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陳貽進 and Yi-chun Mark Tan. "Studies on the turnip mosaic virus." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 1990. http://hub.hku.hk/bib/B31210004.
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Tan, Yi-chun Mark. "Studies on the turnip mosaic virus /." [Hong Kong] : University of Hong Kong, 1990. http://sunzi.lib.hku.hk/hkuto/record.jsp?B12907248.
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Thivierge, Karine. "Host proteins involved in «turnip mosaic virus» life cycle." Thesis, McGill University, 2010. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=86689.
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All viruses are gene poor relative to their host, thus, most steps in virus infection involve interactions between viral components and host factors. Identification of these factors represents one of the major frontiers in current virus research. In this study, protein-protein interaction methodologies were used to find host interactors of Turnip mosaic virus (TuMV) RNA-dependent RNA polymerase (RdRP), VPg-protease (VPg- Pro) and P3 protein.<br>First, eukaryotic elongation factor 1A (eEF1A) was shown to interact with TuMV RdRp and VPg-Pro using tandem affinity purification in Arabidopsis thaliana and/or in vitro assays. Interaction of eEF1A with both viral proteins was shown to take place within 6K-VPg-Pro-induced vesicles. The same vesicles were also shown to contain poly(A)-binding (PABP) and heat shock cognate 70-3 proteins (Hsc70), two previously identified RdRp interactors. To further characterize the content of these vesicles upon TuMV infection, a fluorescently labeled 6K-GFP TuMV infectious clone was constructed and used in confocal microscopy experiments. The inclusion of eEF1A, PABP, Hsc70, eukaryotic initiation factor (iso)4E and VPg-Pro in TuMV-induced vesicles was demonstrated. It is well establish that positive-strand RNA viruses assemble their RNA replication complexes on intracellular membranes, usually in association with vesicle formation. For TuMV, our data suggest that it is the 6K-induced vesicles that house the viral replication complex (VRC). Moreover, the presence of replication and translation elements in these vesicles indicates that both processes might be coupled in TuMV VRC.<br>Secondly, the yeast two-hybrid system was used to identify plant P3-interacting proteins in a cDNA library from A. thaliana. A lipase was recovered from the screen and shown to interact with P3 in vitro. Both proteins were also demonstrated to partially co-localize in the cytoplasm of the cell. Given that lipases play important roles in the plant response to biotic stress, this interaction reinforce the role of TuMV P3 in plant resistance and/or pathogenesis.<br>Les virus ont de petits génomes qui codent pour un nombre limité de protéines et dépendent conséquemment des facteurs de l'hôte pour compléter leur cycle de réplication. Dans ce projet, nous avons utilisé différentes méthodes pour identifier des partenaires protéiques de la polymérase virale à ARN (RdRp), de la VPg-Pro et de la protéine P3 du virus de la mosaïque du navet (TuMV).<br>Premièrement, nous avons trouvé que le facteur eucaryote d'élongation de la traduction 1A (eEF1A) interagit avec la RdRp et la VPg-Pro en utilisant une stratégie de purification en tandem in planta et/ou des essais in vitro. Nous avons montré que ces interactions se produisent en association avec les membranes du réticulum endoplasmique, plus précisément dans les vésicules induites par le polypeptide 6K-VPg-Pro. Nous avons aussi démontré que ces mêmes vésicules contiennent les protéines Hsc70-3 et PABP, deux partenaires connus de la RdRp. Afin de poursuivre la caractérisation du contenu de ces vésicules, nous avons créé un vecteur infectieux du TuMV permettant d'étiqueter les vésicules avec la GFP et d'être utilisé en microscopie confocale. À l'aide de ce vecteur, nous avons observé la présence du facteur eEF1a, de la PABP, de la Hsc70, du facteur eucaryote d'initiation de la traduction (iso) 4E et de la VPg-Pro dans les vésicules induites par le TuMV. Il est bien établit que le complexe de réplication des virus à ARN positif est associé aux membranes cytoplasmiques, généralement sous forme de vésicules. Pour le TuMV, nos données semblent indiquer que les vésicules induites par la protéine 6K contiennent le complexe de réplication viral (VRC). De plus, la présence d'éléments participants à la réplication ainsi qu'à la traduction dans ces vésicules suggère que ces deux processus sont possiblement couplés dans le VRC du TuMV.<br>Deuxièmement, le système du double-hybride en levure a été utilisé pour rechercher des partenaires protéiques de P3. Le criblage de P3 contre une banque d'ADNc d'Arabidopsis thaliana a révélé une interaction entre P3 et une lipase. Lorsque exprimées ensembles dans Nicotiana benthamiana, les deux protéines co-localisent au cytoplasme. Étant donné le rôle des lipases dans les réponses des plantes aux attaques pathogènes, cette interaction renforce le rôle suggéré de la protéine P3 dans la pathogenèse et les mécanismes de résistance des plantes.
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Cotton, Sophie. "Characterization and movement of turnip mosaic virus replication complexes." Thesis, McGill University, 2010. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=86558.
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Viruses are intracellular pathogens that use the host cell to produce new infectious progeny. For all positive-strand RNA viruses that have been investigated so far, viral replication takes place in cytoplasmic virus-induced membrane structures. For turnip mosaic virus (TuMV), the generation of vesicles likely associated with the replication complex depends on the synthesis of the viral protein 6K. To further characterize the vesicles formed during TuMV infection, Nicotiana benthamiana plants were agroinfiltrated with a TuMV infectious clone expressing 6K protein fused to GFP. Using confocal microscopy, cytoplasmic aggregates were observed, corresponding to the 6KGFP-induced vesicles which house the viral replication complexes (VRCs). Intracellular movement of these vesicles was visualized by time-lapse imaging. Vesicle trafficking was inhibited when plants were infiltrated with latrunculin B, an inhibitor of microfilament polymerization. The absence of movement had severe effects on viral accumulation. Viral vesicles also aligned with actin filaments. These results indicate that microfilaments are necessary for VRC trafficking which is important for virus infectivity.<br>The biogenesis of viral vesicles was investigated by infecting cells with two recombinant TuMVs producing 6KGFP or 6KmCherry-labelled vesicles. Individual vesicle within a cell contained unique protein products derived from each recombinant demonstrating the origin of a vesicle from a single viral genome. Green and red sectoring was also observed, meaning that vesicles could fuse together. The presence of the eukaryotic translation factors eIF(iso)4E, PABP and eEF1A enclosed in VRC was demonstrated previously by our group. These data combined to a single-genome origin suggest that viral translation occur within these structures. The same host factors were also found to co-localize with the active replicating sites along with viral proteins VPg-Pro, RdRp and CI using immunofluorescence labelling in infected protoplasts. These data bring accumulating evidence for the possible coupling of viral translation and replication.<br>Les virus sont des parasites intracellulaires qui utilisent la cellule hôte pour produire une nouvelle descendance infectieuse. Pour tous les virus à ARN positif étudiés jusqu'à maintenant, la réplication virale prend place dans des structures membranaires induites par le virus. Chez le virus de la mosaïque du navet (TuMV), la formation de vésicules probablement associées au complexe de réplication dépend de la synthèse de la protéine virale 6K. Afin de caractériser les vésicules formées durant l'infection de TuMV, des plants de Nicotiana benthamiana ont été agroinfiltrés avec un clone infectieux de TuMV exprimant la protéine 6K fusionnée à GFP. Des agrégats cytoplasmiques ont été observés par microscopie confocale, correspondant aux vésicules induites par la protéine 6KGFP et abritant le complexe de réplication virale (VRC). Le mouvement intracellulaire de ces vésicules a été visualisé par imagerie time-lapse. Le trafic des vésicules a été inhibé lorsque les plantes étaient infiltrées avec de la latrunculin B, un inhibiteur de polymérisation des microfilaments. L'absence de mouvement a également conduit à une sévère diminution de l'accumulation virale. Les vésicules colocalisent avec les filaments d'actine. Ces résultats indiquent que les microfilaments sont nécessaires au mouvement des vésicules lequel est important pour l'infection virale.<br>La biogenèse des vésicules virales a été investiguée en infectant les cellules avec deux clones infectieux de TuMV produisant des vésicules étiquetées par 6KGFP ou 6KmCherry. Des vésicules individuelles contenant des protéines uniques dérivant d'un seul clone recombinant a démontré que l'origine des vésicules provient d'un seul génome. Des vésicules ayant des secteurs vert et rouge ont aussi été observé, indiquant que la fusion de vésicules est possible. La présence des facteurs eucaryotes de traduction eIF(iso)4E, PABP et eEF1A à l'intérieur des vésicules a été démontré par notre groupe. Ces données combinées à l'origine unique des vésicules suggèrent que la traduction virale se produit à l'intérieur de ces vésicules. Les mêmes facteurs de traduction ainsi que les protéines virales VPg-Pro, RdRp et CI colocalisent avec les sites de réplication active dans des protoplastes infectés. Ces données apportent des indices supplémentaires sur la possibilité de couplage entre la traduction et la réplication virale chez TuMV.
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Thivierge, Karine. "Protein-protein interactions in turnip mosaic potyvirus replication complex." Thesis, McGill University, 2003. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=80886.
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Interactions between plant and virus proteins play pivotal roles in many processes during the viral infection cycle. Analysis of protein-protein interactions is crucial for understanding virus and host protein functions and the molecular mechanisms underlying viral infection. Several interactions between virus-encoded proteins have been reported. However, few interactions between viral and plant proteins have been identified so far. To examine interactions between Turnip mosaic potyvirus (TuMV) proteins and plant proteins, recombinant proteins were produced and used in ELISA-type assays and in in vitro co-immunoprecipitation experiments. An interaction between TuMV P1 proteinase and wheat poly(A)-binding protein (PABP) was identified. An interaction between P1 protein and the plant Arabidopsis thaliana eukaryotic initiation factor (iso)4E [eIF(iso)4E] was also found. Finally, potential interactions between both TuMV CI and P1 proteins and between TuMV CI protein and eIF(iso)4E were identified.
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Syme, Jennifer. "Characterization of Arabidopsis thaliana (Columbia) infected with turnip mosaic virus (TuMV)." Thesis, McGill University, 1996. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=24043.
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The response of Arabidopsis thaliana (Columbia) to infection with turnip mosaic virus (TuMV) was characterized at the level of: disease symptom expression, cell content and protein composition. Visual symptoms observed were chlorotic and mottled leaf colouring, severely stunted growth, distortion of leaf blades and delayed bolting. All plants died before seed cases dehisced. Electron microscopy revealed three types of cylindrical inclusion bodies: pinwheels, scrolls and laminated aggregates, in the cytoplasm of infected plants similar to those observed in other plants infected with TuMV. Inoculation of Arabidopsis with TuMV resulted in quantitative changes in several proteins in both soluble and membrane proteins, as revealed by electrophoresis on 12% polyacrylamide gels. Antibodies were made to both infected membrane and soluble proteins. Western blots of infected and uninfected, soluble and membrane proteins probed with antibodies revealed quantitative changes in the same proteins identified by polyacrylamide gels. A CNBr 4B activated sepharose column was used to make infection-specific antibodies to infected soluble proteins. No infection-specific host proteins were detected in Arabidopsis infected with TuMV.
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Yang, Chunling. "Functional Genomic analysis of Turnip mosaic virus infection in Arabidopsis thaliana." [Ames, Iowa : Iowa State University], 2007.
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Hughes, Sara Louise. "Interaction of turnip mosaic virus (TuMV) with members of the Brassicaceae." Thesis, University of Birmingham, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.401328.
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Rushholme, Rachel L. "The genetic control of resistance to turnip mosaic virus (TuMV) in Brassica." Thesis, University of East Anglia, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.327536.
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Berthelot, Edwige. "Etude de l'activation de la transmission du Turnip mosaic virus par pucerons." Thesis, Montpellier, SupAgro, 2018. http://www.theses.fr/2018NSAM0039.
