each segment is encapsidated separately. In this situation the viral genome is not recapitulated in a single virus particle but in the viral population. How multipartite viruses manage to efficiently infect individual cells with all segments, thus with the whole genome information, is a long-standing but perhaps deceptive mystery. By localizing and quantifying the genome segments of a nanovirus in host plant tissues we show that they rarely co-occur within individual cells. We further demonstrate that distinct segments accumulate independently in different cells and that the viral system is functional through complementation across cells. Our observation deviates from the classical conceptual framework in virology and opens an alternative possibility (at least for nanoviruses) where the infection can operate at a level above the individual cell level, defining a viral multicellular way of life.

Published
2019
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4. Co-Acquired Nanovirus and Geminivirus Exhibit a Contrasted Localization within Their Common Aphid Vector

Author

Jérémy Di Mattia, Faustine Ryckebusch, Marie-Stéphanie Vernerey, Elodie Pirolles, Nicolas Sauvion, Michel Peterschmitt, Jean-Louis Zeddam, and Stéphane Blanc

Subjects
fluorescent in situ hybridization, qpcr, leaf discs, hemiptera, nanovirus, geminivirus, vector transmission, Microbiology, QR1-502
Abstract

Single-stranded DNA (ssDNA) plant viruses belong to the families Geminiviridae and Nanoviridae. They are transmitted by Hemipteran insects in a circulative, mostly non-propagative, manner. While geminiviruses are transmitted by leafhoppers, treehoppers, whiteflies and aphids, nanoviruses are transmitted exclusively by aphids. Circulative transmission involves complex virus−vector interactions in which epithelial cells have to be crossed and defense mechanisms counteracted. Vector taxa are considered a relevant taxonomic criterion for virus classification, indicating that viruses can evolve specific interactions with their vectors. Thus, we predicted that, although nanoviruses and geminiviruses represent related viral families, they have evolved distinct interactions with their vector. This prediction is also supported by the non-structural Nuclear Shuttle Protein (NSP) that is involved in vector transmission in nanoviruses but has no similar function in geminiviruses. Thanks to the recent discovery of aphid-transmitted geminiviruses, this prediction could be tested for the geminivirus alfalfa leaf curl virus (ALCV) and the nanovirus faba bean necrotic stunt virus (FBNSV) in their common vector, Aphis craccivora. Estimations of viral load in midgut and head of aphids, precise localization of viral DNA in cells of insect vectors and host plants, and virus transmission tests revealed that the pathway of the two viruses across the body of their common vector differs both quantitatively and qualitatively.

Published
2020
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5. Breaking dogmas: the plant vascular pathogen Xanthomonas albilineans is able to invade non-vascular tissues despite its reduced genome

Author

Imène Mensi, Marie-Stéphanie Vernerey, Daniel Gargani, Michel Nicole, and Philippe Rott

Subjects
xanthomonas albilineans, confocal laser scanning microscopy, cytochemistry, transmission electron microscopy, non-vascular plant tissue, Biology (General), QH301-705.5
Abstract

Xanthomonas albilineans, the causal agent of sugarcane leaf scald, is missing the Hrp type III secretion system that is used by many Gram-negative bacteria to colonize their host. Until now, this pathogen was considered as strictly limited to the xylem of sugarcane. We used confocal laser scanning microscopy, immunocytochemistry and transmission electron microscopy (TEM) to investigate the localization of X. albilineans in diseased sugarcane. Sugarcane plants were inoculated with strains of the pathogen labelled with a green fluorescent protein. Confocal microscopy observations of symptomatic leaves confirmed the presence of the pathogen in the protoxylem and metaxylem; however, X. albilineans was also observed in phloem, parenchyma and bulliform cells of the infected leaves. Similarly, vascular bundles of infected sugarcane stalks were invaded by X. albilineans. Surprisingly, the pathogen was also observed in apparently intact storage cells of the stalk and in intercellular spaces between these cells. Most of these observations made by confocal microscopy were confirmed by TEM. The pathogen exits the xylem following cell wall and middle lamellae degradation, thus creating openings to reach parenchyma cells. This is the first description of a plant pathogenic vascular bacterium invading apparently intact non-vascular plant tissues and multiplying in parenchyma cells.

Published
2014
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6. Route of a Multipartite Nanovirus across the Body of Its Aphid Vector

Author

Yannis Michalakis, Jean-Louis Zeddam, Heiko Ziebell, Yahya Z. A. Gaafar, Jeremy Di Mattia, Stéphane Blanc, Mathilde Villegas, Marie-Stéphanie Vernerey, Michel Yvon, Elodie Pirolles, Biologie et Génétique des Interactions Plante-Parasite (UMR BGPI), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Institut Agro - Montpellier SupAgro, Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro), Julius Kühn-Institut (JKI), Evolution Théorique et Expérimentale (MIVEGEC-ETE), Perturbations, Evolution, Virulence (PEV), Maladies infectieuses et vecteurs : écologie, génétique, évolution et contrôle (MIVEGEC), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD [France-Sud])-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD [France-Sud])-Maladies infectieuses et vecteurs : écologie, génétique, évolution et contrôle (MIVEGEC), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD [France-Sud])-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD [France-Sud]), UMR - Interactions Plantes Microorganismes Environnement (UMR IPME), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Université de Montpellier (UM)-Institut de Recherche pour le Développement (IRD [France-Sud]), ANR-14-CE02-0014,Nano,Exploration de la biologie des virus multipartite(2014), ANR-18-CE92-0028,Nanovirus,Aspects moléculaires et cellulaires du cycle de vie des virus multipartites: les nanovirus(2018), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), and Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)

