Dissertations / Theses on the topic 'Nicotiana benthamiana Viruses'
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1
Chewachong, Godwill Mih. "Engineering Plant Virus " Vaccines" Using Pepino mosaic virus as a Model." The Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1384203201.
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Valenzuela, Aguila Sofia. "Transformation of Nicotiana benthamiana with different BWYV (Beet western yellows virus) sequences to test for virus resistance Transformation von Nicotiana benthamiana mit verschiedenen Sequenzen des BWYV (Beet western yellows virus) zur Virus-Resistenztestung /." [S.l. : s.n.], 2000. http://deposit.ddb.de/cgi-bin/dokserv?idn=959528695.
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Varrelmann, Mark. "Begrenzung von heterologer Enkapsidierung und Rekombination bei pathogen-vermittelter Resistenz gegen das Plum pox virus der Pflaume (PPV)." [S.l. : s.n.], 1999. http://deposit.ddb.de/cgi-bin/dokserv?idn=958530033.
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Lin, Junyan. "NONHOST RESISTANCE TO BEAN POD MOTTLE VIRUS IN NICOTIANA BENTHAMIANA." The Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1372723537.
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Fulton, Andrew Dale. "Monoclonal Antibody Expression and Novel Purification in Nicotiana benthamiana." Thesis, Virginia Tech, 2011. http://hdl.handle.net/10919/43361.
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Over the past few decades researchers and industrial professionals alike have realized the vast potential of monoclonal antibodies to treat diseases ranging from arthritis, immune and infectious diseases to cancer. There are a number of antibodies on the market that constitute a large portion of the biopharmaceutical niche in the drug industry. Blockbuster drugs (selling greater than $1 billion/year), include antibodies such as Avastin (bevacizumab), Herceptin (trastuzumab), Rituxan (rituximab), Humira (adalimumab) and Remicade (infliximab), which are cornerstones in this type of sector. With the cost of development to market approval rising astronomically for a new drug, new ways to produce and process these molecules becomes a paramount objective to ultimately help both patients and drug developers.
Plants, such as Nicotiana benthamiana, offer a unique production platform due to their recently found ability to produce large amounts of therapeutic proteins in a quick manner. While production would be simple and cheap, purification would not be due to the presence of toxic compounds in ground plant tissue. The current methods to purify these molecules from plant extract include expensive affinity column steps (Protein A/G) that are difficult to scale-up to bed volumes that would be necessary for this technology.
In the following paper, a method to purify a monoclonal antibody by non-Protein A/G resins is accomplished and compared to purification by Protein A. The modified process involved an UF/DF step, a precipitation of native impurities step using a charged polymer, hydrophobic interaction chromatography and hydrophobic charge induction chromatography. The yield of this modified process was 19.0%. This process compared favorably with Protein A due to the fact that even with washing steps including NaCl and Tween-20, the Protein A elution fraction still contained a large portion of host cell impurities. A chromatography step would need to be included before Protein A to both protect the column resin and provide a more purified immunoglobulin.Master of Science
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Dieterich, Guido. "Molekularbiologische Untersuchungen zur subzellulären Lokalisierung des putativen Transportproteins - P19,5k - des beet western yellows virus (BWYV) und Erarbeitung der Grundlagen für eine gentechnisch zu erzeugende Resistenz gegen das BWYV." [S.l. : s.n.], 2000. http://deposit.ddb.de/cgi-bin/dokserv?idn=960233989.
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Torres, Arzayus Maria Isabel. "Engineering yam mosaic virus resistance in Nicotiana benthamiana using genetic transformation techniques." Thesis, Imperial College London, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.264199.
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Mbewana, Sandiswa. "Development of Rift Valley fever virus candidate vaccines and reagents produced in Nicotiana benthamiana." Doctoral thesis, University of Cape Town, 2017. http://hdl.handle.net/11427/25446.
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Rift Valley fever (RVF) is a haemorrhagic fever agent caused by an infection with an enveloped negative-stranded RNA Rift Valley fever virus (RVFV). It belongs to the genus Phlebovirus in the family Bunyaviridae. The virus is spread by infected mosquitoes and affects ruminants and humans, causing high numbers of neonatal fatalities in animals and occasional fatalities in humans. It is endemic to parts of Africa and the Arabian Peninsula, but is described as an emerging virus due to the wide range of mosquitoes that could spread the disease into non-endemic areas, posing serious health and agricultural problems. The disease can be prevented by vaccination, but there is currently no Food and Drug Administration-approved RVFV vaccine that can be used outside endemic areas, while there are two live attenuated vaccines available for use in endemic areas. These vaccines have the potential for reversion, and are therefore not recommended for use in countries where RVFV is not endemic. This indicates the need for more RVFV vaccine research and development. This work focused on the development of a RVFV vaccine candidate that would allow for differentiation between infected and vaccinated animals as well as humans.
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Wu, Cheng Ying. "Characterization of innate immune response to «Nicotiana benthamiana»-derived Influenza H5 virus-like particles." Thesis, McGill University, 2013. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=119400.
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Current influenza vaccine manufacturing processes using chicken-embryonated egg technology is a time-consuming and laborious process, and is currently the major drawback in counteracting pandemic influenza strain. One solution to that problem is the use of plants to generate vaccine antigen. Virus-like particles (VLP), produced from the tobacco plant Nicotiana benthamiana, represent a cost-effective, alternative platform for influenza vaccine production. Previous studies have shown that the immunization with VLP expressing the hemagglutinin (HA) protein from influenza virus H5N1 (H5-VLP) produced in N. benthamianainduce protective immunity against challenge of cross-clade virus in mice and ferrets. In this study, we used human peripheral blood mononuclear cells (PBMC) to characterize the innate immune response to plant-derived influenza H5-VLP ex vivo. We successfully demonstrate the mitogenic property of H5-VLP on PBMC ex vivo. Furthermore, we detect up-regulation of activation marker in B cells and NK cells, and some T cells. Cytokine profile of the supernatant from VLP-stimulated sample suggests that inflammatory response dominates the innate immunity within first 48 hours and is produced by CD14+ monocytes. Our study demonstrates that tobacco plant-derived influenza VLP are capable of generating innate immune responses in naïve human PBMC, helping us to better understand the immunostimulatory nature of this potential vaccine candidate.A l'heure actuelle, la plupart des vaccins contre les infections par le virus influenza sont produits à partir d'œufs de poule fécondés. Ce procédé long et fastidieux constitue l'un des principaux obstacles à la production rapide d'un vaccin lors d'une pandémie. Une solution à ce problème consiste en l'utilisation de plantes afin de générer les antigènes nécessaires à l'élaboration du vaccin. Les pseudovirus ou Virus-like particles (VLP) produites à partir de la plante de tabac Nicotiana benthamiana représentent une alternative moins couteuse et plus rapide pour la production de vaccins antigrippaux. Des études préalables ont démontré qu'une immunisation avec les VLP exprimant l'hémagglutinine (HA) du virus influenza H5N1 (H5-VLP) induisaient une immunité protective lors d'une infection par ce virus chez la souris et le furet. Dans notre étude, nous avons utilisé les cellules mononuclées du sang périphérique humain (PBMC) afin de préciser la réponse immunitaire innée suite à l'exposition ex vivo aux H5-VLP produites dans N. benthamiana. Nous avons démontré les propriétés mitogéniques des H5-VLP sur les PBMC ainsi qu'une activation des lymphocytes B, des cellules NK et de certaines sous populations de lymphocytes T. L'analyse des cytokines sécrétées dans le surnageant des PBMC exposés ex vivo aux VLP suggère qu'une réponse pro-inflammatoire prédomine 48h après exposition et semble résulter essentiellement d'une activation des monocytes CD14+. Notre étude démontre que les VLP produites à partir de la plante de tabac génèrent une réponse immunitaire innée dans les PBMC provenant de patients naïfs, nous permettant ainsi de mieux comprendre les propriétés immunostimulantes de ce nouveau type de vaccin.