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Beaucoup de virus de plante sont transmis selon le mode non-circulant par vecteurs hémiptères, souvent des pucerons. Ce mode de propagation répand les virus très rapidement d’un hôte à l’autre, ce qui cause d’innombrables dégâts sur de nombreuses cultures d'intérêt agronomique. Actuellement, le manque de connaissances sur leur processus de transmission constitue un frein pour le développement de méthodes de lutte efficaces. Récemment, des résultats ont montré qu’un virus modèle, le Cauliflower mosaic virus (CaMV, genre Caulimovirus), répond à l'arrivée de son vecteur puceron en formant, à ce moment précis et de manière réversible, des complexes transmissibles du virus, obligatoire pour son acquisition par le puceron. Ce phénomène extraordinaire nommé « activation de la transmission (AT) » contrôle l’acquisition du virus et donc sa transmission. Il constitue une découverte majeure pour la transmission non-circulante et ouvre de nombreuses perspectives de recherche dont celle d’élargir ce phénomène à autres virus pour savoir si l’AT est un phénomène généralisé dans la propagation des virus.Dans cette optique, nous nous sommes intéressés à un virus non apparenté au CaMV, le Turnip mosaic virus (TuMV), un Potyvirus, également transmis par pucerons. La transmission du TuMV dépend de l’interaction directe de la particule virale et de la protéine virale facteur assistant de la transmission (HC-Pro). En effet, HC-Pro doit former avec la particule un complexe transmissible, seule forme du virus pouvant être acquis par le vecteur. Nos résultats montrent que le TuMV utilise l’AT et qu’elle est dépendante de la présence d’espèces réactives de l’oxygène (ROS) et de la signalisation calcique. L’AT du TuMV est corrélée avec la formation d’oligomères par ponts disulfures de HC-Pro et avec la formation des complexes transmissibles HC-Pro/particules virales dans les cellules végétales. Notre analyse pharmacologique a montré que l’AT du CaMV dépendait également de la présence des ROS et de la signalisation calcique. Ces deux voies de signalisation sont donc impliquées dans les étapes présumées précoces de l’AT du CaMV et du TuMV. Des expériences avec d’autres composés ont mis en évidence que les étapes suivantes de l’AT sont différentes pour les deux virus. Une recherche d’éliciteurs de l’AT a été initiée mais n’a pour l’instant pas permis d’en identifier un.Ces résultats ont mis en évidence l’existence de l’AT pour un deuxième genre de virus non-circulant, suggérant qu’il pourrait s’agir d’un mode de transmission général. Ces nouvelles données ouvrent, d’un point de vu appliqué, de nouvelles perspectives pour les stratégies de lutte, visant à interrompre spécifiquement le processus de l’AT<br>Many plant viruses are transmitted in non-circulative manner by hemipteran vectors, often aphids. This transmission mode spreads viruses very quickly from one host to another and thereby causes countless damage on many crops of agronomic interest. Currently, the lack of knowledge about their transmission process is a hindrance to the development of effective control methods. Recently, results have shown that the model virus, Cauliflower mosaic virus (CaMV, genus Caulimovirus), responds to the arrival of aphid vectors by forming, at this time and reversibly, virus transmissible complexes, obligatory for the acquisition by the aphid. This extraordinary phenomenon called "transmission activation (TA)" controls the virus acquisition and thus its transmission. It’s a major discovery in non-circulative transmission and opens many research perspectives including expanding this phenomenon to other viruses to see if TA is a widespread phenomenon in the viruses propagation.Having this in mind, we are interested to study a virus unrelated to CaMV, Turnip mosaic virus (TuMV), a Potyvirus, also transmitted by aphids. The TuMV transmission depends on the direct interaction of the virus particle and the viral helper component protease (HC-Pro). Indeed, HC-Pro must form with the virus particle a transmissible complex, the only form of virus that can be acquired by the vector. Our results show that TuMV uses TA and that its TA depends on the presence of reactive oxygen species (ROS) and on calcium signaling. TuMV TA is correlated with the formation of HC-Pro disulfide bond mediated oligomers and the formation of HC-Pro transmissible complexes HC-Pro / viral particle in plant cells. Our pharmacological analysis showed that CaMV TA also depends on the presence of ROS and calcium signaling. These two signaling pathways are therefore involved presumably in the early stages of CaMV and TuMV TA. Experiments with other compounds have shown that the following steps of TA are different for the two viruses. A search for elicitors of the TA has been initiated but has not been successful yet.These results provide evidence of the existence of TA for a second genus of non-circulative virus, suggesting that this might be a general transmission mode. These new data open, from an applied point of view, new perspectives for control strategies, aimed at specifically interrupting the TA process
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Chang, Peta-Gaye Suzette. "Plant Virus Diagnostics: Comparison of classical and membrane-based techniques for immunoassay and coat protein sequence characterization for Cucumber mosaic virus and three potyviruses." Diss., Virginia Tech, 2009. http://hdl.handle.net/10919/28017.
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Diagnostics is important in the development and implementation of pest management strategies. The virus diagnostic capabilities of several plant pathology collaborators within the Integrated Pest Management Collaborative Research Support Program (IPM CRSP) host countries were evaluated with the aid of a survey. Very few plant disease diagnostic clinics had funds to cover daily operations despite over half of the responding clinics receiving an operational budget. Academically and government affiliated clinics within the developing host countries had little access to molecular tools and equipment, relying mostly on biological and serological methods. Clinics affiliated with private companies and within the USA relied more upon molecular assays. Ten CMV isolates identified by tissue blot immunoassay (TBIA) were collected from a garden at the Historic Smithfield Plantation on the Virginia Tech campus, and from Painter, Virginia on the Eastern Shore. Three CMV isolates from Smithfield were biologically compared to six early CMV isolates stored since the 1970s, while all isolates were compared serologically and molecularly. Sequences obtained after reverse transcription-polymerase chain reaction (RT-PCR) assigned the CMV isolates into subgroups, with eleven to subgroup 1A and three to subgroup 2. The subgroup assignments were confirmed by TBIA using CMV subgroup-specific monoclonal antibodies (Agdia Inc). At Smithfield Plantation, another virus, Turnip mosaic virus (TuMV) was identified from Dameâ s Rocket (Hesperis matronalis L.). This is the first report of TuMV in Virginia. In TBIA virus-infected plant samples are blotted onto nitrocellulose membranes, dried, and processed. Membranes can be stored for long periods of time and transported safely across borders without risk of introducing viruses into new environments, but virus remains immunologically active for several months. Methods were developed with CMV and three potyviruses, using the same membranes, for detecting viral RNA by RT-PCR and direct sequencing of PCR products.. Amplification by RT-PCR was possible after membrane storage for up to 15 months. The membranes also performed well with samples sent from IPM CRSP host countries and within the USA. This method should improve molecular diagnostic capabilities in developing countries, as samples can be blotted to membranes and sent to a centralized molecular laboratory for analysis.<br>Ph. D.
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Plante, Daniel 1970. "Interaction of the turnip mosaic potyvirus VPg with the plant translation apparatus." Thesis, McGill University, 2000. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=37812.
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An interaction was recently detected between the potyviral protein, genome-linked (VPg) and the Arabidopsis thaliana translation initiation factor eIF(iso)4E (Wittmann et al., 1997).<br>Here, experiments were undertaken to address biological aspects of the VPg-eIF4E interaction. First, coimmunoprecipitation experiments performed with purified recombinant proteins have shown that VPg not only associates with eIF4E, as was previously published, but also with the larger eIF4F complex, of which eIF4E is a subunit. These results were confirmed by ELISA-type binding assays. It was also shown that there is no direct interaction between VPg and the other subunit of eIF4F, namely eIF4G. Finally, with the same experimental system, it was shown that the presence of eIF4G does not influence the binding affinity of VPg and eIF4E.<br>The interaction of VPg with the plant translation apparatus suggests that potyviral infection may alter the host protein expression profile. This hypothesis was investigated with the use of a protoplast system. We have shown that the global rates of protein synthesis in protoplasts transfected with an infectious TuMV cDNA clone dropped shortly after transfection, by as much as an estimated 70%. Recovery to normal levels occurred within 48 hours.<br>Evidence was obtained that the interaction between VPg and eIF4E is instrumental in this transient down-regulation of protein expression: protoplasts transfected with a mutant TuMV cDNA clone, the VPg of which has no affinity for eIF4E, failed to exhibit the drop in protein synthesis observed with the wild-type clone.
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Nellist, Charlotte F. "The deployment and mechanism of broad-spectrum resistance to turnip mosaic virus in Brassica rapa." Thesis, University of Warwick, 2013. http://wrap.warwick.ac.uk/62029/.
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The potyvirus Turnip mosaic virus (TuMV) is a major constraint on the cultivation of a wide range of plant species worldwide. It causes significant economic losses in brassica species such as Chinese cabbage (Brassica rapa), which is one the most important vegetable crops in the world. The B. rapa line RLR22 has broad-spectrum resistance to TuMV, which is undefeated. Many recessive resistances against plant viruses in the Potyvirus genus are based on mutations in plant eukaryotic translation initiation factor 4E (eIF4E), or its isoform eIF(iso)4E . B. rapa has three eIF4E genes and three eIF(iso)4E genes. Segregation following a cross between RLR22 and the TuMV-susceptible R-o-18 line of the closely related B. rapa ssp. trilocularis revealed the resistance was due to a recessive gene, retr01 that was epistatic to a dominant gene, ConTR01. My research revealed that retr01 is BraA.eIF(iso)4E.a and that ConTR01 is probably BraA.eIF(iso)4E.c. It also showed that the highly sought after broad-spectrum resistance to TuMV is due to a novel, recessive, natural mechanism, based on the mis-splicing of BraA.eIF(iso)4E.a in B. rapa. This results in a range of eIF(iso)4E splice variants, the most common of which retained the whole of intron 1 and appears to be non-functional for the virus. As the susceptible parent in the original cross, R-o-18, was a different sub-species to RLR22 (B. rapa var. pekinensis, Chinese cabbage), the genetic inheritance of resistance was also investigated in crosses with Chinese cabbage lines; F2 segregation ratios were consistent with those predicted for the single recessive gene (retr01 ). Yeast two-hybrid interactions between the viral protein genome-linked (VPg) of TuMV and eIF(iso)4E from B. rapa seem to be TuMV isolate-specific. Aphid transmission experiments to investigate the complementation of an eIF(iso)4E Arabidopsis thaliana knockout line with B. rapa BraA.eIF(iso)4E.a confirmed the earlier results from mechanical inoculation of these plants. The inability of TuMV to access multiple copies of eIF(iso)4E in Chinese cabbage and the broad-spectrum of the resistance, suggest it may prove to be durable.
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Ribeiro, Junior Marcos Roberto 1994. "Caracterização biológica, serológica e molecular de Turnip mosaic virus (TuMV) infectando rúcula, alface e acelga /." Botucatu, 2018. http://hdl.handle.net/11449/153910.
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Orientador: Renate Krause Sakate<br>Banca: Ricardo Gioria<br>Banca: Denise Nakada Nozaki<br>Resumo: A partir do ano de 2016, anormalidades foram observadas em campos de produção de rúcula (Eruca sativa) no interior do estado de São Paulo, região de Botucatu. As plantas acometidas apresentavam mosaico, redução drástica no crescimento e deformação foliar, sintomas típicos da infeção por vírus. Associadas as essas plantas, também foram observadas altas populações de pulgões/afídeos e de plantas daninhas, como nabiça (Raphanus raphanistrum) e nabo forrageiro (Brassica rapa) apresentando sintomas de mosaico. Inicialmente, as plantas foram submetidas ao teste de ELISA indireto usando um antissoro para potyvirus, em seguida foi realizada a extração de RNA total e RT-PCR, utilizando as amostras ELISA-positivas. A sequência correspondente à região codificadora para a proteína capsidial foi obtida e o vírus foi identificado como Turnip mosaic virus (TuMV). Posteriormente outros campos de produção de folhosas foram visitados no Estado de São Paulo (região de Bragança Paulista e Mogi-Mirim) e o TuMV foi novamente identificado em rúcula, porém também em alface (Lactuca sativa) e acelga (Beta vulgaris subsp. vulgaris). Analises biológicas e moleculares agruparam ambos os isolados de TuMV no grupo Brassica-Raphanus (BR), que inclui isolados infectando espécies de Brassica e Raphanus. É apresentado aqui o primeiro relato de TuMV naturalmente infectando rúcula, alface e acelga no Brasil. De acordo com a análise filogenética, pelo menos duas introduções diferentes de isolados de TuMV ocorrer... (Resumo completo, clicar acesso eletrônico abaixo)<br>Abstract: Abnormalities have been observed in rocket (Eruca sativa) fields in Botucatu, Sao Paulo state, Brazil since 2016. Symptoms of mosaic, reduction in foliar growth and deformation, typical symptoms of virus infection were observed in rocket plants. Associated to these plants we also found high populations of aphids, as well as raphanus weeds (Raphanus raphanistrum) and forage turnip (Brassica rapa) with mosaic symptoms. Initially, the plants were submitted to indirect ELISA using a potyvirus antiserum, and then total RNA extraction and RT-PCR were performed using the ELISA-positive samples. The complete coat protein sequence was obtained and the virus was identified as Turnip mosaic virus (TuMV). Later, other fields were visited in Sao Paulo state (cities of Bragança Paulista and Mogi-Mirim) and TuMV was again identified not only in rocket, but also in lettuce (Lactuca sativa) and chard (Beta vulgaris subsp. vulgaris). Biological and molecular analysis grouped both TuMV isolates in the Brassica-Raphanus (BR) clade, which includes isolates infecting Brassica and Raphanus species. Here is the first report of rocket, lettuce and chard naturally infected with TuMV in Brazil. According to the phylogenetic analysis, at least two different introductions of TuMV isolates occurred in Brazil, corresponding to the basal-BR and world-B types, infecting Brassica/Raphanus and Brassica, respectively ...<br>Mestre
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Dufresne, Philippe. "Involvement of poly (A)-binding and heat shock 70 kDa proteins in «Turnip mosaic virus» infection." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=21945.