Subjects
0106 biological sciences, Viral protein, [SDV]Life Sciences [q-bio], Immunology, vector transmission, circulative, plant, Luteoviridae, medicine.disease_cause, 01 natural sciences, Microbiology, Genome, Virus, Plant Viruses, 03 medical and health sciences, Viral Proteins, nanovirus, Viral entry, Virology, Plant virus, medicine, Animals, Geminiviridae, In Situ Hybridization, Fluorescence, 030304 developmental biology, Plant Diseases, Genetics, 0303 health sciences, biology, Nanovirus, DNA Viruses, Virion, biology.organism_classification, 3. Good health, Virus-Cell Interactions, Insect Vectors, aphid, Insect Science, insect vector, Aphids, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, multipartite virus, Nanoviridae, nonpropagative, 010606 plant biology & botany
Abstract

BGPI : Equipe 2 : Interactions Virus Insecte Plante (VIP); International audience; Vector transmission plays a primary role in the life cycle of viruses, and insects are the most common vectors. An important mode of vector transmission, reported only for plant viruses, is circulative nonpropagative transmission whereby the virus cycles within the body of its insect vector, from gut to salivary glands and saliva, without replicating. This mode of transmission has been extensively studied in the viral families Luteoviridae and Geminiviridae and is also reported for Nanoviridae. The biology of viruses within these three families is different, and whether the viruses have evolved similar molecular/cellular virus-vector interactions is unclear. In particular, nanoviruses have a multipartite genome organization, and how the distinct genome segments encapsidated individually transit through the insect body is unknown. Here, using a combination of fluorescent in situ hybridization and immunofluorescence, we monitor distinct proteins and genome segments of the nanovirus Faba bean necrotic stunt virus (FBNSV) during transcytosis through the gut and salivary gland cells of its aphid vector Acyrthosiphon pisum. FBNSV specifically transits through cells of the anterior midgut and principal salivary gland cells, a route similar to that of geminiviruses but distinct from that of luteoviruses. Our results further demonstrate that a large number of virus particles enter every single susceptible cell so that distinct genome segments always remain together. Finally, we confirm that the success of nanovirus-vector interaction depends on a nonstructural helper component, the viral protein nuclear shuttle protein (NSP), which is shown to be mandatory for viral accumulation within gut cells.IMPORTANCE An intriguing mode of vector transmission described only for plant viruses is circulative nonpropagative transmission, whereby the virus passes through the gut and salivary glands of the insect vector without replicating. Three plant virus families are transmitted this way, but details of the molecular/cellular mechanisms of the virus-vector interaction are missing. This is striking for nanoviruses that are believed to interact with aphid vectors in ways similar to those of luteoviruses or geminiviruses but for which empirical evidence is scarce. We here confirm that nanoviruses follow a within-vector route similar to that of geminiviruses but distinct from that of luteoviruses. We show that they produce a nonstructural protein mandatory for viral entry into gut cells, a unique phenomenon for this mode of transmission. Finally, noting that nanoviruses are multipartite viruses, we demonstrate that a large number of viral particles penetrate susceptible cells of the vector, allowing distinct genome segments to remain together.

Published
2020
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7. Localizing Genome Segments and Protein Products of a Multipartite Virus in Host Plant Cells

Author

Anne Sicard, Marie-Stéphanie Vernerey, Elodie Pirolles, Stéphane Blanc, Biologie et Génétique des Interactions Plante-Parasite (UMR BGPI), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut National de la Recherche Agronomique (INRA)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), and Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)

Subjects
Viral protein, Strategy and Management, [SDV]Life Sciences [q-bio], Computational biology, Biology, medicine.disease_cause, Genome, Industrial and Manufacturing Engineering, Virus, Immunolocalization, 03 medical and health sciences, Multipartite virus, medicine, Methods Article, Vicia Faba, 030304 developmental biology, Fluorescence microscopy, 0303 health sciences, medicine.diagnostic_test, 030306 microbiology, Mechanical Engineering, In situ hybridization (ISH), Nanovirus, Metals and Alloys, food and beverages, Plant, FBNSV, Vicia faba, Multipartite, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Viral replication, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Nucleic acid, Fluorescence in situ hybridization
Abstract

International audience; A founding paradigm in virology is that the spatial unit of the viral replication cycle is an individual cell. This concept applied to multipartite viruses-which have a genome composed of two or more nucleic acid segments, each individually encapsulated-implies that all segments constituting a viral genome need to coinfect the same host cell for replication to occur. Would this requirement be verified, it would constitute a major cost for extreme cases of multipartition such as the Faba bean necrotic stunt virus (FBNSV, nanovirus) whose genome is composed of eight complementary segments, each encoding a single gene (Grigoras et al., 2009). To address this question, we followed the distribution of the FBNSV genome segments by fluorescence in situ hybridization combined to immunolocalization of the replication-controlling viral protein within the cells of the host plant: Vicia Faba.A rapid and efficient protocol to localize viral transcripts in plant and insect hosts has been developed earlier (Ghanim et al., 2009). We here improve this method by using random-primed labeled probes and apply it to the detection and quantification of the individual segments composing the FBNSV genome. Moreover, we combine this technique with immunolocalization so that both viral segments and proteins can be visualized within the same samples.

Published
2019
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8. A multicellular way of life for a multipartite virus

Author

Marie-Stéphanie Vernerey, Elodie Pirolles, Michel Yvon, Serafin Gutierrez, Cica Urbino, Stéphane Blanc, Anne Sicard, Yannis Michalakis, Romain Gallet, Michel Peterschmitt, Biologie et Génétique des Interactions Plante-Parasite (UMR BGPI), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut National de la Recherche Agronomique (INRA)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Maladies infectieuses et vecteurs : écologie, génétique, évolution et contrôle (MIVEGEC), Institut de Recherche pour le Développement (IRD [France-Sud])-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), Blanc, Stéphane, Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut National de la Recherche Agronomique (INRA)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro), Evolution Théorique et Expérimentale (MIVEGEC-ETE), Perturbations, Evolution, Virulence (PEV), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD [France-Sud])-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD [France-Sud])-Maladies infectieuses et vecteurs : écologie, génétique, évolution et contrôle (MIVEGEC), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD [France-Sud])-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD [France-Sud]), and Institut National de la Recherche Agronomique : ANR-14-CE02-0014