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De, Figueiredo Pinto Gomes Pera Francisco. "Design and production of a candidate universal influenza A vaccine in Nicotiana benthamiana plants." Master's thesis, University of Cape Town, 2017. http://hdl.handle.net/11427/27063.
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The influenza A virus is responsible for 250,000 to 500,000 deaths every year worldwide and millions more could die in the event of a serious pandemic. Vaccines against influenza have existed for long, but until today they have been limited by extensive production times and reduced cross-protection between different strains of the virus. This leads to a recurrent need to update the vaccine composition every year, which is both costly and inadequate to fight pandemics. An innovative approach that could improve the vaccine efficacy has been recently developed based on the selection of conserved influenza epitopes with potential to induce broader immune responses. The 23-amino acid extracellular domain of the M2 protein (M2e) is highly conserved among different influenza A strains and thus it seems like an ideal candidate for a universal influenza vaccine. However, due to its small size, it is a poor immunogen when used on its own. The aim of this project was to produce M2e-presenting virus-like particles (VLPs) in Nicotiana benthamiana plants via Agrobacterium-mediated transient expression. Plants are increasingly being examined as alternative recombinant protein expression systems due to their safety, scalability and rapid production times. Moreover, numerous studies suggest the use of recombinant virus-like particles (VLPs) to increase the immunogenicity of antigens. Therefore, to obtain VLPs presenting the M2e epitope, I genetically engineered several different M2e-HA fusion proteins by replacing the hemagglutinin (HA) globular head and main epitope with five tandem repeats of M2e epitope sequences (5xM2e) from human, swine, and avian origin influenza A viruses. To increase the chances of obtaining VLPs, M2e-HA fusions either contained the HA stalk domain (5xM2e-HAstalk) or simply the transmembrane region (5xM2e-HAtrans). Furthermore, the tetramerizing leucine zipper derived from the General Control Protein (GCN4) was also included in some of the constructs to promote particle formation. In total, six different M2e-HA fusions were created: 5xM2e-GCN4-HAstalk, 5xM2e-GCN4-HAtrans, 5xM2e-HAstalk, 5xM2e-HAtrans, 1xM2e-HAstalk and 1xM2e-HAtrans. The expression of these proteins was optimized in plants by testing different conditions and using three different expression vectors. Overall, I was able to show expression after only 3 days post-infiltration for most of the M2e-HA v fusion proteins utilizing the pEAQ-HT and pRIC 3.0 expression vectors whereas expression levels with pTRAc were low or non-detectable. Once the expression of the M2e-HA fusions was optimized, the two proteins with the highest potential to form VLPs were selected for further characterization (5xM2e-HAstalk and 5xM2eHAtrans). Using transmission electron microscopy to analyse purified proteins, both 5xM2eHAstalk and 5xM2e-HAtrans were shown to assemble into VLPs resembling the shape and size of native HA VLPs. These VLPs could also be observed in the apoplastic fractions of infiltrated leaves. However, due to the low number of particles observed, the successful incorporation of the M2e peptide on the surface of the particles was inconclusive, as shown by M2e-specific immuno-gold labelling experiments. Furthermore, contrarily to previous studies, co-expression of the M2e-HA fusions with the M1 protein resulted in a decrease in recombinant protein accumulation and VLP formation in our plant system. A possible inhibition mechanism by the M1 protein is discussed. In summary, this research provides preliminary data to produce universal influenza vaccines in plants. I report here for the first time that M2e fused to either the stalk or transmembrane domain of the HA protein, can self-assemble into VLPs without any other proteins, in N. benthamiana plants. Future work on the immunogenicity of the VLPs produced in this study is required to confirm their potential as a universal influenza vaccine that can be rapidly produced.
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Veerapen, Varusha Pillay. "Novel expression and production of Foot-and-mouth disease virus vaccine candidates in Nicotiana benthamiana." Master's thesis, University of Cape Town, 2017. http://hdl.handle.net/11427/27240.
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Foot-and-mouth disease, also known as FMD, is caused by the aphthovirus Foot-and-mouth disease virus (FMDV), which is a highly contagious disease of cloven-hoofed animals. It is endemic in Africa, parts of South America and southern Asia. In South Africa, the disease is controlled essentially through prophylactic vaccination. Current vaccines on the market are chemically inactivated virus strains. However, these are not considered ideal due to the possibly insufficient inactivation which could fail to render the virus harmless. Research on recombinant vaccines which obviate the need for high biosafety requirements for vaccine preparation has shown that recombinant FMDV virus-like particles (VLPs), devoid of viral genetic material, are an ideal vaccine candidate as they are as immunogenic as the virions themselves when administered to animals. These VLPs are formed by the assembly of the FMDV capsid proteins VP0, VP1 and VP3, which are generated upon the proteolytic cleavage of the capsid precursor protein P1-2A, by the FMDV 3C-protease. The expression platforms used to co-express and produce the component capsid proteins and the protease are usually mammalian, insect or E. coli cells. The use of these expression systems requires extensive bioreactor infrastructure and sterile conditions for vaccine preparation which are costly. In addition, some studies have shown how the co-expression of the 3C-protease can prove to be deleterious when expressed at a high concentration in expression systems. In order to circumvent this, and encourage more efficient production of the capsid proteins and subsequent VLP assembly, researchers have shown that the levels of the 3C-protease can be down-regulated by introducing mutations in the 3C gene or a ribosomal frameshift in the gene sequence which subsequently reduce its deleterious effect. Our laboratory has previously shown that similar FMDV VLPs can be assembled by the expression of FMDV P1-2A (referred to as oP1-2A, in this study), in the absence of the 3C-protease in the plant Nicotiana benthamiana, albeit in low amounts. This platform does not require high biocontainment facilities for the production of recombinant proteins and VLPs and the process is easily scalable. This study centers mainly on optimisation of the FMDV capsid protein expression in N. benthamiana in order to increase VLP yields. I first used codon-optimisation as an approach to improve expression of the capsid proteins and compared expression in the presence and absence of the 3C-protease, using mP1-2A-3C (a new codon-optimised construct), mP1-2A and oP1-2A. Electron microscopy (EM) showed that VLPs resulting from the expression of both mP1-2A-3C and mP1-2A were very low in yield, and irregular in shape and size compared to those produced using oP1-2A. The stability of the plant-produced VLPs was assessed by counting numbers of VLPs, when it was seen that expression of mP1-2A-3C compared to oP1-2A produced an average of 1 VLP per field of view versus 3 VLPs per view, for the same magnification. Furthermore, maturation trials at room temperature was performed on the oP1-2A VLPs, whereby a time-point between 30 to 45 minutes was considered ideal to produce stable VLPs. It is also known that FMDV, unlike the other members of the Picornaviridae family, is acid and heat-labile. The second aim of this study was to promote the stability, and hence encourage the amount of the VLPs produced, by engineering acid and heat-resistant mutants, namely, VP1 N17D, VP2 H93C and VP1 N17D/VP4 S73N using site-directed mutagenesis. A fourth mutant, VP3 A118V which is acid sensitive was used as a control in downstream experiments. The mutants were subjected to a lower than normal pH and a higher than normal temperature. Expression of oP1-2A from the pH and heat assays was assessed to be better than its mutants. The optimum VLP count of 3 VLPs per field of view, was achieved from expression of oP1-2A, after treatment at pH 6.2, compared to 2 VLPs or 1 VLP per field of view for the other mutants tested under all the different conditions. The final aim of this study was to test the immunogenicity of the VLPs from expression of oP1-2A in Balb/C mice. Due to the low yields of VLPs obtained from purification through a continuous gradient, a partial purification method was adopted. Two experimental groups of animals were either vaccinated with P1-2A VLPs or with adjuvanted P1-2A VLPs. A control group was administered with partially-purified plant extract, previously infiltrated with pEAQ-HT. The two experimental groups elicited a marginal increase in humoral immune response at 41 days post vaccination (dpv), which increased significantly at 58 dpv. To my knowledge, this is the first study showing that VLPs produced from expression of FMDV P1-2A only, in tobacco plants, can withstand otherwise degradative acidic and heat conditions. This characteristic has potential for extending the shelf-life of such a candidate vaccine. I also implemented maturation steps to further promote the stability of such VLPs. Finally, the partially purified VLPs showed that they stimulate a significant FMDV P1-2A-specific immune response, particularly in combination with the adjuvant Montanide suggesting that it has potential as a candidate FMDV vaccine.