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Viruses are obligate intracellular parasites. Most possess small genomes with a limited coding capacity, which means that they rely on host factors for the completion of their life cycle. The recruitment of cellular proteins is essential as in their absence viruses cannot replicate effectively. Tandem affinity purification was used in Arabidopsis thaliana to identify host interactors of Turnip mosaic virus (TuMV) RNA-dependent RNA polymerase (RdRp). The heat shock cognate 70-3 (Hsc70-3) and poly(A)-binding (PABP) proteins were recovered and shown to interact with the RdRp in vitro. As previously shown for PABP, Hsc70 was redistributed to nuclear and membranous fractions from infected plants and both RdRp interactors were co-immunoprecipitated from a membrane-enriched extract using RdRp specific antibodies. Fluorescently-tagged RdRp and Hsc70-3 localized to cytoplasm and nucleus when expressed alone or in combination in Nicotiana benthamiana. However, when co-expressed with TuMV membrane binding protein 6K-VPg-Pro, they were redistributed to large perinuclear vesicles where replication takes place. Thus, Hsc70-3 and PABP2 are potentially integral components of the replicase complex and could have important roles to play in potyviral RdRp functions. To further characterize the role of PABP in potyvirus infection single and double gene knockouts were isolated and characterized for all high expressing class II PABP genes in Arabidopsis thaliana: PAB2, PAB4 and PAB8, all of which were found to be viable and fertile. Whereas single knockout plants, for the most part, demonstrate a normal phenotype, pab2 pab4 and pab2 pab8 double mutants had important debilitations in regard to the growth and development of both vegetative and reproductive organs. Experiments with these PABP deficient plants indicate that partial PABP depletion in A. thaliana, although not sufficient to confer complete resistance, can impede replicative cycle of TuMV in the cell. TuMV replication in the<br>Les virus sont des parasites intracellulaires stricts. La plupart possèdent de petits génomes ayant une capacité d'encodage limitée et dépendent conséquemment des facteurs de l'hôte pour compléter leur cycle d'infection. Le recrutement de ces protéines est essentiel à leur réplication. Nous avons utilisé une stratégie de purification en tandem dans Arabidopsis thaliana dans le but d'identifier les protéines de l'hôte interagissant avec la polymérase virale à ARN (RdRp) du virus de la mosaïque du navet (TuMV). Les protéines Hsc70-3 et PABP ont été purifiées et leur interaction avec la RdRp confirmée in vitro. Tel que rapporté pour la PABP, la Hsc70 s'est retrouvée redistribuée aux fractions nucléaires et membranaires dans des plants infectés. De plus, nous avons été en mesure de co-immunoprécipiter ces deux protéines dans un extrait membranaire avec un anticorps anti-RdRp. Lorsqu'exprimées seules ou ensemble dans Nicotiana benthamiana, la RdRp et la Hsc70-3 localisèrent au cytoplasme et au noyau. Par contre, lorsque co-exprimées avec le polypeptide 6K-VPg-Pro du TuMV, elles se trouvèrent redistribuées à l'intérieur d'une vésicule périnucléaire là où le virus se réplique. Hsc70-3 et PABP2 sont donc potentiellement des protéines faisant partie du complexe de réplication et ont probablement un rôle important à jouer dans les fonctions de la RdRp. Afin de déterminer le rôle de la PABP dans le cycle de réplication des potyvirus, nous avons généré des mutants d'A. thaliana knockout simple ou double pour tous les gènes de PABP de la classe II; PAB2, PAB4 et PAB8. Alors que les mutants simples présentent un phénotype normal, les mutants double pab2 pab4 et pab2 pab8 ont des déficiences de croissance et de développement. Les expériences menées avec ces plants indiquent qu'une déplétion partielle des niveaux de PABP, quoique étant insuffisante pour mener à un phénotype de résistance complet, inhibe le cycle
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Zaccomer, Bruno. "Stratégie pour interférer avec la réplication virale dans des plantes transgéniques : le système modèle du Tymv (Turnip Yellow Mosaic Virus)." Paris 11, 1993. http://www.theses.fr/1993PA112061.
Full textAbstract:
Le Tymv (Turnip Yellow Mosaic Virus) est un tymovirus dont le génome est composé d'un arn de polarité positive. Nous avons crée des colzas transgéniques exprimant des transcrits contenant l'extrémité 3 non codante du génome viral. Il a été montré in vitro que cette séquence de 100 nucléotides était reconnue par la repli case virale et servait ainsi de site d'initiation pour la réplication. Cette séquence, exprimée dans des plantes, a un effet protecteur vis-à-vis d'une inoculation mécanique de virion ou d'arn viral. Nous pensons que, de même qu'in vitro, elle agit comme compétiteur du génome viral pour la réplication. De plus, nous avons montré que dans des plantes transgéniques infectées, cette séquence est reconnue par la repli case virale et qu'elle permet la synthèse d'un arn complémentaire au transcrit initial. Par la suite, nous avons introduit dans le colza des constructions codant pour des arn défectifs interférents ou di. Les arn di sont de petites molécules d'arn associées naturellement à certaines souches virales. Elles sont dérivées du génome de leur virus assistant dont elles conservent au moins deux origines de réplication qui leur permettent de se répliquer, tout en interférant avec la réplication virale. Nous avons récemment régénéré des plantes exprimant des arn di. Les tests préliminaires d'inoculation que nous avons effectués sur ces plantes semblent montrer que la résistance conférée par l'expression d'arn di est du même type que celle conférée par l'expression de l'extrémité 3 non codante du génome du Tymv
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Ozumit, Alen. "Interaction between turnip mosaic potyvirus (TuMV) cylindrical inclusion protein and Arabidopsis thaliana histone H3 protein." Thesis, McGill University, 2003. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=79060.
Full textAbstract:
Turnip mosaic potyvirus (TuMV) is a single-stranded RNA plant virus. One of its proteins, the cylindrical inclusion (CI) protein, was hypothesized to interfere with host transcription via interaction with histone H3 protein. Interaction between CI and histone H3 was previously observed in Dr. Fortin's laboratory. Based on previous studies that demonstrated the importance of the H3 tail domain in gene regulation and chromosome arrangement, it was hypothesized that CI would interact with the tail rather than the globular domain. The objective of this project was to identify which histone H3 domains CI protein interacts with. The full-length, globular, and tail domains of histone H3 DNA were expressed in E. coli and purified. Based on in vitro interaction experiments, the CI protein was observed to interact with the globular domain of histone H3.
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Sassi, Giovanna. "Relative quantification of host gene expression and protein accumulation upon turnip mosaic potyvirus infection in tobacco." Thesis, McGill University, 2004. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=81433.
Full textAbstract:
Turnip mosaic virus (TuMV) infects a variety of crops, worldwide, including the economically relevant Brassicacea family. It was previously demonstrated that TuMV infection in tobacco protoplasts leads to an overall decrease of host protein. However, it remains unclear whether this phenomenon is due to the repression of plant gene transcription during the infection period or due to viral inhibition of host translation. In this study, quantification of various transcripts and protein products from infected tobacco was performed via real-time RT-PCR and ELISA. In comparison to the gamma-tubulin endogenous control, gene expression for the tobacco H3, HSP70 and granule-bound starch synthase was affected by TuMV infection with time.<br>Tobacco protein accumulation in whole leaf tissues was also significantly affected by increase of virus particles.
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Montesclaros, Luz B. "Mapping of molecular markers surrounding the Tu gene conferring resistance to turnip mosaic virus in Lactuca sativa L." Thesis, McGill University, 1996. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=23923.
Full textAbstract:
In lettuce (Lactuca sativa), the dominant gene Tu confers resistance to turnip mosaic virus (TuMV) infection. In order to eventually clone and characterize the Tu gene using a map-based cloning strategy, the chromosome region in which Tu is located needs to be saturated with molecular markers. Random polymorphic DNA (RAPD) markers were screened using bulked segregant analysis. Nine new RAPD markers, UBC431$ rm sb{420}, UBC431 sb{940}, UBC434 sb{360}, UBC434 sb{1000}, UBC439 sb{520}, UBC448 sb{685:750}, UBC135 sb{240}, OP108 sb{410} and OP108 sb{1305},$ were identified as linked to Tu. Each marker was mapped relative to Tu using F$ sb2$ individuals previously known to be recombinant in the area surrounding the Tu locus. Three new markers, UBC431$ rm sb{420}, UBC439 sb{520} and UBC135 sb{240}$ are within a 5 cM area of Tu. As the number of DNA markers on the map increased map expansion and difficulties in determining a unique order were encountered. To increase the confidence in the estimate of genetic distances, a population of 500 F$ sb2$ plants was screened in order to identify more recombinant individuals around the Tu locus. The population was screened using markers UBC431$ sb{420}$ and UBC135$ sb{240}.$ Thirty-three recombinants were identified in an interval of 6.6 cM. Two markers, UBC346$ sb{1067}$ and OP108$ sb{634},$ tightly flanking Tu were converted to sequence characterized amplified regions (SCAR 346 and SCAR L08). No polymorphism was detected among the SCARs generated. The area surrounding Tu now includes 24 RAPD markers in an interval of 44 cM.
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Robbins, Marjorie. "The location of Tu on the genetic map of Lactuca sativa and the identification of random amplified polymorphic DNA markers flanking and tightly linked to Tu /." Thesis, McGill University, 1993. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=69684.
Full textAbstract:
In Lactuca sativa, the dominant gene Tu confers resistance to infection by turnip mosaic virus (TuMV). Tu and Dm5/8, a gene for resistance to Bremia lactucae, are linked in L. sativa. The area surrounding Dm5/8 on the genetic map of L. sativa contains restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) markers. The orientation of Tu relative to Dm5/8 was not known. Locating Tu would indicate which markers are on the map of lettuce close to Tu. To locate Tu on the L. sativa genetic map, F$ sb3$ families from recombinant F$ sb2$ in the Dm5/8 area of a cross between TuMV-resistant (Cobbham Green) and susceptible (Calmar) cultivars were inoculated with TuMV and phenotyped for Tu by indirect enzyme-linked immunosorbent assay. Polyclonal antibodies for immunodetection were produced using turnip mosaic virus coat protein expressed in E. coli. Phenotypic ratios within F$ sb3$ families were used to determine individual F$ sb2$ genotypes for Tu. With these genotypes, Tu was located on the genetic map of L. sativa relative to data present for Dm5/8 and surrounding markers, between OPM18 and OPY13. Using bulked segregant analysis, bulks created for the Dm5/8 locus were screened for genetic polymorphisms by the RAPD technique. Five new RAPD markers, UBC346, UBC517, UBC563, UBC599, and UBC675 were found linked to Tu after mapping relative to F$ sb2$ genotypes for Tu and other RAPD markers. The resulting three-point mapping information indicates that Tu is flanked by two markers, OPM18/OPL08 and UBC346, at respective genetic distances of 0.4 and 0.7 cM.
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Nettleship, Sarah Bethany. "Molecular analysis of recognition and action of the extreme resistance gene TuRBO1,against turnip mosaic virus in Brassica napus." Thesis, University of Bristol, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.288325.
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Wang, Tongtong. "Resistance to Turnip mosaic virus (TuMV) in Brassica juncea and introgression of resistance from Brassica rapa, Brassica napus and Brassica nigra into Brassica juncea." Thesis, University of Warwick, 2016. http://wrap.warwick.ac.uk/89272/.
Full textAbstract:
Turnip mosaic virus (TuMV, family Potyviridae, genus Potyvirus) has the widest host range amongst potyviruses. Globally it was said to be the second most important virus infecting field vegetables. Brassica juncea (Oriental mustard, family Brassicaceae), is an amphidiploid plant species with the genome AABB, comprising the genomes of the two diploid species, Brassica rapa (AA) and Brassica nigra (BB). It is widely grown and has various uses including as a leaf, stem, or root vegetable, oilseed crop, forage crop, condiment and biofumigant. Most B. juncea cultivars are very susceptible to TuMV, resulting in severe losses. Research on TuMV resistance and the mapping and identification of natural resistance genes would be very useful in order to speed up breeding resistant crops through marker-assisted selection. Sources of resistance to TuMV have been identified in B. juncea. The specificity of the resistances has been determined. A B. juncea DH line for which there is genomic information has been challenged with TuMV and found to be susceptible. This line has been used as a susceptible parent in crosses with resistant plants derived from different sources to develop segregating populations for mapping the resistance gene(s). Two BC1 populations (222 plants and 205 plants) and one F2 population (159 plants) have been phenotyped and segregation ratios were not significantly different from a Mendelian model based on the action of two recessive genes. Parental lines and selected plants in the two BC1 populations have been analysed by SNPs genotyping using the Illumina Infinium Chip. Genetic linkage maps have been constructed and QTLs have been mapped. Additionally, attempts are being made to identify a dominant TuMV resistance gene present in both Brassica napus and B. rapa. Inter-specific crosses have been made in order to introgress this gene into B. juncea. Resynthesised B. juncea plants possessing this dominant resistance have been produced through embryo rescue and polyploidy induction of F1 plants from crosses between resistant B. rapa and susceptible B. nigra plants. BC2 plants have also been developed by crossing B. rapa and B. napus plants possessing the dominant TuMV resistance with a susceptible B. juncea plant line.