Subjects
0301 basic medicine, Virologie, viruses, Cell, plant, Virus Replication, Genome, nanovirus, interaction virus cellule, Biology (General), pathologie végétale, In Situ Hybridization, Fluorescence, Microbiology and Infectious Disease, education.field_of_study, Microscopy, Confocal, General Neuroscience, Microbiology and Parasitology, General Medicine, Microbiologie et Parasitologie, Virus, Vicia faba, Complementation, medicine.anatomical_structure, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Regression Analysis, Medicine, virologie végétale, génome viral, QH301-705.5, Science, 030106 microbiology, Population, Short Report, Genome, Viral, Computational biology, Biology, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, Cellule, Virology, evolution, medicine, education, genome, Plant Diseases, H20 - Maladies des plantes, Génome, General Immunology and Microbiology, virus-host interaction, DNA Viruses, Virion, population genetics, Virus des végétaux, Multipartite, Multicellular organism, 030104 developmental biology, Viral replication, DNA, Viral, multipartite virus, Other
Abstract

A founding paradigm in virology is that the spatial unit of the viral replication cycle is an individual cell. Multipartite viruses have a segmented genome where each segment is encapsidated separately. In this situation the viral genome is not recapitulated in a single virus particle but in the viral population. How multipartite viruses manage to efficiently infect individual cells with all segments, thus with the whole genome information, is a long-standing but perhaps deceptive mystery. By localizing and quantifying the genome segments of a nanovirus in host plant tissues we show that they rarely co-occur within individual cells. We further demonstrate that distinct segments accumulate independently in different cells and that the viral system is functional through complementation across cells. Our observation deviates from the classical conceptual framework in virology and opens an alternative possibility (at least for nanoviruses) where the infection can operate at a level above the individual cell level, defining a viral multicellular way of life., eLife digest Many viruses are small particles consisting of genetic material surrounded by a coat made of proteins. They are unable to multiply on their own and so they must enter a host cell and trick it into reading their genetic information to produce new virus particles. It is generally thought that the process of making new virus particles happens independently in each infected cell. This idea assumes that a given particle contains the entire set of genetic material (known as the genome) of that virus, but this is not always the case. Many so-called ‘multipartite’ viruses have genomes that are split into several segments carried in separate particles: in this case, a single particle only contains a portion of the entire viral genome. Faba bean necrotic stunt virus (or FBNSV for short) is a multipartite virus that infects and causes disease in members of the pea and bean family. There are eight types of FBNSV particle that each carries a distinct genome segment, a small section of the entire viral genome. There is a low probability that a single cell could be infected with all eight different types of particle at the same time and receive the complete FBNSV genome. So how is this virus able to successfully multiply within a plant? To address this question, Sicard et al. used microscopy to study FBNSV genome segments as they infected the cells of faba bean plants. The experiments confirmed that the eight different segments of the FBNSV genome were not necessarily found together within the same cell, but instead accumulated independently in different cells. This means that a cell infected with FBNSV may be unable to make all of the proteins needed to assemble new virus particles. However, additional experiments demonstrated that infected cells may be exchanging virus proteins, which could enable them to create complete virus particles. The findings of Sicard et al. demonstrate that FBNSV hijacks groups of host cells to manufacture new virus particles, rather than relying on individual cells as previously thought. It is possible that other multipartite and non-multipartite viruses work a similar manner. Ultimately, this knowledge may reshape what we know about how viruses infect their hosts.

Published
2019
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10. Development of fluorescence expression tools to study host-mycoplasma interactions and validation in two distant mycoplasma clades

Author

François Thiaucourt, Tiffany Bonnefois, Marie-Stéphanie Vernerey, Philippe Totté, Valérie Rodrigues, Lucia Manso-Silvan, Carinne Puech, Chantal Ripoll, Manso-Silvan, Lucia, Contrôle des maladies animales exotiques et émergentes (UMR CMAEE), Institut National de la Recherche Agronomique (INRA)-Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad), Biologie et Génétique des Interactions Plante-Parasite (UMR BGPI), Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut National de la Recherche Agronomique (INRA), Institut des Neurosciences de Montpellier - Déficits sensoriels et moteurs (INM), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut National de la Recherche Agronomique (INRA)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut National de la Recherche Agronomique (INRA), Institut des Neurosciences de Montpellier (INM), and ProdInra, Archive Ouverte

Subjects
0301 basic medicine, Biodiversité et Ecologie, mycoplasma, fluorescence, whole cell labelling, host-pathogen interactions, flow cytometry, confocal microscopy, L73 - Maladies des animaux, medicine.disease_cause, Applied Microbiology and Biotechnology, law.invention, law, Haemobartonella, Flow cytometry, Cells, Cultured, Genetics, Phagocytes, biology, medicine.diagnostic_test, cytotoxicité, General Medicine, Recombinant Proteins, Molecular Imaging, 3. Good health, Mycoplasma mycoides, host pathogen systems, Biotechnology, Whole cell labelling, Green Fluorescent Proteins, 030106 microbiology, protéine fluorescente, Mutagenesis (molecular biology technique), Bioengineering, Fluorescence, Microbiology, Biodiversity and Ecology, 03 medical and health sciences, Confocal microscopy, medicine, Animals, fluorescent protein, Mycoplasma Infections, clone, Host-pathogen interactions, interaction hôte pathogène, Reproducibility of Results, Mycoplasma, biology.organism_classification, In vitro, [SDE.BE] Environmental Sciences/Biodiversity and Ecology, Spectrometry, Fluorescence, Cytoplasm, Cattle, U30 - Méthodes de recherche, [SDE.BE]Environmental Sciences/Biodiversity and Ecology, mCherry
Abstract