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Agüero, González Jesús. "Desarrollo de vectores virales basados en el virus del manchado foliar de los cítricos (CLBV)." Doctoral thesis, Universitat Politècnica de València, 2013. http://hdl.handle.net/10251/34342.
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Los cítricos son el cultivo frutal económicamente más importante tanto en
España como en el resto de los países productores. La clave para mantener la
competitividad de este sector consiste en obtener material vegetal de alta calidad,
para lo cual son indispensables los programas de mejora. La mejora de cítricos por
métodos clásicos es muy complicada, por lo que hay que recurrir a las nuevas
tecnologías para intentar acelerar y optimizar el procedimiento. La reciente
secuenciación del genoma de dos especies de cítricos ha permitido identificar una
larga lista de genes candidatos a participar en determinados procesos biológicos. Sin
embargo, son necesarios nuevos análisis para asociar cada gen a un fenotipo
específico o función biológica.
El empleo de vectores virales para determinar la función de genes mediante
silenciamiento génico inducido por virus (VIGS) ha demostrado ser una herramienta
muy útil para los estudios de genética reversa realizados en plantas. Este sistema
presenta ventajas respecto a los métodos tradicionales para estudiar la función de
genes como son la mutagénesis o la transformación genética, ya que permite
ensayar la función de numerosos genes en un corto periodo de tiempo. Esto es
especialmente crítico en el caso de los cítricos, que poseen largos periodos juveniles
de entre 6 y 8 años y donde la transformación de plantas adultas es muy difícil.
Además, permite estudiar la función de genes que son esenciales para el crecimiento
o el desarrollo de la planta y cuyo análisis es inviable con los métodos tradicionales.
Al comienzo de la tesis se había desarrollado un vector viral para cítricos
basado en el virus de la tristeza de los cítricos (CTV) con el que se pueden expresar
proteínas pero que no se ha ensayado para estudiar la función de genes mediante
VIGS. En el laboratorio disponíamos de un clon infeccioso de cDNA del genoma
completo del virus del manchado foliar de los cítricos (CLBV), un virus que infecta a
todas las especies y variedades de cítricos ensayadas y es asintomático en la mayoría
de ellas. Este clon infeccioso se ha modificado para obtener vectores virales basados
en el genoma de CLBV que pueden servir tanto para expresar proteínas como para
silenciar mediante VIGS genes de cítricos para la mejora genética de este cultivo.
Para ello, se ha introducido un punto de corte único PmlI en dos zonas del genoma
de CLBV: en el extremo 3¿ no traducible (vector clbv3¿) o en la zona intergénica
localizada entre los genes de las proteínas de movimiento y cápsida (CP) (vector
clbvIN). Para la expresión de secuencias foráneas mediante la formación de un
nuevo RNA subgenómico (sgRNA) se delimitó la secuencia mínima promotora del sgRNA CP mediante clonación de fragmentos de distinta longitud en torno al origen
de transcripción de dicho sgRNA en el vector clbv3'. El fragmento de 92 bases
localizado entre los nt -42 y +50 respecto al inicio de transcripción del sgRNA CP
contenía todos los elementos necesarios para la promoción de un nuevo sgRNA in
vivo. Esta secuencia mínima promotora se clonó en los 2 vectores virales
previamente desarrollados para generar los vectores clbv3¿pr y clbvINpr,
respectivamente. Ambos vectores fueron capaces de producir un nuevo sgRNA y de
expresar proteínas recombinantes.
Para determinar la estabilidad de los vectores obtenidos se clonaron en ellos
fragmentos de secuencias lineales de distinto tamaño, o en tándem invertido para la
formación de una estructura en horquilla, y se inocularon en plantas de N.
benthamiana y cítricos. Todas las construcciones derivadas del vector clbv3' se
mostraron estables a lo largo de las diferentes brotaciones analizadas durante al
menos 3 años, comprobándose la replicación viral e integridad del inserto. Sin
embargo, no se detectó multiplicación viral con ninguna de las construcciones
derivadas del vector clbvIN. La estabilidad de las construcciones derivadas de los
vectores con el promotor duplicado dependía del tamaño del inserto. Con todas
ellas se detectó replicación viral pero se observaron eventos de recombinación
cuando se clonaban fragmentos superiores a 720 nt en el vector clbvINpr o 408 nt en
el vector clbv3'pr.
Un factor importante para determinar la eficiencia y funcionalidad de los
vectores desarrollados es conocer cómo se mueve y se distribuye el virus en los
distintos tejidos de la planta. Para ello se inocularon plantas de N. benthamiana y
cítricos con la construcción clbv3¿pr-GFP, que expresa GFP en los tejidos donde se
localiza el virus. En N. benthamiana, la observación de GFP permitió detectar la
presencia de CLBV en la mayoría de tejidos, acumulándose preferentemente en
óvulos y regiones meristemáticas. En cítricos no se pudo visualizar GFP pero el virus
se detectó en regiones meristemáticas mediante RT-PCR a tiempo real e hibridación
molecular. La acumulación de CLBV en tejidos meristemáticos explicaría la dificultad
de eliminar este virus mediante microinjerto.
Para evaluar la capacidad de los vectores clbv3'pr y clbvINpr para expresar
proteínas se clonó en ellos la secuencia completa del gen gfp y se cuantificó la
cantidad de proteína GFP sintetizada en las plantas infectadas. En N. benthamiana la
cantidad de GFP estimada para el vector clbv3'pr fue de 16 µg de proteína por
gramo de peso fresco, cantidad que resultó entre 5 y 6 veces superior a la estimada para el vector clbvINpr. Sin embargo, en cítricos, debido a la inestabilidad del vector
clbv3'pr, sólo se pudo cuantificar la proteína expresada por la construcción del
vector clbvINpr, estimándose en 0.6 µg de GFP por gramo de peso fresco.
La efectividad de los vectores clbv3', clbv3'pr y clbvINpr para silenciar genes
mediante VIGS se ensayó clonando fragmentos de genes tanto endógenos de
plantas (pds, actina, sulfur) como el gen gfp introducido experimentalmente en
plantas transgénicas. En cítricos todas las construcciones de los tres vectores
indujeron fenotipo de silenciamiento del gen ensayado, aunque el vector clbv3' fue
el más efectivo para el estudio de VIGS en este huésped. Sin embargo, en N.
benthamiana sólo se desencadenó el silenciamiento en las plantas inoculadas con la
construcción clbv3¿pr-hp58PDS, que expresa una horquilla de doble cadena de un
fragmento de 58 nt del gen pds. En todas las plantas silenciadas se detectó una
disminución del correspondiente mRNA del gen ensayado y una acumulación de
siRNAs derivados tanto del mRNA del gen insertado como del RNA genómico del
virus. Por otro lado, el fenotipo de silenciamiento de los genes ensayados se observó
en sucesivas brotaciones, lo que confirma la gran estabilidad de los vectores basados
en el genoma de CLBV.
Los vectores virales desarrollados en esta tesis constituyen una herramienta
eficiente para el estudio de la función de genes mediante genética reversa utilizando
la técnica VIGS. También pueden ser útiles para estudio de genética directa
mediante expresión de proteínas o para la protección del cultivo frente a
enfermedades producidas por virus, bacterias y hongos o frente a plagas de
invertebrados.Agüero González, J. (2013). Desarrollo de vectores virales basados en el virus del manchado foliar de los cítricos (CLBV) [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/34342
TESIS
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Verbeek, Matthew James Robert. "The Expression of Shuni Virus Nucleocapsid Protein in Nicotiana benthamiana for Use as a Diagnostic Reagent." Master's thesis, Faculty of Science, 2019. http://hdl.handle.net/11427/31017.