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Rubio, Bernadette. "Réponse d’Arabidopsis thaliana au Turnip mosaic virus (TuMV) en conditions extérieures et en conditions contrôlées : phénotypage fin de traits de maladie et métaboliques et architecture génétique associée." Thesis, Bordeaux, 2017. http://www.theses.fr/2017BORD0758/document.
Full textAbstract:
Les plantes sont des organismes immobiles qui doivent répondre et s’adapter à des contraintes abiotiques et biotiques. Parmi les stress biotiques, les maladies virales, établies ou émergentes, peuvent être responsables de pertes de rendement majeures aux conséquences économiques importantes. Face aux phytovirus la lutte génétique constitue le moyen de lutte le plus efficace, le plus respectueux de l’environnement et du consommateur. Comprendre l’interaction entre les plantes et les virus reste indispensable pour rechercher de nouvelles sources de résistances. Ce travail de thèse s’intéresse à l’étude du pathosystème naturel Arabidopis thaliana/Turnip mosaic virus (TuMV). Les essais ont été menés majoritairement en conditions extérieures permettant une analyse de l’interaction dans un environnement multistress. La réponse d’A. thaliana a été explorée par l’étude de traits liés à la maladie et par la variation en métabolites primaires et secondaires. Ce travail a permis i) de caractériser de façon fine la réponse d’A. thaliana au TuMV en conditionsmultistress en exploitant la diversité naturelle d’une population mondiale et française ii) de déterminer l’architecture génétique de cette interaction par des approches de génétique d’association et de QTL mapping. Plusieurs nouveaux loci potentiellement impliqués dans la réponse ont été identifiés iii) de montrer l’intérêt du phénotypage métabolique pour discriminer les accessions en fonction de leur sensibilité au TuMV. La multidisciplinarité des approches constitue la richesse de ce travail de thèse qui contribue à une meilleure caractérisation et compréhension de la réponse des plantes lors d’une infection virale<br>Plants are immobile organisms which have to adapt to abiotic and biotic constraints. Among bioticstress, established or emerging viral diseases, may be responsible for major yield losses withsignificant consequences. Genetic control is the most effective, environmentally and consumerfriendlyway to control viral infections. Understanding plant/virus interactions remains essential tosearch for new sources of resistance. This work, focuses on the study of the natural pathosystemArabidopsis thaliana/Turnip mosaic virus (TuMV). Most of the trials were conducted in commongarden conditions allowing the analysis of the interaction in a multistress environment. A. thaliana’sresponse was explored through the study of disease-related traits and the variations in primary andsecondary metabolites. This work allows i) the fine characterization of A. thaliana’s response toTuMV in multistress conditions through the exploration of the natural diversity of a world and Frenchpopulation ii) to determine the genetic architecture of this interaction by genome wide associationsand QTL mapping. Several new loci potentially involved in the response have been identified iii) tohighlight the interest of metabolic phenotyping to discriminate accessions according to theirsusceptibility to TuMV. The multidisciplinary approaches contribute to a better characterization andunderstanding of plant-virus interaction
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Dupin, André. "Le virus de la mosaique de l'aubergine : sequence de sa proteine capsidale et apports a la connaissance des tymovirus." Université Louis Pasteur (Strasbourg) (1971-2008), 1987. http://www.theses.fr/1987STR13180.
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Morch, Marie-Dominique. "Organisation et expression genetiques du virus de la mosaique jaune du navet : contribution a l'etude des strategies d'expression des virus a arn, a la discussion de leur variabilite et a l'elaboration de strategies de protection." Paris 7, 1988. http://www.theses.fr/1988PA077124.
Full textAbstract:
On procede au sequencage du genome complet du virus constitue d'un arn de polarite positive a 6318 nucleotides et contenant 3 cistrons. Parmi les strategies mises en oeuvre pour synthetiser ses proteines, on presente la maturation proteolytique de la proteine 206 k codee par ce virus. Une approche "arn-sens" pour la lutte anti-virale est testee experimentalement: des arn "pseudogenomiques" rentrent en competition avec l'arn du virus et sont capables d'inhiber sa replication in vitro
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FLORENTZ, EGELE CATHERINE. "L'extremite 3'oh aminoacyable du rna du virus de la mosaique jaune du navet : relations entre structure et fonctions." Université Louis Pasteur (Strasbourg) (1971-2008), 1987. http://www.theses.fr/1987STR13181.
Full textAbstract:
Analyse structurale (structure secondaire et tertiaire) d'un fragment contenant la sequence "trna like" (extremite 3' oh possedant plusieurs caracteristiques d'un arn de transfert). Modelisation de la structure sur ecran graphique. Analyse des zones de contact entre ce fragment d'arn et la valyl-trna synthetase, a l'aide de differentes sondes structurales. Discussion sur le role de l'extremite "trna like" dans le cycle de developpement du virus (regulation de la traduction de l'information genetique portee par l'arn viral)
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Weiland, John J. "The roles of turnip yellow mosaic virus genes in virus replication." Thesis, 1992. http://hdl.handle.net/1957/36154.
Full textAbstract:
Turnip yellow mosaic virus is a monopartite, plus sense RNA virus
infecting the Cruciferae, and is a model system for the study of RNA virus
replication. A cDNA clone (pTYMC) representing an infectious RNA
genome of the European isolate of TYMV was constructed and used to assess
the importance of virus genes in virus infectivity.
Derivatives of pTYMC with alterations in open reading frame 69 (ORF-
69) were made. The mutations disrupted the expression of ORF-69 in vitro as
predicted. Although the ORF-69 mutants were competent for replication in
protoplasts, none of the mutants detectably infected turnip or Chinese cabbage
plants, except where reversion mutations led to the restoration of an
uninterrupted ORF-69. The data suggest a role for ORF-69 expression in the
cell-to-cell movement of the virus.
Mutant RNAs with a deletion or frameshift in the coat protein ORF
infected protoplasts and plant leaves. No systemic infection symptoms were
generated by these mutants, and no viral products were detected in young,
expanding tissue of infected plants. When the coat protein deletion mutant
and an ORF-69 mutant were co-inoculated onto plants, only a virus
producing a coat protein of wild type size was detected in symptomatic,
systemic tissue in these inoculations, emphasizing a requirement for the
expression of native size coat protein for the systemic translocation of TYMV
infection.
The role of ORF-206 expression in TYMV replication was examined.
Three classes of mutants were made in ORF-206: those affecting the synthesis
of the 150 kDa protein, those affecting the synthesis of the 70 kDa protein, and
those affecting the synthesis of both the 150 and the 70 kDa proteins. All ORF-
206 mutations eliminated RNA infectivity. Protoplast inoculations using
mixtures of individual ORF-206 mutant RNAs and a helper genome
demonstrated that co-replication of defective genomes could occur.
Moreover, inoculations in which individual 150 kDa and 70 kDa protein
mutant RNAs were combined showed that complementation between these
two classes of mutants was possible. The data indicate that RNAs expressing
wild type 150 kDa protein are favored replication substrates in mixed
infections, and suggest that the 150 kDa protein functions preferentially in cis.<br>Graduation date: 1993
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Chen, Tsui-Miaw, and 陳翠妙. "Characterization of a Turnip Mosaic Virus Isolate Causing Albinic Mosaic Disease of Garland Chrysanthemum." Thesis, 1997. http://ndltd.ncl.edu.tw/handle/98367360107761049077.
Full textAbstract:
碩士<br>國立中興大學<br>植物病理學系<br>85<br>A disease of garland chrysanthemum ( Chrysanthemum coronarium
var. spatiosumBailey ) showing symptoms of albinism, mosaic,
leaf-cleavage and stunting wasfound in the central part of
Taiwan. A filamentous virus ( named GCV-Am )about 775 × 13
nm in size was isolated from the infected plants and
wasproved to be the causal agent of the disease. In the host
range test, thevirus caused systemic mosaic symptoms on
many cruciferous crops includingChinese mustard ( Brassica
campestris ssp. shinensis ), Chin ese white cabbage( B.
campestris ssp. chinese var communis ), radish ( Raphanus
sativas L. ) ,rape ( B. campestris ), and mustard ( B. juncea
), and induced necrotic locallesions on Chenopodium quinoa,
C. amaranticolor, Gomphrena globosa andNicotiana tabacum (
TT5 ). It was transmitted by aphids ( Myzus persicaeSultz.
) in a non-persistent manner. Numerous cytoplasmic inclusions
withboundles or irregular shapes surrounding nuclei, were
observed in epidermalcells of infected garland chrysanthe
mum and rape when the epidermis wasstained with calcomine
orange-Luxol brilliant green BL. Electron microscopicanalysis
of ultrathin sections of infected leaf cells of rape and
mustardrevealed inclusions of laminated aggregates and
scrolls in the cytoplasm.Virions of GCV-Am were purified from
infected leaves of rape by polyethyleneglycol precipitation
followed by CsCl isopycnic centrifugation. Relativemolecular
weight of the coat protein of GCV-Am was estimated as 37 kDa.
Anantiserum was produce d from New Zealand white rabbit
immunized with virusparticles. The titer of the antiserum was
1/512 and 1/8 as determined by ringtest and immunodiffusion
test, respectively. Results of immunodiffusion testsand gold
immuno-labelling indicated that GCV-Am is
serologicallyindistinguishable from turnip mosaic virus ( TuMV
). Based on host reactions,particle morphology, transmission
properties, molecular weight of coat proteinand serological
reactions, GCV-Am was identified as an isolate of TuMV. Current
study also indicates that the garland chrysanthemum is a new
natural hostof TuMV. GCV-Am induced symptoms on Brassica
campestris subsp. pekinensiscultivars Tropical delight,
Crusader, PI 418957 and PI 419105, similar tothose induced
by the TuMV C5 strain originated from Taiwan. The
antiserumproduced for GCV-Am gave patterns of coalescence
when GCV-Am was tested inimmunodiffusion tests with C1, C2,
C3, C4, and C5 strains of TuMV indicatingreactions of
identity. In indirect ELISA and western blott ing tests,
theantisera to GCV-Am or TuMV-B ( from German ) also reacted
with the GCV-Amisolate and the other five strains,
indistinguishably. The molecular weight ofthe coat protein of
GCV-Am was also similar to those of other five strains.All
isolates of the TuMV strains used in current studies induced
cylindricalinclusions in host cytoplasm, in various shapes
of pinwheels, laminatedaggregates, scrolls and bundles. In
order to further characterize the virus atthe molecular level,
CP genes of GCV- Am, TuMV C4, and TuMV C5 were cloned,
sequenced and analyzed. Comparison of nucleotide identities of
the CP readingframes and the 3* non-coding regions of the
three isolates of TuMV revealedthat the three isolates are
closely related strains of the same virus. Thethree Taiwan
isolates were further compared with other reported TuMV
isolatesof MV, MUT, and RN ( from UK ), NIAP, CQS( from Korea ),
CAPP1 ( from Canada )and CPP ( from China ). Results showed
that the CP genes of the GCV-Am andTu-C5 is olates from Taiwan
share the highest amino acid identity of 99.31 ％with both MV
and MUT strains ( from UK ), and the Tu-C4 isolate shares
thehighest identity of 99.31 ％ with the NIAP strain ( from
Korea ). A comparisonof the 3* non-coding regions of the seven
TuMV isolates showed that the threeTuMV isolates from Taiwan
share higher percentages ( 98.56 - 99.04 ％ ) ofhomology with
the CAPP1 strain ( from Canada ). Our results indicate that
thedegree of the homology of the CP genes is related to the
major difference inhost specificity, but not to geographic
distribution. This investigationindicates that GCV-Am is a
TuMV isolate causing albinic mosaic disease ofgarland
chrysanthemum. The virus is indistinguishable from other TuMV
strainsin Taiwan both in serological tests and
cytopathological observations. Themolecular analysis of the
coat protein genes of GCV-Am and TuMV strainsfurther
sugests that GCV-Am is a variant of TuMV C5 strain.
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Pei-Yin, Chen. "Development of Turnip Yellow Mosaic Virus Particles as Nanomaterial for Biosensor." 2006. http://www.cetd.com.tw/ec/thesisdetail.aspx?etdun=U0005-2007200622302600.
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Chen, Pei-Yin, and 陳佩吟. "Development of Turnip Yellow Mosaic Virus Particles as Nanomaterial for Biosensor." Thesis, 2006. http://ndltd.ncl.edu.tw/handle/87739518542017335266.