Fluorescence expression tools for stable and innocuous whole mycoplasma cell labelling have been developed. A Tn4001-derivative mini-transposon affording unmarked, stable mutagenesis in mycoplasmas was modified to allow the constitutive, high-level expression of mCherry, mKO2 and mNeonGreen. These tools were used to introduce the respective fluorescent proteins as chromosomal tags in the phylogenetically distant species Mycoplasma mycoides subsp. mycoides and Mycoplasma bovis. The production, selection and characterisation of fluorescent clones were straightforward and resulted in the unprecedented observation of red and green fluorescent mycoplasma colonies in the two species, with no apparent cytotoxicity. Equivalent fluorescence expression levels were quantified by flow cytometry in both species, suggesting that these tools can be broadly applied in mycoplasmas. A macrophage infection assay was performed to assess the usefulness of mNeonGreen-expressing strains for monitoring mycoplasma infections, and notably cell invasion. The presence of fluorescent mycoplasmas inside live phagocytic cells was detected and quantified by flow cytometry and corroborated by confocal microscopy, which allowed the identification of individual mycoplasmas in the cytoplasm of infected cells. The fluorescence expression tools developed in this study are suitable for host-pathogen interaction studies and offer innumerable perspectives for the functional analysis of mycoplasmas both in vitro and in vivo.

Published
2016
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11. Co-Acquired Nanovirus and Geminivirus Exhibit a Contrasted Localization within Their Common Aphid Vector

Author

Michel Peterschmitt, Stéphane Blanc, Marie-Stéphanie Vernerey, Faustine Ryckebusch, Elodie Pirolles, Nicolas Sauvion, Jean-Louis Zeddam, Jeremy Di Mattia, Biologie et Génétique des Interactions Plante-Parasite (UMR BGPI), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Département Systèmes Biologiques (Cirad-BIOS), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad), UMR - Interactions Plantes Microorganismes Environnement (UMR IPME), Institut de Recherche pour le Développement (IRD [France-Sud])-Université de Montpellier (UM)-Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad), CIRAD, INRAE, IRD, ANR-18-CE92-0028,Nanovirus,Aspects moléculaires et cellulaires du cycle de vie des virus multipartites: les nanovirus(2018), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Institut Agro - Montpellier SupAgro, Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro), and Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Université de Montpellier (UM)-Institut de Recherche pour le Développement (IRD [France-Sud])

Subjects
0301 basic medicine, geminivirus, [SDV]Life Sciences [q-bio], viruses, 030106 microbiology, lcsh:QR1-502, vector transmission, Article, lcsh:Microbiology, Plant Viruses, Hemiptera, 03 medical and health sciences, nanovirus, Virology, Plant virus, leaf discs, Animals, Geminiviridae, Intestinal Mucosa, Saliva, In Situ Hybridization, Fluorescence, Virus classification, Plant Diseases, Genetics, Aphid, biology, Coinfection, fungi, food and beverages, biochemical phenomena, metabolism, and nutrition, biology.organism_classification, Insect Vectors, 3. Good health, qPCR, Phenotype, 030104 developmental biology, Infectious Diseases, Aphids, Vector (epidemiology), DNA, Viral, fluorescent in situ hybridization, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Nanoviridae, Aphis craccivora
Abstract

Single-stranded DNA (ssDNA) plant viruses belong to the families Geminiviridae and Nanoviridae. They are transmitted by Hemipteran insects in a circulative, mostly non-propagative, manner. While geminiviruses are transmitted by leafhoppers, treehoppers, whiteflies and aphids, nanoviruses are transmitted exclusively by aphids. Circulative transmission involves complex virus&ndash, vector interactions in which epithelial cells have to be crossed and defense mechanisms counteracted. Vector taxa are considered a relevant taxonomic criterion for virus classification, indicating that viruses can evolve specific interactions with their vectors. Thus, we predicted that, although nanoviruses and geminiviruses represent related viral families, they have evolved distinct interactions with their vector. This prediction is also supported by the non-structural Nuclear Shuttle Protein (NSP) that is involved in vector transmission in nanoviruses but has no similar function in geminiviruses. Thanks to the recent discovery of aphid-transmitted geminiviruses, this prediction could be tested for the geminivirus alfalfa leaf curl virus (ALCV) and the nanovirus faba bean necrotic stunt virus (FBNSV) in their common vector, Aphis craccivora. Estimations of viral load in midgut and head of aphids, precise localization of viral DNA in cells of insect vectors and host plants, and virus transmission tests revealed that the pathway of the two viruses across the body of their common vector differs both quantitatively and qualitatively.

Published
2020
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12. A mulicellular lifestyle for a multipartite virus

Author

Stéphane Blanc, Elodie Pirolles, Romain Gallet, Anne Sicard, Jérémy Di Mattia, Marie-Stéphanie Vernerey, Michel Yvon, Serafin Gutierrez, Yannis Michalakis, Biologie et Génétique des Interactions Plante-Parasite (UMR BGPI), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut National de la Recherche Agronomique (INRA)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro), Evolution Théorique et Expérimentale (MIVEGEC-ETE), Perturbations, Evolution, Virulence (PEV), Maladies infectieuses et vecteurs : écologie, génétique, évolution et contrôle (MIVEGEC), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD [France-Sud])-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD [France-Sud])-Maladies infectieuses et vecteurs : écologie, génétique, évolution et contrôle (MIVEGEC), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD [France-Sud])-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD [France-Sud]), and Société Française de Microbiologie (SFM). FRA. American Sorciety for Microbiology (ASM), USA.