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Many devastating zoonotic viruses such as West Nile and Rift Valley fever viruses are endemic to South Africa, affecting livestock and ultimately, through their arthropod vectors, also infecting humans. One such zoonotic virus that is of interest is Shuni virus (SHUV). SHUV belongs to the viral genus Orthobunyavirus, family Peribunyaviridae., and order Bunyavirales. Discovered in arthropods and humans in Nigeria, it was soon identified as a possible cause for cases of neurological disease in horses within South Africa. Studies have shown South African veterinarians who had come into contact with such cases tested positive for antibodies against the virus. Therefore, SHUV is being further investigated as a potential cause of neurological disease within humans and there is a need to develop appropriate quick and effective diagnostic reagents to allow for surveillance of the virus. The main focus for this study was the development of diagnostic reagents centred around the nucleocapsid (N) protein of the SHUV. The N proteins of closely related members of the order Bunyavirales have shown to be highly abundant in infection and cause an immune response in the infected hosts thus making it the ideal target. Using available SHUV genome sequences and data, the N protein gene was designed and synthesised to be expressed in both Escherichia coli and plant expression systems. The expression of the N protein in E. coli, followed by subsequent washing with BugBuster, led to a final mass of 5.1 mg of the SHUV N protein from a 1000 ml culture. This led to a SHUV N yield of 5.1 µg/ml of culture and was measured to make up 69.5% of the total soluble protein. The immunisation of rabbits with this recombinantly expressed SHUV N allowed for the development of polyclonal antibodies which were successfully used in immunoblot studies to detect plant produced SHUV N protein. Plants are an effective and possibly cheaper alternative production system to bacterial, mammalian, or insect cell cultures and thus the N protein was transiently expressed in N. benthamiana plants using Agrobacterium tumefaciens-mediated infiltration. The recombinant protein produced underwent purification using nickel affinity chromatography. This led to yields of 2.248 mg of SHUV N protein from 35 plants which gave a yield of 9.9 mg/kg of raw plant material. This purified plant produced N protein acted as an antigen for diagnostic assays such as ELISA, which was used to screen known SHUV infected sera. This led to mixed results due to the limited sera samples available. However, as a proof of concept, it has shown great potential and thus opens the door to a possible inexpensive dual-use assay for use in the diagnoses of both animal and human SHUV infection.
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Bruckner, Fernanda Prieto. "Aspectos da interação entre a proteína TCTP e o potyvírus PepYMV na infecção de tomateiro e Nicotiana benthamiana." Universidade Federal de Viçosa, 2012. http://locus.ufv.br/handle/123456789/5355.
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Viruses are organisms with small genomes of simple organization, which coding about 3 to encode 10 viral proteins. The success of the infection depends on the manipulation of the cell by the virus, by means of complex interactions occurring between viral factors and host factors. The induced changes by virus infection include cell morphology changes, cell cycle changes and alterations in gene expression, among others. Understanding the processes that favor viral infection necessarily involves the study of virus-host interactions. In order to better understand the processes related to infection by tomato potyvirus Pepper yellow mosaic virus (PepYMV) a subtractive library was built 72 hours after infection. Several genes were identified as induced or repressed by viral infection. Among the induced genes, is the gene encoding the Translationally controlled tumor protein (TCTP). TCTP protein is highly conserved in all eukaryotes. Its functions are related to growth control and cell cycle, anti-apoptotic activity, and response to different biotic and abiotic stresses. The involvement of this protein in infection PepYMV has not been established, but studies in a strain of transgenic tomato plants silenced for TCTP showed that the silenced plants have a lower accumulation of PepYMV, indicating that TCTP promotes viral infection. In this study, we sought to advance the understanding of mechanisms involving TCTP in the process of infection by PepYMV. N. benthamiana plants silenced by VIGS TCTP were used to study the effect of silencing in viral infection, and the silenced plants accumulate fewer viruses in early stages of virus infection. Individual expression of viral proteins in N. benthamiana identified P3 and CP as capable of inducing TCTP expression at similar levels to those induced during PepYMV infection, and expression of NIb reduced expression of TCTP. The verification of direct interactions occurrence between viral proteins and TCTP by double-hybrid assay showed that TCTP not interact separately with any of the proteins of viral origin. Purification of proteins of health and infected N. benthamiana plants by affinity with TCTP identified several proteins that putativaly interacts with TCTP. As in two hybrid assay, interactions involving PepYMV proteins were not detected. These results sugests that TCTP actuation must involve the formation of protein complexes involving viral and plant proteins or contribute indirectly to PepYMV infection, without involving direct interactions between TCTP and viral proteins.
Os vírus são organismos com genomas pequenos, de organização simples, que codificam em média 3 a 10 proteínas. O sucesso da infecção depende da manipulação da célula pelo vírus, por meio de interações complexas que ocorrem entre fatores virais e fatores do hospedeiro. As modificações induzidas na célula incluem alterações morfológicas, alteração do ciclo celular e na expressão gênica, entre outras. A compreensão dos processos que favorecem a infecção viral passa necessariamente pelo estudo de interações vírus-hospedeiro. No intuito de compreender melhor os processos relacionados à infecção de tomateiros pelo potyvírus Pepper yellow mosaic virus (PepYMV) uma biblioteca subtrativa foi construída 72 horas após a infecção. Diversos genes cuja expressão foi alterada pela infecção foram identificados. Dentre os genes induzidos, se encontra o gene que codifica a Translationally controlled tumor protein (TCTP). A proteína TCTP é altamente conservada em todos os eucariotos. Suas funções estão relacionadas a controle do crescimento e ciclo celular, atividade anti-apoptótica, e resposta a diferentes tipos de estresses abióticos e bióticos. O envolvimento desta proteína na infecção pelo PepYMV ainda não foi estabelecido, porém estudos em uma linhagem de tomateiro transgênica silenciadas para a TCTP, mostraram que as plantas silenciadas apresentam um menor acúmulo de PepYMV, indicando que a TCTP favorece a infecção por este vírus. Neste trabalho, buscou-se avançar na compreensão dos mecanismos que envolvem a TCTP no processo de infecção pelo PepYMV. Plantas de Nicotiana benthamiana silenciadas para TCTP por VIGS (Virus Induced Gene Silence) foram utilizadas para estudar o efeito do silenciamento na infecção viral, sendo que as plantas silenciadas acumularam menos vírus no início da infecção. A expressão individual das proteínas de origem viral em N. benthamiana identificou a P3 e a CP como capazes de induzir a expressão de TCTP em níveis semelhantes aos observados durante a infecção pelo PepYMV, sendo que a expressão da proteína NIb reduziu a expressão de TCTP. A verificação da ocorrência de interações diretas entre a TCTP e as proteínas virais, por ensaio de duplo híbrido, mostrou que a TCTP não interage separadamente com as proteínas de origem viral. A purificação de proteínas de plantas de N. benthamiana, sadias e infectadas, por afinidade com a TCTP identificou diversas proteínas que possivelmente 7 interagem com a TCTP. Assim como no ensaio de duplo híbrido, a interação com proteínas virais não foi detectada. Estes resultados sugerem que o papel da TCTP deve envolver a formação de complexos proteicos entre proteínas virais e da planta, ou favorecer a infecção de forma indireta.
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Stephan, Dirk. "Molekulare Charakterisierung von beet mild yellowing virus (BMYV) und beet chlorosis virus (BChV) sowie Selektion von BMYV Amplicon-transgenen Nicotiana benthamiana." [S.l. : s.n.], 2005. http://deposit.ddb.de/cgi-bin/dokserv?idn=974988146.