Full textAbstract:
碩士<br>國立中興大學<br>生物科技學研究所<br>94<br>Turnip yellow mosaic virus is an icosahedral plant virus, 28nm in diameter. The viral particle, composed of a 6.3 kb single-strand positive-sense RNA genome encapsidated by 180 copies of capsid protein, offers a uniquely programmable biological nanoscaffold for multiplexed conjugation. Our interest is to create a multivalent display system which would dramatically enhance the sensitivity of conventional ELISA. To achieve this objective, we used molecular techniques to create a recombinant virus that has an extra cysteine residue which chemically is highly active, at the C-terminus of the coat protein. Fluorescein-5-maleimide and Fluorescein N-hydroxysuccinimide were used to determine the number of the exposed thiol-groups and amine-groups, respectively, on the surface of the virus particles. Fluorescein-5-maleimide did not label the wild-type particles suggested that there are no cysteines on the surface, whereas the surface of the mutant particles were strongly labeled at least higher than 100 thiol-groups per particle. Fluorescein N-hydroxysuccinimide labeled the wild-type and the mutant particles with similar efficiency 400 amine-groups on the particle surface were estimated from the labeling intensity. Fluorescence labeling of fluorescein N-hydroxysuccinimide on the virus particle was successful, and the conjugation of an antibody to the virus particle is in progress. The efficiency of this nanobiosensor will be tested in the future.
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Tsai, Ching-Hsiu. "Characterization of the role of the 3' noncoding region of turnip yellow mosaic virus RNA." Thesis, 1993. http://hdl.handle.net/1957/36247.
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Wu, Zi-Rong, and 吳姿蓉. "A New Virus Architecture : Self-assembly of Genetically Modified Turnip Yellow Mosaic Virus as Nanomaterials." Thesis, 2004. http://ndltd.ncl.edu.tw/handle/74614357836908041786.
Full textAbstract:
碩士<br>國立中興大學<br>生物科技學研究所<br>93<br>貳、英文摘要
The field of nanotechnology has created immense interests and high expectations. Recently biotechnology has become a promising contributor to development of nanotechnology, creating a new discipline - nanobiotechnology. Among various biomolecules candidates, macro molecular assemblages, like viruses, seem to be favorable contenders for most applications due to their stability and flexibility of manipulation. Here we have used Turnip yellow mosaic virus (TYMV), a non-enveloped icosahedral virus of 28 nm diameter, as a candidate by modifying its coat protein to carry a C-terminal cysteine residue alone (TYMV-Cys) or with six histidine residues preceding the Cys residue (TYMV-(His)6Cys). These constructs are expected to generate novel virus particles with about a hundred sulfur functional groups that can be used to link with gold particles or other linkers, such as fluorescien-5-maleiminde and N-(3- maleimidylpropiobyl)biocytin. The surface properties of these genetically modified viruses can also be applied to form desired patterns under highly controlled conditions. The results of the modification of virus genome show that the mutant viruses form intact virions the result shows the morphology of the mutant virions is intact and similar to the wild type virion. The TEM images results seem like the binding efficiency of mutant type TYMV-Cys with nanogold was higher than the wild type TYMV. In order to further demonstrate that the dark particles distributed on the virus surface were metal nano-gold, we will confirm it by using Energy Dispersive Spectrum (EDS). TYMV (His)6Cys was unable to produce virions both in infected plants and in protoplasts derived from Chinese cabbage. We will also compare and confirm the binding ability of TYMV-(Ala)6Cys with nanogold.
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"The study of anti-viral properties of trichosanthin on turnip mosaic virus." Chinese University of Hong Kong, 1994. http://library.cuhk.edu.hk/record=b5895463.
Full textAbstract:
by Lam Ying Hoo.<br>Thesis (M.Phil.)--Chinese University of Hong Kong, 1994.<br>Includes bibliographical references (leaves 145-149).<br>Acknowledgements --- p.i<br>Abstract --- p.ii<br>Contents --- p.iv<br>Abbreviations --- p.x<br>Chapter Chapter 1 --- Introduction<br>Chapter 1.1. --- Trichosanthin --- p.1<br>Chapter 1.2. --- Anti-plant viral and fungal properties of RIPs --- p.3<br>Chapter 1.3. --- Agrobacterium-mediated transformation --- p.5<br>Chapter 1.3.1. --- Ti (tumor inducing) plasmid --- p.6<br>Chapter 1.3.2. --- Role of vir proteins in T-DNA transfer --- p.6<br>Chapter 1.3.3. --- Integration of T-DNA into plant genome --- p.11<br>Chapter 1.3.4. --- Use of Agrobacterium plasmid as transformation vectors --- p.13<br>Chapter 1.4. --- Objective and strategy of producing transgenic plants that express TCS --- p.15<br>Chapter Chapter 2 --- Materials and Methods<br>Chapter 2.1. --- Bacterial Strains used --- p.19<br>Chapter 2.2. --- General Techniques --- p.20<br>Chapter 2.2.1. --- Growth of bacterial strains --- p.20<br>Chapter 2.2.2. --- Restriction Enzyme Digestion of DNA --- p.21<br>Chapter 2.2.3. --- Agarose Gel Electrophoresis of DNA --- p.21<br>Chapter 2.2.4. --- Purification of DNA fragments from Agarose Gel using GeneClean II® ( BIO 101 Inc.) kit --- p.22<br>Chapter 2.2.5. --- Purification of DNA fragments by Phenol/Chloroform Extraction --- p.23<br>Chapter 2.2.6. --- Ligation of DNA fragments --- p.24<br>Chapter 2.2.7. --- Preparation and Transformation of Escherichia coli Competent Cells --- p.24<br>Chapter 2.2.8. --- Minipreparation of Plasmid DNA --- p.26<br>Chapter 2.2.9. --- Preparation of Plasmid DNA using Magic´ёØ Minipreps DNA Purification kit from Promega --- p.27<br>Chapter 2.2.10. --- Preparation of Plasmid DNA using Qiagen-pack 100 Cartridge --- p.29<br>Chapter 2.2.11. --- SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.30<br>Chapter 2.2.12. --- Western Blot detection of TCS --- p.33<br>Chapter 2.2.13. --- Polymerase Chain Reaction (PCR) --- p.34<br>Chapter 2.3. --- Construction of Plant Transformation Vectors --- p.36<br>Chapter 2.3.1. --- Construction of pSLJ58210 --- p.36<br>Chapter 2.3.2. --- Construction of pSLJ TCS1 and pSLJ TCS2 --- p.38<br>Chapter 2.3.3. --- Conjugation of pSLJ TCS1 and pSLJ TCS2 into A. tumefaciens by Triparental Mating --- p.41<br>Chapter 2.4. --- Transformation of Tobacco Leaf Explants by Agrobacterium tumefaciens --- p.43<br>Chapter 2.4.1. --- Growth of A. tumefaciens LBA4404 (pSLJ TCS1) --- p.43<br>Chapter 2.4.2. --- Surface Sterilization of tobacco leaves --- p.43<br>Chapter 2.4.3. --- Inoculation of tobacco leaf explants with A. tumefaciens LBA4404 (pSLJ TCS1) --- p.44<br>Chapter 2.4.4. --- Regeneration of shoots from Transformed explants --- p.45<br>Chapter 2.4.5. --- Rooting of Transformed shoots --- p.45<br>Chapter 2.4.6. --- Re-establishment of cultured Plantlets in soil --- p.45<br>Chapter 2.5. --- Analysis of the Regenerated Transgenic Tobacco --- p.46<br>Chapter 2.5.1. --- Isolation of plant leaf protein --- p.46<br>Chapter 2.5.2. --- SDS-PAGE and Western blot detection of TCS --- p.48<br>Chapter 2.5.3. --- Anti-viral assay of Transgenic tobacco against TuMV --- p.48<br>Chapter 2.6. --- Bioassay of Inhibitory activity of TCS protein against TuMV --- p.49<br>Chapter 2.6.1. --- Preparation of biologically active TCS protein --- p.49<br>Chapter 2.6.2. --- Purification of TuMV from infected plant leaves --- p.51<br>Chapter 2.6.3. --- Mechanical Inoculation of virus onto host plant --- p.53<br>Chapter 2.6.4. --- Anti-viral assay on Local Lesion host --- p.54<br>Chapter 2.6.5. --- Anti-viral assay on Systemic host --- p.55<br>Chapter 2.7 --- Establishment of the plant culture medium for efficient Regeneration from tissue explants of Brassica parachinensis --- p.56<br>Chapter 2.7.1. --- Preparation and Sterilization of culture medium --- p.57<br>Chapter 2.7.2. --- Preparation of Sterile seedlings of B. parachinensis --- p.57<br>Chapter 2.7.3. --- Regeneration from Cotyledon petiole and Hypocotyl segment explants --- p.58<br>Chapter 2.7.4. --- Regeneration from Internode stem segment explants of shoot culture --- p.60<br>Chapter 2.8. --- Reagents and Buffers --- p.61<br>Chapter 2.8.1. --- Media for Bacterial culture --- p.61<br>Chapter 2.8.2. --- Media for Plant tissue culture --- p.64<br>Chapter 2.8.3. --- Restriction Enzymes --- p.66<br>Chapter 2.8.4. --- Buffers for Agarose Gel Electrophoresis --- p.66<br>Chapter 2.8.5. --- DNA ligation Buffer --- p.67<br>Chapter 2.8.6. --- Reagents for preparation of E.coli competent cells --- p.67<br>Chapter 2.8.7. --- Reagents for preparation of Plasmid DNA --- p.68<br>Chapter 2.8.8. --- Reagents for Qiagen-pack 100 Cartridge --- p.69<br>Chapter 2.8.9. --- Reagents for SDS-PAGE --- p.70<br>Chapter 2.8.10. --- Reagents for Western Blotting --- p.71<br>Chapter Chapter 3 --- Construction of Plant Transformation Vectors<br>Chapter 3.1. --- Introduction --- p.73<br>Chapter 3.2. --- Results --- p.74<br>Chapter 3.2.1. --- Construction of pSLJ58210 --- p.74<br>Chapter 3.2.2. --- Construction of the recombinant binary vectors pSLJ TCSl and pSLJ TCS --- p.78<br>Chapter 3.2.3. --- Conjugation ofpSLJ TCS 1 and pSLJ TCS 2 into Agrobacterium tumefaciens via Triparental Mating --- p.82<br>Chapter 3.3. --- Discussion --- p.90<br>Chapter Chapter 4 --- Transformation of Tobacco Leaf Explants by Agrobacterium tumefaciens<br>Chapter 4.1. --- Introduction --- p.94<br>Chapter 4.2. --- Results --- p.95<br>Chapter 4.2.1. --- Regeneration of leaf explants after transformation --- p.95<br>Chapter 4.2.2. --- The level of expression of TCS in transgenic tobacco leaf --- p.100<br>Chapter 4.3. --- Discussion --- p.104<br>Chapter 4.3.1. --- Regeneration of transgenic tobacco plants --- p.104<br>Chapter 4.3.2. --- Expression of TCS in transgenic tobacco plants --- p.108<br>Chapter Chapter 5 --- Two approaches to study the Inhibitory effect of TCS on TuMV<br>Chapter 5.1. --- Introduction --- p.112<br>Chapter 5.2. --- Results --- p.113<br>Chapter 5.2.1. --- Expression and purification of recombinant TCS --- p.113<br>Chapter 5.2.2. --- Purification of TuMV --- p.119<br>Chapter 5.2.3. --- Anti-viral assay on local lesion host --- p.119<br>Chapter 5.2.4. --- Anti-viral assay on Systemic host --- p.124<br>Chapter 5.2.5. --- Anti-viral assay of Transgenic tobacco against TuMV --- p.126<br>Chapter 5.3. --- Discussion --- p.129<br>Chapter Chapter 6 --- Establishment of plant culture conditions for efficient shoot regeneration from tissue explants of B.parachinensis<br>Chapter 6.1. --- Introduction --- p.133<br>Chapter 6.2. --- Results --- p.133<br>Chapter 6.3. --- Discussion --- p.137<br>Chapter Chapter 7 --- Conclusion<br>Appendix<br>Chapter A.1. --- Size of molecular weight markers --- p.143<br>Chapter A.2. --- References --- p.145
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Powell, Joshua D. "Biophysical properties of the turnip yellow mosaic virus explored by coat protein mutagenesis." Thesis, 2012. http://hdl.handle.net/1957/29113.
Full textAbstract:
Plant viruses have been instrumental in our understanding of the biophysical properties pertaining to non-enveloped icosahedral virus particles. A substantial amount of research has been performed over five decades on Turnip yellow mosaic virus (TYMV), arguably one of the most extensively studied icosahedral plant viruses and the type-member of the Tymovirus plant virus genus. Even with a substantial body of published scientific literature, little is known about the role of specific coat protein (CP) residues in TYMV assembly, disassembly and disencapsidation.