Subjects
[SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, ComputingMilieux_MISCELLANEOUS
Abstract

International audience

Published
2018

13. Multipartite viruses: a decentralized mode of functioning

Author

Anne Sicard, Marie-Stéphanie Vernerey, Ioannis Michalakis, Serafin Gutierrez-Simon, Stéphane Blanc, Biologie et Génétique des Interactions Plante-Parasite (UMR BGPI), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut National de la Recherche Agronomique (INRA)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro), Centre National de la Recherche Scientifique (CNRS), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (CIRAD). Montpellier, FRA., ProdInra, Migration, Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut National de la Recherche Agronomique (INRA)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), and Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)

Subjects
[SDV.BV]Life Sciences [q-bio]/Vegetal Biology, [SDV.BV] Life Sciences [q-bio]/Vegetal Biology, ComputingMilieux_MISCELLANEOUS
Abstract

National audience

Published
2015

14. Three Infectious Viral Species Lying in Wait in the Banana Genome

Author

Marie-Stéphanie Vernerey, Franc-Christophe Baurens, Valérie Barbe, Marie-Line Iskra-Caruana, Matthieu Chabannes, Pierre-Olivier Duroy, Stéphanie Bocs, Marguerite Rodier-Goud, Philippe Gayral, Laboratoire des interactions plantes micro-organismes (LIPM), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad), Amélioration génétique et adaptation des plantes méditerranéennes et tropicales (UMR AGAP), Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro)-Institut National de la Recherche Agronomique (INRA)-Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro), Biologie et Génétique des Interactions Plante-Parasite (UMR BGPI), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut National de la Recherche Agronomique (INRA)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Génomique métabolique (UMR 8030), Genoscope - Centre national de séquençage [Evry] (GENOSCOPE), Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS), Institut de recherche sur la biologie de l'insecte UMR7261 (IRBI), Université de Tours-Centre National de la Recherche Scientifique (CNRS), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS), Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Recherche Agronomique (INRA)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut National de la Recherche Agronomique (INRA)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université d'Évry-Val-d'Essonne (UEVE), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Université d'Évry-Val-d'Essonne (UEVE)

Subjects
0106 biological sciences, Genes, Viral, Gene Dosage, 01 natural sciences, Genome, virus des végétaux, F30 - Génétique et amélioration des plantes, Plant Viruses, banana B genome, Musa balbisiana, Gene Order, Banana streak virus, In Situ Hybridization, Fluorescence, Genomic organization, Genetics, Recombination, Genetic, 0303 health sciences, education.field_of_study, Vegetal Biology, banane, musa balbisiana, infectious, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Genome, Plant, sélection végétale, Genotype, Immunology, Population, endogenous Banana streak virus (eBSV), Genomics, Relation hôte pathogène, Biology, Microbiology, evolution 20 21, 03 medical and health sciences, musa acuminata, Virology, education, Gene, 030304 developmental biology, H20 - Maladies des plantes, amélioration génétique, génome, Haplotype, interaction hôte pathogène, Musa, séquence nucléotidique, biology.organism_classification, maladie des plantes, [SDV.BV.PEP]Life Sciences [q-bio]/Vegetal Biology/Phytopathology and phytopharmacy, Genetic Diversity and Evolution, Insect Science, Biologie végétale, 010606 plant biology & botany
Abstract

Plant pararetroviruses integrate serendipitously into their host genomes. The banana genome harbors integrated copies of banana streak virus (BSV) named endogenous BSV (eBSV) that are able to release infectious pararetrovirus. In this investigation, we characterized integrants of three BSV species— Goldfinger (eBSGFV), Imove (eBSImV), and Obino l'Ewai (eBSOLV)—in the seedy Musa balbisiana Pisang klutuk wulung (PKW) by studying their molecular structure, genomic organization, genomic landscape, and infectious capacity. All eBSVs exhibit extensive viral genome duplications and rearrangements. eBSV segregation analysis on an F1 population of PKW combined with fluorescent in situ hybridization analysis showed that eBSImV, eBSOLV, and eBSGFV are each present at a single locus. eBSOLV and eBSGFV contain two distinct alleles, whereas eBSImV has two structurally identical alleles. Genotyping of both eBSV and viral particles expressed in the progeny demonstrated that only one allele for each species is infectious. The infectious allele of eBSImV could not be identified since the two alleles are identical. Finally, we demonstrate that eBSGFV and eBSOLV are located on chromosome 1 and eBSImV is located on chromosome 2 of the reference Musa genome published recently. The structure and evolution of eBSVs suggest sequential integration into the plant genome, and haplotype divergence analysis confirms that the three loci display differential evolution. Based on our data, we propose a model for BSV integration and eBSV evolution in the Musa balbisiana genome. The mutual benefits of this unique host-pathogen association are also discussed.

Published
2013
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15. Virus factories of Cauliflower Mosaic Virus are virion reservoirs that engage actively in vector transmission

Author

Marie-Stéphanie Vernerey, Aurélie Bak, Jean Luc Macia, Enrick Malouvet, Daniel Gargani, Martin Drucker, Stéphane Blanc, Biologie et Génétique des interactions Plantes-parasites pour la Protection Intégrée, Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut National de la Recherche Agronomique (INRA)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad), SPE Department of INRA, Region Languedoc-Roussillon, and ANR (Agence National de la Recherche, France) [ANR12-BSV7-005-01]

Subjects
0106 biological sciences, Organite cellulaire, [SDV]Life Sciences [q-bio], viruses, Virologie, Myzus persicae, Fluorescent Antibody Technique, Caulimovirus mosaïque du chou fleur, Virus Replication, 01 natural sciences, Microtubules, Inclusion bodies, Caulimovirus, bodies, product, 2. Zero hunger, 0303 health sciences, biology, Protoplasts, Brassica rapa, food and beverages, plant-cells, Virus-Cell Interactions, Vecteur de maladie, Virion assembly, microscopy, leaves, Développement biologique, Immunology, gene-VI, Microtubule, Relation hôte pathogène, inclusion-body protein, Microbiology, Virus, 03 medical and health sciences, Viral Proteins, Virology, expression, Viroplasm, Animals, aphtransmission, Transmission des maladies, 030304 developmental biology, Plant Diseases, H20 - Maladies des plantes, Brassica napus, Virion, DNA, Virus des végétaux, biochemical phenomena, metabolism, and nutrition, biology.organism_classification, Viral replication, Cytoplasm, Insect Science, Aphids, Cauliflower mosaic virus, 010606 plant biology & botany
Abstract