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Ghoshal, Basudev. "Symptom recovery in Tomato ringspot virus infected Nicotiana benthamiana plants : investigation into the role of plant RNA silencing mechanisms." Thesis, University of British Columbia, 2014. http://hdl.handle.net/2429/49984.
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Symptom recovery in virus-infected plants is characterized by the emergence of asymptomatic leaves after a systemic symptomatic phase of infection and has been linked with the clearance of the viral RNA due to the induction of RNA silencing. However, the recovery of Tomato ringspot virus (ToRSV)-infected Nicotiana benthamiana plants is not associated with viral RNA clearance in spite of active RNA silencing triggered against viral sequences. ToRSV isolate Rasp1-infected plants recover from infection at 27°C but not at 21°C, indicating a temperature-dependent recovery. In contrast, plants infected with ToRSV isolate GYV recover from infection at both temperatures. In this thesis, I studied the molecular mechanisms leading to symptom recovery in ToRSV-infected plants.
I provide evidence that recovery of Rasp1-infected N. benthamiana plants at 27°C is associated with a reduction of the steady-state levels of RNA2-encoded coat protein (CP) but not of RNA2. In vivo labelling experiments revealed efficient synthesis of CP early in infection, but reduced RNA2 translation later in infection. Silencing of Argonaute1-like (NbAgo1) genes prevented both symptom recovery and RNA2 translation repression at 27°C. Also, translation repression was compromised in Rasp1-infected wild-type (WT) plants grown at 21°C. NbAgo1 and NbAgo2 mRNAs accumulated to similar levels at 21°C and 27°C in mock-inoculated WT plants. Both genes were induced during Rasp1 infection. Interestingly, the effect of silencing NbAgo2 on Rasp1 infection was only evident at low temperatures resulting in higher accumulation of CP. Taken together, our results suggest that although both NbAgo1 and NbAgo2 genes are induced, recovery of Rasp1-infected plants at 27°C is associated with an NbAgo1-dependent mechanism that represses the translation of viral RNA2.
In contrast, recovery of GYV-infected plants is associated with a reduction of viral RNA and CP levels at both temperatures. Moreover, silencing of either NbAgo1 or NbAgo2 did not prevent recovery of GYV-infected plants at 21°C. However, both GYV-infected NbAgo1 and NbAgo2-silenced plants accumulated higher level of CP in recovered leaves compared to control plants. In conclusion, this study identifies translation repression as a novel regulatory mechanism in recovery and suggests that different mechanisms may operate during recovery in an isolate and/or temperature-dependent manner.Science, Faculty of
Botany, Department of
Graduate
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Naude, Jason Christopher Delville. "The Expression of Chikungunya Virus Envelope 2 Glycoprotein Variants in Nicotiana benthamiana for the Development of a Diagnostic Reagent." Master's thesis, Faculty of Science, 2021. http://hdl.handle.net/11427/32942.
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Chikungunya fever is a non-fatal but highly debilitating disease that affects primates, birds and humans. The causative agent is the chikungunya virus (CHIKV), an arbovirus of the Alphavirus genus. CHIKV is responsible for the largest epidemic recorded for an Alphavirus, infecting an estimated 1.4 to 6 million patients worldwide. Furthermore, it has been recognised by the United States army as a potential biological weapon used for bioterrorism owing to the potential for infection via aerosol. CHIKV is primarily transmitted by infected Aedes aegypti mosquitos and is currently distributed in Africa, parts of Asia and South, Central and North America. As a result of the virus genetically adapting to infect the Aedes albopictus mosquito, its recent and rapid spread to non-endemic regions has occasioned increasing anxiety as well. Infection in humans presents as a sudden onset of fever, rash and severe arthralgia that persists for years. At present, there is no fast and effective diagnostic test to distinguish CHIKV from other similar viruses. This is a problem because viral infection displays the same symptoms as that of dengue, Zika, Ebola and yellow fevers while prognosis, patient care, and persistent symptoms of these viruses are very different. Usually, during the development of a diagnostic reagent, Biosafety Level 3 (BSL3) containment is required for purifying antigens from live viruses. These lab diagnostic tests are expensive to perform and, in regions facing a CHIKV epidemic, are inefficient due to their long waiting periods. This results in patients going undiagnosed or misdiagnosed and/or falling outside the window of prophylactic treatment. As such, a cheap and rapid diagnostic reagent to detect the presence of CHIKV antibodies would be most advantageous. In this study, two recombinant variants of the CHIKV E2 glycoprotein were expressed in Nicotiana benthamiana plants to assess their viability for use in a diagnostic reagent for CHIKV infection. Two versions of a tobacco sp. codon-optimised, 6xhis-tagged CHIKV E2 envelope glycoprotein gene were synthesised and cloned into the plant expression vector, pTRAkc-ERH. The E2 glycoprotein is a desirable protein candidate used for a diagnostic reagent as it is a major target for neutralizing antibody production against CHIKV during early infection. One variant contained a ~52 kDa full length E2 glycoprotein (CHIKV E2-HIS) while the other contained a ~49 kDa truncated E2 glycoprotein lacking its transmembrane domain (CHIKV E2ΔTM-HIS). Following this, an expression time trial was performed whereby the recombinant proteins were expressed in N. benthamiana plants via Agrobacterium-mediated small-scale 6 syringe-infiltration at different optical densities, OD600 = 1.0 and 0.5. To improve expression, both genes were co-infiltrated and co-expressed with a human chaperone proteins calreticulin (CRT) or calnexin (CNX), or a plant silencing-suppressor protein NSs. Expression of the recombinant protein variants alone showed low to undetectable levels of expression in plant leaves across 7 days post infiltration (dpi) for both ODs tested. CHIKV E2ΔTM-HIS yielded the highest levels of all combinations tested at an OD600 = 1.0 when co-expressed with CRT and harvested at 3 dpi. These parameters were used for subsequent scaling up and production of E2 using vacuum infiltration. Attempts at purifying CHIKV E2ΔTM-HIS proteins using Ni-NTA affinity chromatography and further investigation into the exposure of the 6xHis-tag on the native conformation of CHIKV E2ΔTM-HIS indicated that the 6xHis-tag was insufficiently exposed on E2 and thus inaccessible to facilitate purification by Ni-NTA affinity chromatography. Further attempts at purifying recombinant CHIKV E2ΔTM-HIS proteins by pH purification were also unsuccessful as large amounts of plant-protein contaminants present in all samples prevented adequate separation from CHIKV E2ΔTM-HIS. A different approach utilising ammonium sulphate precipitation facilitated separation of recombinant CHIKV E2ΔTM-HIS from some of the contaminating plant proteins in the 30 - 60% ammonium sulphate fraction; however, large amounts of recombinant CRT were co-purified with E2 in this fraction. Although expression of a candidate diagnostic reagent in plants for detecting CHIKV antibodies in the form of E2 glycoprotein was achieved, further research needs to be done to optimise a purification strategy for CHIKV E2ΔTM-HIS proteins.
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Varennes-Jutras, Philippe. "Protection des protéines recombinantes sécrétées chez l'hôte d'expression "Nicotiana benthamiana" par expression hétérologue du canal ionique M2 du virus de l'Influenza." Doctoral thesis, Université Laval, 2017. http://hdl.handle.net/20.500.11794/28235.