We have shown through our mutagenesis studies that the N-terminal region of the CP that is involved in the formation of an annulus structure and is disordered in A-subunit pentamers is not essential in vivo, but annulus-forming residues are critical in ensuring virion stability and low accessibility after virus is purified (Chapter 2). We have shown that a range of amino acid residue types is tolerated within the CP N-terminus in vivo, although they can greatly affect the stability of virions and empty particles, most notably at low pH (Chapter 3). Unlike full-length CP, N-terminal deletion and substitution mutants fail to reassemble into particles in vitro (Chapter 2, 3) suggesting a critical determinant for the N-terminus in reassembly (discussed Chapter 7). This is the first documented in vitro reassembly reported for a member of the Tymoviridae family and should provide a framework for further studies. We have identified a new way to create empty artificial top component (ATC)-particles through treatment with EDTA (Chapter 6) and we also show that tymoviruses can be engineered with altered pH-dependent enhanced stability (Chapter 4). In collaboration with the Qian Wang laboratory from the University of South Carolina we have shown that an RGD (Arg-Gly-Asp) motif can be genetically engineered within the CP of TYMV, resulting in infectious particles with attractive stem-cell adhesion properties (Chapter 5). With focus on basic viral mechanisms, we have crystallized the TYMV virion and ATC particle at pH 7.7 and collected data to less than 5 Å resolution (Chapter 4, supplementary). These structures represent the first tymovirus-based structures solved above pH 5.5 and will provide insight into the N-terminal conformations within the TYMV particle. Finally, we have characterized an N-terminal CP cleavage seen after ATC formation (Chapter 4) suggesting an additional and yet uncharacterized feature associated with decapsidation.<br>Graduation date: 2012
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Wu, Chun-I., and 吳純宜. "Molecular characterization and antibody preparation of a Turnip mosaic virus isolate infecting calla lily." Thesis, 2004. http://ndltd.ncl.edu.tw/handle/83619781109952089792.
Full textAbstract:
碩士<br>國立臺灣大學<br>植物病理與微生物學研究所<br>92<br>A calla lily leaf sample infected by several viruses was collected from the field in 2002. When Chenopodium quinoa was inoculated by the leaf extract, it appeared chlorotic local lesions. After three successive single lesion isolation, a virus isolate was obtained. The yellow spot and stripe symptoms showed when the isolate was transferred back to tissue-cultured transplants of calla lily. The virus isolate reacted positively with the monoclonal antibody specific against potyvirus group by indirect enzyme-linked immunosorbent assay (ELISA). Flexuous virus particles about 750 nm in length were observed by the transmission electron microscopy. All of above evidences revealed that the isolate is a potyvirus but different from those calla lily-infected potyviruses studied in our laboratory, including Dasheen mosaic virus (DsMV), Zantedeschia mosaic virus (ZaMV) and Zantedeschia mild mosaic virus (ZaMMV). To further characterize the viral molecular nature, a pair of degenerate primers designed according to the conserved sequences of the reported potyviruses was used to amplify 1.7 kb fragment by RT-PCR. After cloning and sequencing, viral genomic sequences from partial NIb gene to 3’UTR were obtained and they are similar to Turnip mosaic virus (TuMV) by the database alignment. Therefore, this virus isolate was named as TuMV-ZAN. Using the same clonging strategy, we obtained the full length genomic sequences of TuMV-ZAN including 5’ and 3’ UTR, P1, HC-Pro, P3, 6K1, CI, 6K2, NIa, NIb and CP genes. Another TuMV isolate infecting calla lily was found and sequenced in 2004, but its nucleotide sequences of CP was only 89％ identity with those of TuMV-ZAN. This isolate was named TuMV-ZAN2. Phylogenetic analysis of the CP gene indicates that the ZAN and ZAN2 isolates are located at world-B and basal-BR group, respectively.
In order to obtain the antiserum of TuMV for further study and field survey, we cloned the CP gene of TuMV-ZAN into pET-29a(+) expression vector. After transformation to Escherichia coli BL21 (DE3) and induction by IPTG, the recombinant CP of TuMV-ZAN was expressed and purified to immunize rabbits. The titer and specificity of the prepared TuMV antiserum was tested by indirect-ELISA and western blot analysis. Besides, we used the same kind of expressed CP to immunize mouse and obtained a hybridoma cell line－Mab403 after screening. We found that the monoclonal antibody (Mab403) could reacte not only with TuMV but with at least eight other potyviruses.
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Tseng, Chun-Hao, and 曾君豪. "Construction of in vitro infectious transcripts of Turnip mosaic virus TW and C1 strains." Thesis, 2003. http://ndltd.ncl.edu.tw/handle/08375984139017493517.
Full textAbstract:
碩士<br>國立高雄師範大學<br>生物科學研究所<br>91<br>Turnip mosaic virus（TuMV）, a member of Potyvirus genus in Potyviridae family, is one of the most important viruses infecting cruciferous crops, and spreads worldwide including Africa, Asia, Australia, Europe, and North and South America. TuMVs are classified into four strains - C1, C2, C3, and C4 according to various symptoms developed on different Chinese cabbages varieties. A fifth strain, C5, and another special strain, TW, were reported in Taiwan. TuMV-TW and C1 were sequenced in our laboratory in 2001. In this study, modified Moloney Murine Leukemia Virus reverse transcriptase was used to synthesize the full-length first strand cDNAs of TuMV-TW and C1. The 5'' terminal primer was designed to include a T7 promoter sequence and partial viral 5'' NTR sequence. The 3'' terminal primer was designed to include a 14-T tail and a restriction enzyme cutting site. Both primers were used to synthesize full-length double-stranded viral cDNA by PCR. With the completion of the full-length dsDNA, TuMV-TW and C1 transcripts were obtained by in vitro transcription with T7 RNA polymerase. Purificated transcripts were inoculated onto mustard MT1 and confirmed by ELISA 14 and 24 days after inoculation. Mottlings were observed on mustard inoculated leaves with capped TuMV-TW transcripts at day 24. No symptom was observed for TuMV-TW transcripts without cap and TuMV-C1 transcripts with or without cap. ELISA test showed positive reaction on plant 14 and 24 days after inoculation with TuMV-TW transcripts with cap. A 10kb RT-PCR product was confirmed from plants infected with TuMV-TW transcripts with cap.
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Matsuda, Daiki. "Two new roles for the TYMV tRNA-like structure : translation enhancement and repression of minus strand synthesis." Thesis, 2004. http://hdl.handle.net/1957/30728.
Full textAbstract:
Some positive-strand RNA plant viruses possess a transfer RNA-like structure
(TLS) at the 3'-terminus of their genomic RNAs. The closest mimicry to tRNA is
exhibited by the valylatable TLSs from tymoviruses and furo-like viruses, which are able
to interact with key cellular tRNA enzymes: [CTP, ATP]:tRNA nucleotidyltransferase
(CCA NTase), valyl-tRNA synthetase (ValRS), and translation elongation factor 1A
(eEF1A). In this thesis, I report the discovery of two new roles of the Turnip yellow
mosaic tymovirus TLS, in translation enhancement (Chapter 2) and repression of minus
strand initiation (Chapter 4).
Placement of the 3'-terminal 109 nts of TYMV RNA in a luciferase reporter RNA
with a generic 5'-UTR enhanced translation by about 20-fold in cowpea protoplasts.
Exhibiting a synergistic relationship with the 5'-cap, the 3'-translation enhancement was
largely dependent on the aminoacylatability of the TLS and apparently on eEF1A
interaction. In the presence of the 5'-UTR from genomic TYMV RNA, translation of
both the overlapping proteins p69 and p206 was strongly dependent on a 5'-cap structure,
and was enhanced by the 3'-enhancer. These in vivo results contradict the proposed
model in which translation initiation of p206, but not p69, is cap-independent and TLS-dependent (Barends et al. Cell 112(2003):123-9).
In vitro experiments with a partially purified preparation of TYMV replicase have
investigated the phenomenon of minus strand repression. Interaction of purified
eEF1A���GTP specifically with the valylated TLS decreased the template activity for
minus strand to near-background levels. eEF1A���GTP acts by making the 3'-CCA minus
strand initiation site unavailable to the replicase. The influence of eEF1A in
simultaneously enhancing translation and repressing minus strand synthesis can be
considered a regulation that ensures robust translation early in the infection and that
offers a coordinated transition from translation to replication.
Previously shown to be critical for TYMV infectivity, a valylatable TLS was
investigated for its role in the replication and infectivity of the bipartite Peanut clump
pecluvirus. A valylatable TLS provided a small competitive advantage in protoplasts and
whole plants. The advantage was more apparent in protoplasts than in whole plants, and
more so in the replication protein-encoding RNA1 than in the trans-replicating RNA2.<br>Graduation date: 2004
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Wallace, S. Ellen. "Search for protein-protein interactions underlying the cis-preferential replication of turnip yellow mosaic virus." Thesis, 1997. http://hdl.handle.net/1957/34184.
Full textAbstract:
Coreplication experiments have revealed that replication of
turnip yellow mosaic virus (TYMV) RNA in turnip protoplasts is cis-preferential.
Genomes encoding mutant p141 or p66, proteins
essential for virus replication, were inefficiently rescued by a
helper genome. One model for the cis-preferential replication of
TYMV is that p66 and p141 form a complex that associates with the
RNA from which they are translated, limiting their availability in
trans. Three types of experiments were used in this study in an
attempt to obtain physical evidence for the hypothetical interaction
between p66 and p141. Immunoprecipitations from in vitro
translation reactions using antiserum that recognizes p66 (and its
progenitor, p206) coprecipitate p141, indicating that the proteins
form a complex in vitro. The results of coimmunoprecipitations of
translation products with in-frame deletions did not lead to
definitive information about interaction domains. p66 and the
helicase domain of p141 do not detectably interact in the yeast two-hybrid
system or in GST fusion interaction assays. Problems with
the expression of full length p141 fusions make conclusions about
the interaction of other p141 domains with p66 not possible at this
time. Since the helicase domain of p141 does not appear to interact
with p66, future experiments will focus on obtaining expression of
smaller domains of p141, outside the helicase domain, and
determining if they interact with p66. Variations to the model that
do not necessitate the direct interaction between p66 and p141 are
also considered.<br>Graduation date: 1997
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Peňázová, Eliška. "Studium rezistence perspektivních genotypů zelenin z čeledi Brassicaceae =:Study of the resistance of perspective vegetable genotypes from the Brassicaceae family /." Doctoral thesis, 2018. http://www.nusl.cz/ntk/nusl-426281.
Full textAbstract:
The topic of this thesis is focused on the testing of resistance of selected Brassica species to the black rot infection and viral mosaics caused by economically important pathogens of Brassicaceae family. The theoretical part describes characteristics of causal pathogens - Xanthomonas campestris pv. campestris (Xcc), Turnip mosaic virus (TuMV) and Turnip yellow mosaic virus (TYMV), and summarize the current state of a resistance study of these pathogens in the Brassicaceae family. The thesis also describes modern molecular methods used for the detection of bacterial and viral pathogens. In the experimental part, the detections of Xcc, TuMV and TYMV pathogens were optimized by PCR and RT-PCR. For bacterium Xcc, the Real-time PCR targeting a part of the zur gene sequence was designed using a TaqMan® probe. This detection system was subsequently processed in the form of a certified methodology for use in diagnostics. To increase the specificity, Real-time PCR targeting zur gene was involved in the Multiplex Real time PCR reaction. Then the dynamics of the Xcc infection was monitored in 6 hybrid cabbage cultivars. The testing of resistance to the black rot disease was optimized by the procedure including artificial inoculations using the suspension of the Xcc isolates HRIW 3811, 3971A and 1279A and the SU1 isolate originated from the Czech Republic. In a four-year experiment, the total of 42 homozygous breeding lines and 4 hybrid cultivars were tested, where 5 lines were recommended for breeding for resistance to the black rot disease. For the detection of TuMV and TYMV viruses, Real-time RT-PCR approaches based on the TaqMan® probe and SYBR Green dye were tested. The target region of both detections was the coat protein. The TuMV detection has been optimized for SYBR Green approach; for the TYMV detection, the use of the TaqMan® probe has been recommended. Detection systems were used to evaluate artificial inoculations of 6 cabbage cultivars by individual viruses. The tested plants did not show visual symptoms of infection therefore the presence of viruses was evaluated by Real-time RT PCR. The system designed for TYMV detected the presence of virus in all tested samples, TuMV was detected only in two samples. Negative detection results are probably in connection with the absence of TuMV symptoms which indicates unsuccesful plant inoculation. For both detection systems, it was recommended the verification on a wider range of viral isolates prior to standard use in diagnostics
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Tsai, Shih-Ming, and 蔡世閔. "Development of Turnip Yellow Mosaic Virus as Fluoresent Tracer to Improve the Sensitivity of Immunoassay." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/00698326229047906428.
Full textAbstract:
碩士<br>中興大學<br>生物科技學研究所<br>95<br>Natural polymers and virus capable of self-replicating and assembly draw many scientists into the development and application of these materials in nanotechnology. Many applications and material development are based on the manipulation of various peptides on the surface of virus particle. Turnip yellow mosaic virus (TYMV) is an icosahedral virus, 28 nm in diameter. It comprises a positive-stranded RNA genome and 180 copies of capsid protein. In a previously study, a mutant virus (TYMV/Cys) which displays one cysteine residue at the C terminus of viral coat protein was constructed showed a similar infectivity to that of wild type TYMV. TYMV/Cys provides 110 and 400 of thiol and amine groups, respectively, on the surface which could conjugate with different kinds of fluorescent markers. Since the C terminus of coat protein on the virus surface is predicted to be in a cleft, the location may be sterically hindered for further applications. A new construct, TYMV/A6C, containing 6 alanine residues with a C-terminal cysteine is predicted to extend the thiol group from the cleft. Characterization of TYMV/A6C indicates that it could provide about 90 and 400 of thiol and amine groups, respectively, on the viral surface. TYMV/A6C was further used to be conjugated with 1.4 nm-diameter nano-gold particles through the thiol groups and the labeling was observed under transmission electron microscope. Purified antibody and fluorescein were conjugated to the thiol and amine groups, respectively, on TYMV/A6C and subsequently purified with an ion-exchange column. This fluorescently labeled viral particle conjugated with antigen specific antibody was tested in a fluorescent immumosorbent assay and compared to the conventialthe sensitivity of conventional ELISA (enzyme-link immunosorbent assay) system.