Cauliflower mosaic virus (CaMV) forms two types of inclusion bodies within infected plant cells: numerous virus factories, which are the sites for viral replication and virion assembly, and a single transmission body (TB), which is specialized for virus transmission by aphid vectors. The TB reacts within seconds to aphid feeding on the host plant by total disruption and redistribution of its principal component, the viral transmission helper protein P2, onto microtubules throughout the cell. At the same time, virions also associate with microtubules. This redistribution of P2 and virions facilitates transmission and is reversible; the TB reforms within minutes after vector departure. Although some virions are present in the TB before disruption, their subsequent massive accumulation on the microtubule network suggests that they also are released from virus factories. Using drug treatments, mutant viruses, and exogenous supply of viral components to infected protoplasts, we show that virions can rapidly exit virus factories and, once in the cytoplasm, accumulate together with the helper protein P2 on the microtubule network. Moreover, we show that during reversion of this phenomenon, virions from the microtubule network can either be incorporated into the reverted TB or return to the virus factories. Our results suggest that CaMV factories are dynamic structures that participate in vector transmission by controlled release and uptake of virions during TB reaction.

Published
2013
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16. Toward the deciphering of chromosome structure in Vitis vinifera

Author

Anne-Françoise Adam-Blondon, Marie-Stéphanie Vernerey, M. C. Le Paslier, Dominique Brunel, Aurélie Bérard, A. Chauveau, Aurélie Canaguier, I. Le Clainche, Cécile Guichard, Olivier Coriton, G. Di Gaspero, Unité de recherche en génomique végétale (URGV), Institut National de la Recherche Agronomique (INRA)-Université d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS), Etude du Polymorphisme des Génomes Végétaux (EPGV), Institut National de la Recherche Agronomique (INRA), Amélioration des Plantes et Biotechnologies Végétales (APBV), Institut National de la Recherche Agronomique (INRA)-Université de Rennes (UR)-AGROCAMPUS OUEST, Biologie et Génétique des interactions Plantes-parasites pour la Protection Intégrée, Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut National de la Recherche Agronomique (INRA)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Università degli Studi di Udine - University of Udine [Italie], Institut National de la Recherche Agronomique (INRA)-Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-AGROCAMPUS OUEST, Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro), and ProdInra, Migration

Subjects
0106 biological sciences, Genetics, Genome evolution, Contig, sequence assembly, [SDV]Life Sciences [q-bio], food and beverages, Sequence assembly, Chromosome, SNP, Context (language use), 04 agricultural and veterinary sciences, Horticulture, Biology, 01 natural sciences, Genome, grapevine, [SDV] Life Sciences [q-bio], Centromere, 040103 agronomy & agriculture, 0401 agriculture, forestry, and fisheries, genetic map, Gene, 010606 plant biology & botany
Abstract

International audience; In the context of the sequencing project for the grapevine reference genome sequence, two genetic maps were completed with SSR markers in order to align the sequence contigs of the Vv12X.0 version of the genome assembly along the chromosomes. For this purpose, two full-sib families, 'Syrah' x 'Grenache' (SxG, 192 individuals) and 'Chardonnay' x 'Bianca' (CxB, 358 individuals) were respectively scored with 223 and 401 SSR. These two maps, supplemented with former data, allowed anchoring over 90% of the reference genome sequence. SNP markers were then defined from 37 anchored but not oriented and 44 not oriented and not anchored scaffolds of sequence. So far 110 new markers have been mapped, allowing anchoring 31 not yet anchored scaffolds (similar to 9 Mb) and orienting 35 scaffolds (similar to 15.5 Mb). In parallel, the recombination rate along the chromosomes is under study, in relation with sequence features (gene/repeat density, %GC, distance to the centromere/telomere). Finally, we are developing a cytogenetic map in Vitis vinifera using BAC-FISH. All these approaches will be combined to study the structure of the Vitis vinifera genome, including the factors affecting recombination rates and the dynamics of the genome evolution.

Published
2010

17. Patterns of sequence polymorphism in the fleshless berry locus in cultivated and wild Vitis vinifera accessions

Author

Thierry Lacombe, Patrice This, Laurent Torregrosa, Olivier Coriton, Amidou N'Diaye, Aurélie Canaguier, Dominique Brunel, Anne-Françoise Adam-Blondon, Marie-Stéphanie Vernerey, Jamila Chaïb, Cléa Houel, Cécile Guichard, Roberto Bacilieri, Rémi Bounon, Marie-Christine Le Paslier, Alexis Dereeper, Jean-Pierre Péros, Unité de recherche en génomique végétale (URGV), Institut National de la Recherche Agronomique (INRA)-Université d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS), Etude du Polymorphisme des Génomes Végétaux, Institut National de la Recherche Agronomique (INRA), Commonwealth Scientific and Industrial Research Organisation [Canberra] (CSIRO), Diversité et adaptation des plantes cultivées (UMR DIAPC), Université Montpellier 2 - Sciences et Techniques (UM2)-Institut National de la Recherche Agronomique (INRA)-Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad), Biologie et Génétique des Interactions Plantes-Agents Pathogènes, Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Supérieure Agronomique de Montpellier (ENSA M)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro), Unité mixte de recherche amélioration des plantes et biotechnologies végétales, AGROCAMPUS OUEST, Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut National de la Recherche Agronomique (INRA), Amélioration des Plantes et Biotechnologies Végétales (APBV), Institut National de la Recherche Agronomique (INRA)-Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-AGROCAMPUS OUEST, Amélioration génétique et adaptation des plantes méditerranéennes et tropicales (UMR AGAP), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut National de la Recherche Agronomique (INRA)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut National de la Recherche Agronomique (INRA)-Université Montpellier 2 - Sciences et Techniques (UM2), Institut National de la Recherche Agronomique (INRA)-AGROCAMPUS OUEST, and Institut National de la Recherche Agronomique (INRA)-Université de Rennes (UR)-AGROCAMPUS OUEST