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Les systèmes d’expression végétaux sont utilisés couramment pour la production hétérologue de protéines recombinantes complexes. Des contraintes biochimiques dans le système de sécrétion cellulaire, comme l’abondance de protéases peu spécifiques ou des variations de pH d’un organite à l’autre, compromettent toutefois l’expression de plusieurs protéines d’intérêt. Des approches ont été développées pour améliorer l’environnement intracellulaire dans la plante hôte de manière à accroître la qualité et le rendement des protéines sécrétées. Dans ce projet, nous avons évalué l’impact de l’homéostasie du pH dans le système de sécrétion cellulaire des feuilles du Nicotiana benthamiana sur l’expression et la stabilité des protéines recombinantes. Nous démontrons le potentiel du canal ionique M2 du virus de l’Influenza comme nouvel outil pour augmenter le pH des compartiments acides du système de sécrétion cellulaire, une approche éventuellement utile pour la stabilisation des protéines sensibles aux milieux acides. En lien avec l’influence bien documentée du pH sur l’activité des protéases cellulaires, nous montrons ensuite qu’une modification du pH induite par l’expression du canal M2 influence les profils de dégradation de protéines de fusion sensibles à la protéolyse, des observations qui confirment l’impact du pH sur l’activité protéolytique des cellules végétales et qui suggèrent le potentiel du canal ionique M2 comme protéine accessoire pour augmenter la stabilité et le rendement des protéines recombinantes in planta. Finalement, nous abordons l’impact du canal ionique M2 sur l’expression des protéines endogènes à l’échelle de la cellule. Nous démontrons qu’une altération du pH dans le système de sécrétion en réponse à l’expression du canal ionique a des effets étendus sur le protéome foliaire, affectant la teneur de protéines retrouvées dans plusieurs compartiments cellulaires incluant les chloroplastes, le cytosol et la vacuole. Nous rapportons aussi l’établissement d’un ‘protéome hybride’ dans les plantes exprimant M2, composé de protéines caractéristiques aussi bien de plantes témoins non infectées que de plantes agroinfectées exprimant activement des protéines de défense. En résumé, nos données mettent en évidence le rôle de l’homéostasie du pH sur le protéome des cellules végétales et l’impact significatif du gradient de pH dans le système de sécrétion cellulaire sur la stabilité et le rendement des protéines recombinantes sensibles à l’acidité ou à la protéolyse.Plant expression systems are commonly used for the heterologous production of complex recombinant proteins. However, biochemical conditions in the plant cell secretory pathway, such as the presence of poorly-specific proteases or pH variations from one organelle to another, impair the expression of several potentially useful proteins. Approaches have been developed to improve the cellular environment of the host plant in such a way as to increase the quality and yield of secreted recombinant proteins. In this project, we assessed the impact of pH homoeostasis in the leaf cell secretory pathway of wild tobacco Nicotiana benthamiana on the expression and stability of recombinant proteins. We demonstrate the potential of Influenza virus proton channel M2 as a new tool to increase pH in acidic compartments of the cell secretory pathway, eventually useful to stabilize acid-labile recombinant proteins. In line with the well-established influence of pH on cell protease activities, we then show that pH modification induced by the expression of the M2 channel influences the degradation profile of fusion proteins susceptible to proteolysis, thus confirming the impact of pH on protease activities in plant cells and highlighting the potential of M2 as an accessory protein to increase the stability and yield of recombinant proteins in planta. Finally, we describe the impact of the M2 proton channel on the expression of endogenous proteins at the cellular scale. We show that pH alteration in the secretion system upon M2 expression has cell-wide effects on the leaf proteome, affecting the content of proteins found in various cell compartments including the chloroplast, the cytosol and the vacuole. We report the establishment of a ‘hybrid proteome’ in leaf cells expressing the proton channel, composed of protein clusters characteristic of both control, non-infected plants and agroinfected plants actively expressing defense-related proteins. Overall, our data highlight the central role of pH homeostasis on the proteome of plant cells and the strong impact of pH gradient in the cell secretory pathway on the stability and yield of acidic pH-labile and protease-susceptible recombinant proteins.
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Charlesworth, Steven Roy. "Investigation into resistance strategies against geminiviruses by understanding and adapting RNA interference." Thesis, Queensland University of Technology, 2018. https://eprints.qut.edu.au/121482/2/Steven%20Roy%20Charlesworth%20Thesis.pdf.
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This project investigated new strategies to improve viral resistance in crops against the increasing global threat of DNA viruses. The research showed that a combination of protective strategies is more likely to be effective and durable than a single approach. A gene from a wild relative was identified as a new potential source of DNA virus resistance.
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Xavier, André da Silva. "Efeito do silenciamento dos genes DnaJ e TCTP na infecção de tomateiro e Nicotiana benthamiana pelo potyvírus Pepper yellow mosaic virus (PepYMV)." Universidade Federal de Viçosa, 2012. http://locus.ufv.br/handle/123456789/4413.
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Previous issue date: 2012-07-20Conselho Nacional de Desenvolvimento Científico e Tecnológico
During coevolution, plant viruses have developed the ability to modulate the expression of several host genes, or to alter the function of cognate protein to succeed in their multiplication and perpetuation. These virus-induced changes might lead to a high level of dependency, creating an indissoluble link between virus and host. The objective of this study was to investigate the contribution of a DnaJ of Nicotiana benthamiana (homologous of the tomato protein) and of the TCTP protein from tomato during PepYMV infection. These two genes were identified as differentially expressed in a cDNA library constructed from tomato plants infected by PepYMV. Kinetic studies of DnaJ expression in PepYMV-infected N. benthamiana demonstrated that the induction occurs at both 72 hours post-inoculation (hpi) and 14 days post-inoculation (dpi). Plants of N. benthamiana silenced for DnaJ by means of VIGS and tomato plants cv. Moneymaker silenced by transgenesis for TCTP were obtained and mechanically inoculated with PepYMV. Viral infection was confirmed by ELISA and viral load determined by qRT-PCR. Silencing of the DnaJ gene in N. benthamiana interfered with the early stages of viral infection (72 hpi) but its effect on established infections (14dpi) was inconclusive. Non-transformed tomato plants showed severe symptoms of the disease, while TCTP-silenced transgenic plants showed greatly attenuated symptoms or remained asymptomatic. Viral load was dramatically reduced in silenced plants. The subcellular localization of a TCTP-GFP fusion protein in healthy or PepYMV-infected N. benthamiana plants was analyzed by confocal microscopy. In healthy plants TCTP was nuclear and cytoplasmic, while in infected plants at 14 dpi, its subcellular localization was exclusively cytoplasmic. Together, these results suggest that both TCTP and DnaJ are proteins which positively regulate the infection cycle of PepYMV, being required for disease development in the case of TCTP or for the rapid establishment of viral infection in the case of DnaJ. Further studies should be conducted in order to unravel the mechanisms by which these host factors are used to benefit the viral infection.
Durante a coevolução, os vírus de plantas desenvolveram a capacidade de modular a expressão de alguns genes do hospedeiro, ou alterar a função cognata de proteínas para obter sucesso em sua multiplicação e perpetuação. Essas alterações induzidas pelos vírus podem culminar em elevados níveis de especialização, tornando o vínculo com seus hospedeiros indissociável. O objetivo deste trabalho foi investigar a contribuição de uma DnaJ de Nicotiana benthamiana homóloga de tomateiro e da proteína TCTP de tomateiro durante a infecção pelo potyvírus PepYMV. Esses dois genes foram identificados como diferencialmente expressos em uma biblioteca de cDNA construída a partir de tomateiro infectado pelo PepYMV. Estudos de cinética de expressão do gene DnaJ em N. benthamiana infectadas pelo PepYMV demonstraram que a indução do gene ocorre 72 horas pós-inoculação (hpi) e aos 14 dias pós-inoculação (dpi). Plantas de N. benthamiana silenciadas para DnaJ por meio de VIGS e de tomateiro cv. Moneymaker silenciadas por transgenia para o gene TCTP foram obtidas e inoculadas mecanicamente com o PepYMV. A infecção viral foi confirmada por ELISA e a carga viral determinada por qRT-PCR. O silenciamento do gene DnaJ em N. benthamiana interferiu nos estágios iniciais da infecção viral (72 hpi), porém seu efeito em infecções já estabelecidas (14dpi) foi inconclusivo. Plantas não-transformadas de tomateiro exibiram sintomas severos da doença, enquanto as plantas transgênicas silenciadas para o gene TCTP apresentaram sintomas muito atenuados ou permaneceram assintomáticas. Nas plantas silenciadas a carga viral foi drasticamente reduzida. A localização subcelular de TCTP fusionada à proteína GFP em plantas de N. benthamiana sadias ou infectadas pelo PepYMV foi analisada por microscopia confocal. Em plantas sadias a localização de TCTP foi nuclear e citoplasmática, porém em plantas infectadas aos 14dpi, a localização de TCTP foi exclusivamente citoplasmática. Os resultados obtidos sugerem que tanto TCTP quanto DnaJ são proteínas que regulam positivamente o ciclo de infecção do PepYMV, sendo necessárias para o desenvolvimento da doença no caso de TCTP, ou para o rápido estabelecimento da infecção no caso de DnaJ. Estudos posteriores devem ser conduzidos afim de descobrir o mecanismo pelo qual esses fatores do hospedeiro são utilizados em benefício da infecção viral.