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Chiu, Ming-Tzu, and 邱敏慈. "The stability of P1 of Turnip mosaic virus impacts the HC-Pro suppression of miRNA pathway." Thesis, 2012. http://ndltd.ncl.edu.tw/handle/41524347460714478945.
Full textAbstract:
碩士<br>國立臺灣大學<br>植物病理與微生物學研究所<br>100<br>The P1/HC-Pro of potyvirus is the first discovered viral suppressor and HC-Pro needs to work with P1 for enhancing the suppression effect against post transcriptional gene silencing (PTGS) and inhibiting the microRNA (miRNA) pathway; however, the mechanism of P1/HC-Pro remains unclear. Furthermore, P1 is the most divergent protein in length and amino acid sequence among potyviruses. For studying the function of P1/HC-Pro, the antisera specific to P1, HC-N (1-267 a.a. of HC-Pro), HC-C (249-458 a.a. of HC-Pro) or CP were developed. In order to improve the quality of antiserum, fast protein liquid chromatography (FPLC) was used to purify immunoglobulin G (IgG) and to concentrate IgGs by protein centricon for enhancing the sensitivity. In this study, we demonstrated that the rapid turnover rate of P1 in vivo and the stability of P1 is crucial for miRNA pathway. Moreover, TuMV-infected plants and P1/HC-Pro transgenic Arabidopsis were treated with MG132 (26S proteasome inhibitor) to demonstrate that P1 is under an ubiquitin-independent degradation process. Interestingly, the P1 protein became stabilized and detectable while its N-terminus is fused with YFP (yellow fluorescent protein) or Flag tags and it caused a mild phenotype with serrated leaves in transgenic plants. Surprisingly, the P1dN600/HC-Pro transgenic Arabidopsis, which retained the conserved regions at the C-terminus of P1, became detectable and showed mild leaf serration in Arabidopsis. It suggests that the N-terminus of P1 regarding its stability is crucial for inhibiting miRNA pathway. We conclude that the stability of P1 affects the efficiency of miRNA suppression, and the N-terminus of P1 regulates the turnover rate. Study on the instability of P1will help clarify the role of P1/HC-Pro in miRNA pathway.
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Wu, Meng-Fang, and 吳孟芳. "Analysis of the Sequence Diversity of the Viral Genomic Regions of Different Turnip Mosaic Virus Strains." Thesis, 1999. http://ndltd.ncl.edu.tw/handle/49642581514872413710.
Full textAbstract:
碩士<br>國立高雄師範大學<br>生物科學研究所<br>87<br>TuMV is a member of the potyvirus group, with a flexuous, rod-shaped particle of 720 nm in length. It is transmitted by mechanical inoculation and several species of aphids in a non-persistent manner but not through seeds. TuMV infects many economically important crops, and is one of the most important viruses in the world affecting Brassica species including Chinse cabbage, radish and smooth-leaf mustard, it includes mottling, mosaic, leaf distortion and stunting in infected plants. Provvidenti discribed four strains, C1、C2、C3、C4 of TuMV based on Chinese cabbage differerntial varieties. Green and Deng reported a fifth strain, C5, in Taiwan. The TuMV-TW strain was isolated from a mustard field in Taiwan. Virons of six TuMV strains were purified from infected leaves of mustard by polyethylene glycol precipitation followed by a Cs2SO4 isopycnic centrifugation. Electrophoresis revealed presence of a single RNA band about 10 Kb for each of six strains of TuMV. Relative molecular weight of coat proteins from six TuMV strains were about as 37 KDa. They reacted with an antiserum prepared to TuMV-TW in Western blot assay. The cDNAs of coat protein (CP) genes of the six TuMV strains were prepared by using reverse transcription polymerase chain reaction (RT-PCR). Analysis of nucleotide sequences and amino sequences of CP genes and 3'' non-coding regions were showed that the CP genes of TuMV-C2 and C3, and those of TuMV-C5 and TW had close relationships. The CP genes and 3'' non-coding regions of the six TuMV strains were also compared with other reported TuMV strains of CPP (from China), CAPP (from Canada), CQS、 NIAP (from Korea), CP、CPRN、 MUT (from UK) and JAP (from Japan). Results showed that the CP genes of TuMV-C5 and TW had close relationships with NIAP strain (from Korea). The six TuMV strains were serologically indistinguishable. The cDNAs of coat protein (CP) upstream genes including nuclear inclusion b protein ( NIb ), nuclear inclusion a protein ( NIa ), genome-linked viral protein ( VPg ), 6K2 protein ( p6K2 ) and cytoplasmic inclusion protein ( CI ) of TuMV strains, C1 and TW, were also prepared by RT-PCR. Nucleotide sequences and amino sequences of NIb genes of TuMV strains, C1 and TW were compared with other reported TuMV strains of CAPP (from Canada), NIAP (from Korea) and JAP (from Japan). Results showed that the NIb gene of TuMV-TW had a close relationship with that of JAP strain (from Japan). Comparsion of NIa genes of TuMV strains, C1 and TW were compared with other reported TuMV strains of CAPP (from Canada), TMU18654 (from Korea) and JAP (from Japan). Results indicated that the NIa gene of TuMV-TW had a close relationship with that of TMU18654 strain (from Korea). Comparsion of VPg genes showed that TuMV-TW had a close relationships with JAP strain (from Japan), whereas the p6K2 gene and CI gene of TuMV-C1 strain had close relationships with that of JAP strain (from Japan).
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MRÁZKOVÁ, Ivana. "Virocidní účinnost ribavirinu a acyklických nukleosid fosfonátů na virus žluté mozaiky vodnice." Master's thesis, 2010. http://www.nusl.cz/ntk/nusl-53042.
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A new method was developed for testing antiviral compounds against plant viruses based on rapidly growing brassicas in vitro on liquid medium. While using ribavirin as a standard for comparison, phytotoxicity and ability of the acyclic nucleotide analogues(R)-PMPA, PMEA, PMEDAP, and (S)-HPMPC to eliminate ssRNA Turnip yellow mosaic virus (TYMV) were evaluated by this method. Double antibody sandwich ELISA was used for relative quantification of viral protein in plants. Ribavirin had the most powerful antiviral effect against TYMV. On the other hand, (R)-PMPA and PMEA had no antiviral effect and almost no phytotoxicity compared to the control. (S)-HPMPC and PMEDAP showed moderate antiviral effect, accompanied by higher phytotoxicity.
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Huang, Shi-Hua, and 黃詩華. "Studies of pathogenic determinant site on the genome of Turnip mosaic virus C1 strain to mustard cultivar 5780." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/21470557096439855832.
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碩士<br>國立高雄師範大學<br>生物科學研究所<br>93<br>Turnip mosaic virus (TuMV) is a member of Potyvirus genus in the family of Potyviridae and worldwide spread including Africa, Asia, Australia, Europe, and the north and south America. It causes huge economic loss in yields which is next to Cucumber mosaic virus. According to the reports of Provvidenti, Green and Wang, six strains, TuMV-C1, C2, C3, C4, C5 and TW, were identified in Taiwan. Potyvirus is a positive RNA virus with a length of 10 kb containing a single open reading frame which encoding a polyprotein precursor. In our laboratory full length and near full length of nucleic acid of TuMV-C1 (accession no. AF394601) and TW (accession no. AF394602) had been sequenced and analyzed. A mustard cultivar 5780, which is cytoplasmic resistant to strains of TuMV-C2, C3, C4 and TW, was screened. However mustard 5780 is susceptible to TuMV-C1. The purpose of this study is to explore the resistant mechanism of mustard cultivar 5780 to TuMV-TW strain. In order to construct TuMV hybrid transcripts four kinds of long-length dsDNA were amplified by RT-PCR with primers designed for TuMV-TW and C1, and then spliced by BstPI and ligased by T4 DNA ligase. Mustard 5780 and MT1 were inoculated with TuMV C1-TW and TuMV TW-C1 hybrid transcripts, respectively, and then confirmed the infection by ELISA. The results showed that cultivar MT1 could be infected by TuMV C1-TW and TuMV TW-C1 hybrid transcripts. However, cultivar 5780 only could be infected by TuMV C1-TW hybrid. The results revealed that the key region of TuMV-C1 viral RNA for its infectivity to mustard 5780 is located in the 6.6 kb region from the 5' end.
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Tze-Huang, Shen. "Expression of a single-chain variable fragment against the NSs protein of Watermelon silver mottle virus in tobacco plants by transgenic approach and Turnip mosaic virus vector." 2006. http://www.cetd.com.tw/ec/thesisdetail.aspx?etdun=U0005-2808200616093300.
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Shen, Tze-Huang, and 沈澤煌. "Expression of a single-chain variable fragment against the NSs protein of Watermelon silver mottle virus in tobacco plants by transgenic approach and Turnip mosaic virus vector." Thesis, 2006. http://ndltd.ncl.edu.tw/handle/41736856730448129110.
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碩士<br>中興大學<br>植物病理學系所<br>94<br>Antibodies or antibody fragments expressed by transgenes or viral vectors in plants can directly target crucial antigens and accumulate in the right cell compartments, they are able to effectively trigger pathogen resistance and to modulate plant metabolism. Watermelon silver mottle virus (WSMoV) is a member of the genus Tospovirus in the Family Bunyaviridae, causing severe damages for the production of watermelon, wax gourd, melon and other cucurbits in Taiwan. The NSs protein of tospoviruses is a gene-silencing suppressor and is a pathogencity determinant for the severity of symptoms. In our previous report, the monoclonal antibodies (MAbs) against the WSMoV NSs protein were produced and the recognition site of the MAbs was identified at the amino acid 98-120 of the N-terminal region of the WSMoV NSs protein. The MAbs-recognized region is also conserved among the NSs proteins of the WSMoV-serogroup tospoviruses, including WSMoV, Peanut bud necrosis virus (PBNV), Capsicum chlorosis virus (CaCV) and Calla lily chlorotic spot virus (CCSV), and it is considered to play an important role in functions of NSs protein. In this investigation, we cloned a single-chain variable fragment (scFv reading frame) of the MAb, produced from a hybridoma cell line 239F1B9, against the common epitope of WSMoV NSs protein. Subsequently, the scFv reading frame was constructed in the severe and mild strains of Turnip mosaic virus (TuMV) vectors and binary vector pBI121 of Agrobacterium for expression of scFv in Nicotiana benthamiana plants. Expression of the free-form scFv protein in the N. benthamiana and Chenopodium quinoa plants infected with the severe TuMV recombinant TYC5-WNSssf or the mild TuMV recombinant THCIG5-WNSssf was detected by western blotting using the monoclonal antibody against the hexa histidine tag. Moreover, the transgenic N. benthamiana plants carrying the scFv transgene were also generated and the presence of the scFv reading frame was confirmed by polymerase chain reaction (PCR). The WNSs scFv transgenic tobacco plants were challenged with WSMoV under greenhouse conditions and symptom development was significantly delayed for 7 to 20 days.
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Chin-Chih, Chen. "Development of Turnip mosaic virus as a Plant Viral Vector for Expressing Foreign Proteins and Generation of Attenuated Strains for Cross Protection." 2006. http://www.cetd.com.tw/ec/thesisdetail.aspx?etdun=U0005-1708200613393500.
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Chen, Chin-Chih, and 陳金枝. "Development of Turnip mosaic virus as a Plant Viral Vector for Expressing Foreign Proteins and Generation of Attenuated Strains for Cross Protection." Thesis, 2006. http://ndltd.ncl.edu.tw/handle/83842984055341980680.
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博士<br>國立中興大學<br>植物病理學系所<br>94<br>Turnip mosaic virus (TuMV), a member of the genus Potyvirus, is the most important virus infecting field-grown crucifers world-wide, and with a wide host range including 318 plants species in 156 genera of 43 families. In this study, the molecular characterizations of two TuMV isolates, YC5 and RC4, isolated from calla lily (Zan- tedeschia spp., Araceae), and compared their genetic variablitiy to those TuMV strains isolates available in GenBank based on the full-length RNA genomic sequences and coat protein coding regions were analyzed. The infectious cDNA clone of TuMV-YC5 driven by the 35S promoter was constructed, and modified as a viral vector to express the open reading frame (ORF) of GFP or a dust mites allergen Der p 5 (from Dermatophagoides pteronyssinus Trouessart). Five different insertion sites in the TuMV- YC5 vector were created. The effectiveness and stability of the expressed heterologous proteins in plants by TuMV-YC5 vector at these different insertion sites were compared. The ORFs of GFP and Der p 5 were successfully expressed by TuMV-YC5 vector in various host plants. Finally, the attenuated mild strains with the ability to protect plants of Nicotiana benthamiana Domin and Brassica campestris L. var. chinese from the infection of severe-strain TuMV-YC5 were constructed.