Subjects
0106 biological sciences, Mutant, Plant Science, 01 natural sciences, Linkage Disequilibrium, génétique, Nucleotide diversity, Loss of heterozygosity, tomate, lcsh:Botany, Genotype, Vitis, évolution, In Situ Hybridization, Fluorescence, 2. Zero hunger, Genetics, 0303 health sciences, Vegetal Biology, tomato fruit size, Chromosome Mapping, food and beverages, lcsh:QK1-989, lycopersicon esculentum, Research Article, origins, genetic-map, grapevine genome, Locus (genetics), Single-nucleotide polymorphism, Biology, linkage disequilibrium quantitative trait, Polymorphism, Single Nucleotide, Synteny, Chromosomes, Plant, 03 medical and health sciences, Species Specificity, evolution, qtls, mutation, Genetic variation, [SDV.BV]Life Sciences [q-bio]/Vegetal Biology, Gene, 030304 developmental biology, Polymorphism, Genetic, génome, Genetic Variation, fruit, légume, Genetic Loci, Biologie végétale, Microsatellite Repeats, 010606 plant biology & botany
Abstract

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; Background: Unlike in tomato, little is known about the genetic and molecular control of fleshy fruit development of perennial fruit trees like grapevine (Vitis vinifera L.). Here we present the study of the sequence polymorphism in a 1 Mb grapevine genome region at the top of chromosome 18 carrying the fleshless berry mutation (flb) in order, first to identify SNP markers closely linked to the gene and second to search for possible signatures of domestication. Results: In total, 62 regions (17 SSR, 3 SNP, 1 CAPS and 41 re-sequenced gene fragments) were scanned for polymorphism along a 3.4 Mb interval (85,127-3,506,060 bp) at the top of the chromosome 18, in both V. vinifera cv. Chardonnay and a genotype carrying the flb mutation, V. vinifera cv. Ugni Blanc mutant. A nearly complete homozygosity in Ugni Blanc (wild and mutant forms) and an expected high level of heterozygosity in Chardonnay were revealed. Experiments using qPCR and BAC FISH confirmed the observed homozygosity. Under the assumption that flb could be one of the genes involved into the domestication syndrome of grapevine, we sequenced 69 gene fragments, spread over the flb region, representing 48,874 bp in a highly diverse set of cultivated and wild V. vinifera genotypes, to identify possible signatures of domestication in the cultivated V. vinifera compartment. We identified eight gene fragments presenting a significant deviation from neutrality of the Tajima's D parameter in the cultivated pool. One of these also showed higher nucleotide diversity in the wild compartments than in the cultivated compartments. In addition, SNPs significantly associated to berry weight variation were identified in the flb region. Conclusions: We observed the occurrence of a large homozygous region in a non-repetitive region of the grapevine otherwise highly-heterozygous genome and propose a hypothesis for its formation. We demonstrated the feasibility to apply BAC FISH on the very small grapevine chromosomes and provided a specific probe for the identification of chromosome 18 on a cytogenetic map. We evidenced genes showing putative signatures of selection and SNPs significantly associated with berry weight variation in the flb region. In addition, we provided to the community 554 SNPs at the top of chromosome 18 for the development of a genotyping chip for future fine mapping of the flb gene in a F2 population when available.

Published
2010
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18. Breaking dogmas: the plant vascular pathogen Xanthomonas albilineans is able to invade non-vascular tissues despite its reduced genome

Author

Philippe Rott, Marie-Stéphanie Vernerey, Imène Mensi, Daniel Gargani, Michel Nicole, Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad), Biologie et Génétique des Interactions Plante-Parasite (UMR BGPI), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut National de la Recherche Agronomique (INRA)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Institut de Recherche pour le Développement (IRD [Nouvelle-Calédonie]), Cirad, Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut National de la Recherche Agronomique (INRA)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro), and Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)

Subjects
Xanthomonas albilineans, L73 - Maladies des animaux, Tissu végétal, Immunologie, Microscopie électronique, Microscopie, lcsh:QH301-705.5, pathologie végétale, Pathogen, Vascular tissue, confocal laser scanning microscopy, Vegetal Biology, Saccharum officinarum, Microscopy, Confocal, non-vascular plant tissue, General Neuroscience, food and beverages, xanthomonas albilineans, cytochemistry, transmission electron microscopy, Bulliform cell, Saccharum, Épidémiologie, Research Article, Xanthomonas, Immunology, Laser, Biology, General Biochemistry, Genetics and Molecular Biology, Microbiology, Xylem, Parenchyma, [SDV.BV]Life Sciences [q-bio]/Vegetal Biology, microbiologie, Technique analytique, Plant Diseases, Research, Vascular bundle, Plant Leaves, lcsh:Biology (General), Phloem, Biologie végétale, Genome, Bacterial
Abstract

Xanthomonas albilineans , the causal agent of sugarcane leaf scald, is missing the Hrp type III secretion system that is used by many Gram-negative bacteria to colonize their host. Until now, this pathogen was considered as strictly limited to the xylem of sugarcane. We used confocal laser scanning microscopy, immunocytochemistry and transmission electron microscopy (TEM) to investigate the localization of X. albilineans in diseased sugarcane. Sugarcane plants were inoculated with strains of the pathogen labelled with a green fluorescent protein. Confocal microscopy observations of symptomatic leaves confirmed the presence of the pathogen in the protoxylem and metaxylem; however, X. albilineans was also observed in phloem, parenchyma and bulliform cells of the infected leaves. Similarly, vascular bundles of infected sugarcane stalks were invaded by X. albilineans . Surprisingly, the pathogen was also observed in apparently intact storage cells of the stalk and in intercellular spaces between these cells. Most of these observations made by confocal microscopy were confirmed by TEM. The pathogen exits the xylem following cell wall and middle lamellae degradation, thus creating openings to reach parenchyma cells. This is the first description of a plant pathogenic vascular bacterium invading apparently intact non-vascular plant tissues and multiplying in parenchyma cells.