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Solofoharivelo, Marie Chrystine. "Molecular Characterizations of Transgenic Nicotiana Benthamiana Plants Resistant to Red Clover Necrotic Mosaic Virus and Effects of Mixed Infections with Potato Virus Y on RNAi-Mediated Resistance." Diss., The University of Arizona, 2008. http://hdl.handle.net/10150/194799.
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Engineered resistance mediated by RNA interference to control viral diseases in plants has shown great promise. However, the discovery that most known plant viruses encode RNAi suppressors which interfere with RNAi raised the issue to whether this type of engineered resistance can be durable in the presence of heterologous viruses in mixed infection. The overall goal of this study was to investigate the mechanism of suppression of RNAi-mediated resistance in transgenic plants in the presence of a virus carrying a strong suppressor of RNAi. Nicotiana benthamiana plants were transformed with a 1.2 kb from the 5' end of RCNMV RNA-1. Transgenic resistant lines were obtained. Resistance in two different transgenic lines was shown to be mediated by two different types of RNAi: constitutive RNAi in D2 line induced by doubles-stranded (ds) transgene transcripts and virus-induced RNAi in B1 line. We demonstrated that PVY differentially affected RNAi-mediated resistance in the two lines. D2 line is completely immune to RCNMV infection. D2 line contained multiple copies of the 1.2 kb transgene which are rearranged and produced dsRNAs. PVY did not break the resistance in this transgenic line however data showed that PVY interfered with RNAi which correlated to an increase of the 1.2 kb transgene mRNA. In addition, PVY infection induced accumulation of 21 nt siRNAs and did not alter the transcription of the transgene. In contrast, PVY infection suppressed resistance mediated by virus-induced RNAi in B1 line. B1 contains a single copy of the1.2 kb transgene and is initially susceptible to RCNMV infection however became resistant to RCNMV in newly merging leaves after 14 days post inoculation. PVY infection did not affect the accumulation of the 1.2 kb transgene mRNA nor the accumulation of 21 nt siRNA corresponding to the transgene. The differential effect of PVY infection on the two RNAi-mediated resistances in the two transgenic lines suggests that properly designed resistant plants might withstand mixed virus infections and the presence of a strong suppressor of RNAi. In addition, the differential effect of PVY on RNAi suggests that parallel but distinct pathways are involved in dsRNA-induced, virus-induced, and sense RNAi.
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Yu, Ming. "Characterization of cDNAs from Nicotiana benthamiana that encode proteins which interact with tomato golden mosaic virus AL2 protein in the yeast two-hybrid system." NCSU, 2003. http://www.lib.ncsu.edu/theses/available/etd-07182003-150714/.
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The AL2 protein of tomato golden mosaic virus (TGMV) is multifunctional. It is required for derepression of the TGMV AR1 gene in phloem tissue, and for trans-activating the AR1 and BR1 genes in mesophyll cells. It also enhances virus susceptibility when expressed in transgenic plants. It is thought that TGMV AL2 protein accomplishes these functions by interactions with unknown host factors. In this study, cDNAs from Nicotiana benthamiana plants that encode proteins which interact with TGMV AL2 were characterized. A yeast two-hybrid assay identified two cDNA clones, Nb#51 and Nb#62, that specifically interacted with TGMV AL2, but not with negative control proteins. Sequences of these two cDNA clones were determined by primer walking, which revealed that Nb#51 appears to be a 3?-coterminal truncated version of Nb#62. Inspection of the amino acid sequences encoded by Nb#62 found the presence of both ankyrin-repeats and tetratricopeptide repeats (TPR). Deletion analysis showed that the TPR motif, together with its flanking regions, was sufficient to confer on Nb#62 the ability to interact with TGMV AL2, whereas the ankyrin-repeats were not required for this interaction. Nb#62-specific mRNAs were detected in N. benthamiana plants by northern hybridization in potato virus X (PVX) infection experiments, but not in heat-shock experiments. Virus-induced gene silencing (VIGS) assays were used to investigate the possible function(s) of the Nb#62-encoded protein in normal plants and in the context of a TGMV infection. When PVX carrying a 618-bp fragment from the 5?-end of the Nb#62 cDNA was used as a silencing trigger in N. benthamiana plants, VIGS effectively targeted the transcripts which contained sequence similarity with the trigger fragment. However, the infected plants didn?t have difference in the phenotype when compared to the PVX vector infection. When TGMV carrying a 93-bp fragment from the 5?-end of the Nb#62 cDNA infected plants, viral DNA accumulation in the upper leaves was reduced, when compared to wild-type TGMV or a control TGMV construct containing a 95-bp fragment from the tobacco Sulfur gene.
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O'Connor, Steven Patrick. "The production of foot-and-mouth disease virus-like particles in the plant Nicotiana benthamiana: a potential candidate vaccine for foot-and-mouth disease." Master's thesis, University of Cape Town, 2018. http://hdl.handle.net/11427/29378.
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Foot and mouth disease virus (FMDV) infects cloven-hoofed animals causing the highly contagious foot and mouth disease. It is spread by contact or through aerosol. The disease is often debilitating for infected animals and can be fatal. Severe measures are taken to contain outbreaks; quarantine and trade restrictions are imposed and herds with infected individuals are culled to prevent the spread of the disease. Consequently, outbreaks of the disease have drastic implications for agriculture and social economies which can be devastating for affected countries. There are seven serotypes of the virus; of which SAT1, SAT2, and SAT3 are endemic to Africa. South African buffalo populations such as those in the Kruger National Park, are natural carriers of FMDV (Thomson 1995). Careful monitoring and regular vaccination are necessary to detect and prevent outbreaks and the spread of the disease to livestock of neighbouring areas and farms. The vaccines currently used are inactivated FMDV virions. These are produced in cell culture, an expensive process that requires high levels of biosafety. Furthermore, inactivated virions present non-structural proteins (NSPs) and thus cannot be distinguished from the infectious virus by imported ELISA kits that utilise the NSPs as coating antigens and conventionally produced detecting antibodies. We aimed to use recombinant constructs encoding the FMDV capsid and protease genes, cloned into the different vectors; pRIC, pEAQ and pTRAc, for transient expression in Nicotiana benthamiana to generate virus-like particles as an alternative vaccine candidate. Using a plant based expression system presents numerous advantages over the traditional cell culture production of the vaccine currently used. After having synthesised the FMDV genes P12A and 3C, the fusion gene P1-2A-3C (required for the vaccine) was cloned into these different plant expression vectors available in our laboratory. With Agrobacteria mediated infiltration of N. benthamiana, we demonstrated expression of recombinant protein by western blotting; and Coomassie stain, for each of the different constructs. Analytical ultra-centrifugation through a sucrose gradient was used to purify protein extracts. Comparison against a dilution series of bovine serum albumin was used to quantify the yield for each respective vector construct by densitometry. Transmission Electron Microscopy (TEM) imaging was used to qualitatively determine virus-like particle (VLP) assembly. In conclusion, we demonstrate proof of concept for a viable alternative approach for the production of a candidate vaccine for FMDV.