The Chapter 1 reviews all relevant references for this study. This chapter is written in Chinese. The other chapters are written in English for publication in journal.
In the Chapter 2, the complete nucleotide sequences of TuMV-RC4 and TuMV-YC5 isolates were determined from overlapped viral cDNA clones derived from reverse-transcription polymerase chain reaction (RT-PCR) using the TuMV specific primers. Both TuMV-RC4 and TuMV-YC5 showed similar genomic features. When compared with TuMV isolates available in GenBank,sequences coding for P1, P3 and 6k2 proteins were identified as the most varible regions. Based on the variablilty of amino acid sequences at the amino acids positions of 471-490 in CIP region, TuMV isolates were divided into two groups. Among 42 TuMV isolates compared, only Q-Ca (Acc. No. D10927) and C1-isolate (Acc. No. AF394601) have a sequence of GSQPVxM- xDxVxMxKIGVTL, while RC4, YC5, and other 38 isolates with a sequence of WLTASEYARLGANVEDRRDV. The protease recognition tetrapeptides for P1 protease and nuclear inclusion a protease (NIa-Pro) are variable among TuMV isolates, but both of RC4 and YC5 are identical. The phylogenetic analyses based on the comparison of the deduced amino acid sequences of the complete polyproteins with those of other 30 TuMV isolates, and the coat protein regions with those of other 59 TuMV isolates revealed that RC4 and YC5 are both classified in the group that contains isolates belonging to the Brassica pathotype.
In Chapter 3, a dust mite allergen Der p 5 expressed in edible crucifers by TuMV- YC5 vector was investigated in this study. Our previous study indicated that oral feeding of mice with dust mite allergen Der p 5 produced by Zucchini yellow mosaic virus (ZYMV) vector in squash provides a novel approach for immunotherapy of allergic asthma. However, the non-edibility of squash leaves sparked a biosafety concern. In this investigation, we expressed the dust mite allergen by TuMV vector in crucifers that are consumed as fresh edible vegetables to minimize the biosafety concerns for humans. An infectious cDNA clone driven by a 35S promoter was successfully constructed by ligating overlapping cDNA clones generated from RT-PCR amplified fragments of TuMV-YC5. The ORFs of GFP or Der p 5 allergen were in-frame inserted at the N-terminal region of the HC-Pro of TuMV-YC5, respectively, to generate a free-form or fused-form proteins, and expressed in Chenopodium quinoa Willd., N. benthamiana, or Brassica spp. Expression of free-form Der p 5 by TuMV-YC5 vector in crucifers (such as B. campestris L. var. chinese and B. campestris L. var. ching-geeng) is 1.7- to 4.6-folds higher than those by ZYMV vector in squash. Since oral feeding of high dose allergen is essential for effective immunotherapy, we consider that the expression of the dust mite allergen by TuMV vector in cruciferous crops that are consumed as fresh edible vegetables is a more feasible approach than the ZYMV-squash system.
In Chapter 4, the effectiveness and stability of heterologous proteins expressed in plants by TuMV-YC5 vector at five different insertion sites was analyzed. The N-terminal regions (NT) of HC-Pro and CP reading frames are generally used for the in-frame insertion of large heterologous ORFs in potyviral vectors for protein expression in plants. The infectious cDNA clone of TuMV-YC5, driven by a 35S promoter, was modified to carry foreign ORFs at the possible sites to investigate the expression efficiency. A sequence cassette including directional cloning sites and the coding sequence for the NIa protease recognition site was separately constructed in the NT regions of HC-Pro, P3, CIP, NIb and CP proteins in TuMV-YC5, by a PCR-based site-directed mutagenesis or PCR-modification. The ORFs of GFP and Der p 5 allergen were individually in-frame inserted at these sites in TuMV-YC5 vector to generate free-form proteins. The results revealed that the five insertion sites are effective for the expression of heterologous ORFs of GFP and Der p 5, but the CIP-NT site is not feasible for the latter. The significant higher yields of GFP expressed by CP-NT are 1.2-3.6 folds higher than that expressed by HC-Pro-NT in plants of C. quinoa, N. benthamiana, and several species of Brassica crops. Lower yields of GFP expressed by P3-NT and CIP-NT, 0.2-0.9 and 0.3-0.8 folds, respectively, were compared to that expressed by HC-Pro-NT in most of the tested hosts. Yields of Der p 5 expressed by CP-NT and HC-Pro-NT were similar and significantly higher than those expressed by other sites, however, lower yields of Der p 5 were expressed when its ORF was inserted at P3-NT (0.2-0.6 folds to HC-Pro-NT) or NIb-NT (0.6-0.9 folds to HC-Pro-NT). Moreover, Der p 5 ORF inserted at NIb-NT did not produce detectable protein in plants of Brassica spp. Symptoms on most of the tested hosts infected by the TuMV-GFP or TuMV-Derp5 recombinant, expressing free-form GFP or Der p 5 by the HC-Pro-NT, P3-NT, and CIP-NT, were delayed or milder than those caused by other recombinants. Our results revealed that the engineered TuMV-YC5 is an efficient vector for the expression of foreign proteins by inserting their ORFs at the five different NT sites. However, the yield and the stability of the foreign ORFs, and the symptomology of TuMV recombinant-infected hosts are affected by the insertion sites, host plants, and the foreign ORFs themselves.
In Chapter 5, HC-Pro protein has pronounced effects on replication and symptom expression of potyviruses. In this study, a PCR-modified mutant derived from p35SYC5 was recognized as a mild strain, for it only induced mild symptoms on the hosts of Chenopodium quinoa Willd., Nicotiana benthamiana Domin, and Brassica spp. The recombination analyses between the mild mutant and the parental strain indicated that the changes in HC-Pro region were critical for the attenuation. Further analyses by different constructs with amino acid changes in the N-terminal region of HC-Pro indicated that the amino acids (I7) and (G171), and the additional NIa protease recognition site (CVYHQA) added between P1 and HC-Pro regions of TuMV-YC5 were critical for the attenuation. When only (F7) was changed to (I7), the recombinant pHCIE9 still induced severe symptoms on tested host plants. However, when this change was combined with the added NIa cleavage site to generate the infectious construct pHCNIE16, the recombinant became a mild strain. Another construct pHCFG47 with the only change of E171 to G171 also induced mild symptoms. If this construct contained the NIa protease site in addition, the recombinant pHCNFG8 showed even milder symptoms on plants of N. benthamiana. Based on this, the construct pHCIG5 containing double point mutations of F7 to I7 and E171 to G171 in the HC-Pro region, without the NIa cleavage site, was generated and it induced symptoms on plants of N. benthamiana milder than those induced by the construct with only mutation of E171 to G171. In cross-protection assay, the infectious TuMV-YC5 clone carrying a reporter GFP gene was used as severe-strain challenger. By western blotting and RT-PCR at 14 days after the challenge inoculation, GFP was not detected from the plants of N. benthamiana protected with the mild strain TuMV-HCIG5 derived from pHCIG5 and subsequently challenged by severe strain at 10 days after the protective inoculation. Similar results were shown in the plants of Brassica sp. protected with the mild strain TuMV-HCIG5. Moreover, our results demonstrate that various mild mutants provide effective cross-protection on plants of N. benthamiana against the infection of the severe-strain TuMV-YC5.
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KASALOVÁ, Tereza. "Kvantifikace progrese virové infekce virů RaMV a TuRSV pomocí real-time PCR." Master's thesis, 2008. http://www.nusl.cz/ntk/nusl-45378.
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The relative amount of viral RNA of two comoviruses (RaMV and TuRSV) in different parts of plant during infection was determined. The two viruses were compared according to their ability to spread and multiply in plants.
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Liu, Han-Lin, and 劉漢麟. "Study of a broad-spectrum monoclonal antibody, single-chain variable fragments and epitope of potyviruses, and the pathogenicity determinants of Turnip mosaic virus infectious clone." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/64384162768281805451.
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博士<br>國立臺灣大學<br>植物病理與微生物學研究所<br>104<br>The genus Potyvirus is one of the important plant virus genera, and comprises 158 formal species based on ICTV Virus Taxonomy. Potyviruses can be transmitted by aphids and mechanical inoculation. Some potyviruses have broad host range and cause serious economic losses. In Taiwan, calla lily is reported to be infected by five potyviruses, Calla lily latent virus (CLLV), Dasheen mosaic virus (DsMV), Konjak mosaic virus (KoMV), Turnip mosaic virus (TuMV), and Zantedeschia mild mosaic virus (ZaMMV). To reduce the cost and time of virus indexing, the conserved 121-amino-acids core regions of the capsid protein (CP) of DsMV, KoMV, and ZaMMV were concatenated and expressed. The recombinant protein was used as an antigen to prepare and screen the potyvirus group-specific monoclonal antibody (MAb). The selected C4 MAb could detect nine potyviruses in addition to the five calla lily potyviruses. In the first part of this study, we cloned the variable regions of the heavy (VH) and light (VL) chains of the C4 MAb, and then constructed them as C4 single-chain variable fragments (scFvs). In E. coil expression system, a new PelE secretory signal peptide was used to help the secretion of C4 scFv. The data showed not only long but also short PelE signal peptide could secrete C4 scFv to medium and reduce inclusion body formation. According to western blot and I-ELISA, the soluble C4 scFv showed a binding specificity similar to that of the C4 MAb. In the second part, to identify which epitope is recognized by C4 MAb, the phage display peptide library was used to screen the C4 MAb-reactive peptides. The sequence alignment of C4 MAb-reactive peptides with potyviral CP sequences indicated that a conserved 12-amino acid (WxMMDGxxQxxY/F) sequence may be recognized by C4 MAb, and thus it was named as the C4 epitope. The results of amino acid substitution analysis indicated that tryptophan and tyrosine residues of C4 epitope are crucial for reacting with C4 MAb. Furthermore, sequence alignment of Hippeastrum mosaic virus (HiMV), which could not be detected by C4 MAb, and amino acid substitution analysis also showed the aspartic acid is also involved in binding with C4 MAb. These results of epitope mapping demonstrated the C4 epitope is a common CP epitope of potyviruses. We also tried to develop the C4 epitope as a new epitope tag. The epitope sequences of ZaMMV, KoMV, and DsMV were separately fused to the C-terminus of CP of Odontoglossum ringspot virus (ORSV), and then epitope-tagged ORSV CPs were expressed in a bacterial system and purified. The results of Western blotting and ELISA showed the C4 epitope of KoMV (Ko) had the strongest binding affinity to C4 MAb. To examine the applicability of Ko tag in planta, the transiently expressed Ko-tagged GFP and ORSV CP could be successfully detected by C4 MAb, and the Ko-tagged P19 of Tomato bushy stunt virus (TBSV) still maintained its silencing suppressor function. Furthermore, Ko-tagged EGFP could be successfully detected and subsequently immunoprecipitated by C4 MAb in a mammalian cell system. These data proved that Ko tag has the potential to become a new epitope tag in bacterial, plant, mammalian cell systems. In the third part, TuMV was used as an experimental material to develop a reverse genetic system. We obtained four full-length clone of TuMV by a two-step cloning method. In the infectivity assay, the p35S-TuMV-27 clone which had similar infectivity to p35S-TuMV-1 revealed much better infectivity than p35S-TuMV-5 and p35S-TuMV-6. The sequence comparison of TuMV-5, TuMV-6 and TuMV-27 clones indicated that they have only 1~3 nucleotide difference at the extreme 5'' end of viral genome. After 5’ replacement and 5’ G deletion analyses, these data verified that the lacking of the adenine at position 7 of 5’ UTR could reduce the infectivity. Besides, the infectious TuMV-27 clone was used to analyze which amino acid residues responsible for the lack of infectivity of pTuMV-T100 clone. Based on unique restriction enzyme sites, three fragments (AB, BH, and HX) of TuMV-T100 were used to separately replace the corresponding fragments of TuMV-27. The result of infectivity assay indicated all of these fragments contain the amino acid mutations affected infectivity. TuMV-AB and TuMV-BH could replicate in protoplasts, but could not infect N. benthamiana and C. quinoa plants. These results indicated that they may be defective in cell-to-cell movement. In contrast, TuMV-HX could induce small local lesions on C. quinoa, but its CP accumulation was lower than that of TuMV-27. The point mutation assay confirmed that CP mutation of Y219N decreased the infectivity of TuMV-27. In addition, other tyrosine mutants (TuMV-Y191A, TuMV-Y219A, and TuMV-Y224A) had similar phenotype as TuMV-Y219N. In transient expression assay, the tyrosine-mutated CP was less stable than wild-type CP. Thus, we suggested that the stability of TuMV CP may be affected by tyrosine phosphorylation.