Published
2014
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19. Localizing Genome Segments and Protein Products of a Multipartite Virus in Host Plant Cells

Author

Marie-Stephanie Vernerey, Elodie Pirolles, Stephane Blanc, and Anne Sicard

Subjects
Biology (General), QH301-705.5
Abstract

A founding paradigm in virology is that the spatial unit of the viral replication cycle is an individual cell. This concept applied to multipartite viruses–which have a genome composed of two or more nucleic acid segments, each individually encapsulated–implies that all segments constituting a viral genome need to coinfect the same host cell for replication to occur. Would this requirement be verified, it would constitute a major cost for extreme cases of multipartition such as the Faba bean necrotic stunt virus (FBNSV, nanovirus) whose genome is composed of eight complementary segments, each encoding a single gene (Grigoras et al., 2009). To address this question, we followed the distribution of the FBNSV genome segments by fluorescence in situ hybridization combined to immunolocalization of the replication-controlling viral protein within the cells of the host plant: Vicia Faba.A rapid and efficient protocol to localize viral transcripts in plant and insect hosts has been developed earlier (Ghanim et al., 2009). We here improve this method by using random-primed labeled probes and apply it to the detection and quantification of the individual segments composing the FBNSV genome. Moreover, we combine this technique with immunolocalization so that both viral segments and proteins can be visualized within the same samples.

Published
2019
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20. The question of modularity in the genome of multipartite viruses

Author

Anne Sicard, Marie-Stéphanie Vernerey, Michel Yvon, Michalakis, Y., Serafin Gutierrez-Simon, Stéphane Blanc, Biologie et Génétique des interactions Plantes-parasites pour la Protection Intégrée, Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut National de la Recherche Agronomique (INRA)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro), Centre National de la Recherche Scientifique (CNRS), International Union of Microbiological Societies (IUMS). Montréal, CAN., and ProdInra, Migration

Subjects
[SDV.BV]Life Sciences [q-bio]/Vegetal Biology, [SDV.BV] Life Sciences [q-bio]/Vegetal Biology, ComputingMilieux_MISCELLANEOUS
Abstract

International audience

21. Association genetics in relation with berry size variation in grapevine (Vitis vinifera)

Author

Cléa Houel, Le Clainche, Isabelle I., Canaguier, Aurelie A., F Adam-Blondon, Anne-Francoise A., Rémi Bounon, Berard, A., Marie-Christine Le Paslier, Brunel, Dominique D., Laurent Torregrosa, Roberto Bacilieri, Stéphane Nicolas, Thierry Lacombe, Alexis Dereeper, Jean-Pierre Peros, Chaïb, J., Thomas, M., Marie-Stéphanie Vernerey, Coriton, Olivier O., Unité de recherche en génomique végétale (URGV), Institut National de la Recherche Agronomique (INRA)-Université d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS), Diversité et adaptation des plantes cultivées (UMR DIAPC), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut National de la Recherche Agronomique (INRA)-Université Montpellier 2 - Sciences et Techniques (UM2), Plant Industry, Commonwealth Scientific and Industrial Research Organisation [Canberra] (CSIRO), Amélioration des Plantes et Biotechnologies Végétales (APBV), Institut National de la Recherche Agronomique (INRA)-Université de Rennes (UR)-AGROCAMPUS OUEST, Institut National de la Recherche Agronomique (INRA)-Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-AGROCAMPUS OUEST, Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro), and Université Montpellier 2 - Sciences et Techniques (UM2)-Institut National de la Recherche Agronomique (INRA)-Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)

Subjects
[SDV]Life Sciences [q-bio], taille des baies, Solanacées
Abstract

Corresponding author: houel@evry.inra.fr; International audience; Berry size is an important trait both for yield (table grapes) and in relation to quality (wine grapes). The final size of an organ is dependent on cell number, cell size and/or number of carpel. Indeed it has been shown that cell number is a major determinant of fruit size variability in Solanaceae and Prunus, whereas a preliminary study on a small sample of grapevine genotypes suggested that the variability of berry size resulted from cell volume enlargement. At present only few genes involved in fruit size variation have been identified, mainly in Solanaceae and none in grapevine. Our aims are: (1) to check, in a large sample of grapevine genotypes, the relative importance of cell volume enlargement, number of cell and carpel in berry size variation, (2) to develop association genetics approaches with candidate genes for berry size and (3) to study the DNA polymorphism along the region carrying the fleshless berry locus (flb) in order to detect possible selection pressures in cultivated varieties. The flb locus was identified in a grapevine natural mutant which present berries with a size reduced by 20 times and circular seeds similar to wild grapevine berries. The ovaries and the berries of 3 subpopulations of cultivated Vitis vinifera genotypes (279), which represent the genetic and phenotypic variability of berry size, have been phenotyped. Preliminary results on a small sample of individual (27) suggested that the final berry size was determined at early stage of berry development. The ovarian size just before fertilization was variable and proportional to the final berry size. However the ovarian cell number or size seemed to explain the final berry size differently according to sub-populations. Several mechanisms and probably several genes would be involved in the variation of the berry size in these sub-populations. These data are currently under validation on the whole set of 279 individuals. In parallel, 44 candidate genes, orthologous to genes known to be involved in flower or fruit development and/or co-localizing with berry size QTLs (Quantitative Trait Locus) and/or expressed during grapevine berry development, have been partially or totally sequenced in 47 genotypes which reflect all the genetic diversity of grapevine. Several thousands of SNPs (Single Nucleotide Polymorphims) have been identified. A subset of these SNPs will be used for genotyping the 3 sub-populations using the SNPlex technology, in order to detect a possible correlation between genotypic and phenotypic variation. Finally, in order to test the hypothesis that flb is one of the genes selected during grapevine domestication, 69 gene fragments along flb locus (1Mbp) were sequenced in a sample of wild and cultivated Vitis vinifera. Our results revealed a small zone showing more polymorphism in wild genotypes than in cultivated genotypes and a large zone of 171Kb showing either more polymorphism in cultivated genotypes than in wild genotypes. These intervals will be studied more in details using two approaches: gene expression studies and an improvement of the genotype sampling for the DNA polymorphism studies.

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