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Gil, Capitán María Teresa. "Inmunomodulación de la infección de Plum pox virus mediante la expresión estable y transitoria de anticuerpos recombinantes contra la NIb replicasa viral en plantas de Nicotiana benthamiana." Doctoral thesis, Universitat Politècnica de València, 2013. http://hdl.handle.net/10251/34204.
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Plum pox virus (PPV) es miembro del género Potyvirus, causa la enfermedad de la sharka que tiene un serio impacto agrícola y económico sobre el cultivo de frutales de hueso. Puesto que la utilización de variedades resistentes es la estrategia más duradera y definitiva en la lucha contra virosis de plantas. El objetivo general de esta tesis doctoral sería evaluar la expresión de anticuerpos recombinantes específicos de proteínas de PPV para interferir o inmunomodular la infección viral y servir así como estrategia para obtener resistencia.
Para ello se ha utilizado el fragmento de anticuerpo recombinante scFv2A (del inglés "single chain Fv variable fragment) que reconoce específicamente a la proteína NIb replicasa de PPV. Esta proteína se localiza principalmente en el núcleo de células de plantas infectadas, aunque su función de replicación, presumiblemente, tiene lugar en estructuras membranosas derivadas del retículo endoplásmico (RE) en el citoplasma.
Se transformaron plantas de Nicotiana benthamiana con tres versiones del fragmento recombinante scFv2A dirigidas a diferentes compartimentos celulares. Una versión citosólica (scFv2A), una versión nuclear (NLS-scFv2A) y otra dirigida a membranas del RE (6K2-scFv2A) por la cara citosólica. Se obtuvieron diversas líneas transgénicas: líneas A, líneas N y líneas K transformadas respectivamente con las construcciones scFv2A, NLS-scFv2A y 6K2-scFv2A. Se realizaron ensayos independientes de desafío, mediante inoculación mecánica con extractos de plantas infectadas con virus o bien inoculando con un clon recombinante de PPV fusionado a GFP (PPV-GFP). Tras los ensayos, se comprobó que las plantas de las diversas líneas transgénicas de N. benthamiana que expresaban los fragmentos scFv2A, 6K2scFv2A o NLSscFv2A presentaban diferentes grados de protección frente a la infección por PPV. Además, se determinó que varias de las líneas transgénicas, tanto líneas A como K y N, presentaban porcentajes de infección significativamente mGil Capitán, MT. (2010). Inmunomodulación de la infección de Plum pox virus mediante la expresión estable y transitoria de anticuerpos recombinantes contra la NIb replicasa viral en plantas de Nicotiana benthamiana [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/34204
Palancia
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Martínez, Priego Lucía. "Actividad antiviral de pequeños RNAs endógenos y supresión de silenciamiento génico por la proteína 16K del virus del cascabeleo del tabaco (TRV)." Doctoral thesis, Universitat Politècnica de València, 2016. http://hdl.handle.net/10251/61464.
Full textAbstract:
[EN] During viral infections, the outcome of the infective process is a net balance between the compatible and defence interactions. When a virus infects the eukaryotic cell, it must deal with different host defence mechanisms among which RNA silencing is part of the initial plant innate defence response. RNA silencing in plants has the role of restraining viral proliferation in the infected cell and therefore regulates the equilibrium between viral load and plant cell integrity that is key for the plant-virus compatibility. The virus itself is inductor, target and suppressor of the RNA silencing in plants. Viral silencing suppressor proteins (VSR) counteract host antiviral silencing and modify the host gene expression programme to generate a permissive environment for compatible infections.
In this PhD thesis we have studied the interface between viral and plant RNA silencing in the context of a compatible infection. Using Tobacco rattle virus (TRV) as a model viral system and Nicotiana benthamiana and Arabidopsis thaliana as host model systems, we have dissected the role of endogenous small RNAs to promote gene silencing responses to viral sequences. Our results point to possible functional interactions between miRNAs and complementary sequences in viral genomes even though the role of those interactions as a viral proliferation controls mechanisms is not part of this thesis. We have found that TRV 16K silencing suppressor protein effects play a central role to dictate the way the TRV and plant RNA silencing interact. The 16K protein avoids, partially, the assembly of silencing effectors complexes and thus compromises the impact of antiviral vsiRNAs-mediated and endogenous small RNAs-mediated RNA silencing. The suppressor effect of TRV does not have a significant impact on the miRNAs content, relative composition and activity although we cannot discard an effect on the metabolisms of some particular miRNA species.[ES] En las infecciones por virus, el desenlace del proceso infectivo debe entenderse como el resultado neto de las interacciones compatibles y de defensa entre el virus y la planta hospedadora. Cuando un virus entra en una célula eucariota debe lidiar con la activación de diferentes mecanismos de defensa del huésped. El silenciamiento génico mediado por RNA constituye una primera línea de defensa innata de la planta, siendo los propios virus inductores, dianas y supresores de este sistema de defensa. Las plantas a través del silenciamiento génico son capaces de limitar la proliferación viral en las células infectadas permitiendo un delicado equilibrio entre la multiplicación del virus y la integridad celular. Sobre este equilibrio se fundamenta la relación de compatibilidad en la interacción planta-virus. En este escenario, los virus utilizan sus proteínas supresoras de silenciamiento (VSR) para modular los efectos antivirales del silenciamiento y reprogramar la expresión génica del huésped proporcionando un entorno favorable para el desarrollo de la infección compatible Con este trabajo hemos abordado el modo en que los virus interaccionan con el silenciamiento génico en el contexto de una infección compatible. Empleando el virus del cascabeleo del tabaco (TRV) como sistema viral y Nicotiana. benthamiana y Arabidopsis thaliana como modelos de huésped, hemos indagado en el potencial de los pequeños RNAs (sRNAs) endógenos para guiar procesos de silenciamiento sobre secuencias virales. Nuestros resultados suLa manera en que TRV interacciona con la ruta de silenciamiento está condicionada gieren la posibilidad de interacciones funcionales entre microRNAs (miRNAs) y secuencias complementarias en el genoma del virus, si bien su relevancia como mecanismo de control de la proliferación viral no se ha estudiado en este trabajo. por el efecto supresor de la proteína 16K. Esta proteína impide, al menos parcialmente, el ensamblaje de los complejos efectores de silenciamiento y puede por tanto comprometer el efecto del silenciamiento antiviral dependiente de sRNAs tanto virales (vsiRNAs) como endógenos. El efecto supresor de TRV no parece perturbar globalmente el contenido, composición relativa y actividad de los miRNAs, si bien no es descartable que induzca alteraciones en el metabolismo de especies concretas.
[CAT] En les infeccions per virus, el desenllaç del procés infectiu ha d'entendre's com el resultat net de les interaccions compatibles i de defensa entre el virus i la planta hoste. Quan un virus entra a una cèl·lula eucariota ha de lluitar amb l'activació de diferents mecanisme de defensa de l'hoste. El silenciament gènic per RNA constitueix una primera línia de defensa innata de la planta, i els propis virus son inductors, dianes i supressors d'aquest sistema de defensa. Les plantes a través d'aquest silenciament, són capaces de limitar la proliferació viral a les cèl·lules infectades, permetent un delicat equilibri entre la multiplicació del virus i la integritat cel·lular. En aquest equilibri es fonamenta la relació de compatibilitat existent a la interacció planta-virus. En aquest escenari, els virus utilitzen les seues proteïnes supressores de silenciament (VSR) per tal de modular els efectes antivirals del silenciament i reprogramar l'expressió gènica de l'hoste, proporcionant un entorn favorable per al desenvolupament de la infecció compatible. Amb aquest treball hem abordat la manera en la que els virus interaccionen amb el silenciament gènic en el context d'una infecció compatible. Emprant el virus del cascavelleig del tabac (TRV) com a sistema viral i Nicotiana. benthamiana i Arabidopsis thaliana com a hostes models, hem indagat en el potencial dels RNAs endògens curts de doble cadena (sRNAs) per guiar processos de silenciament sobre seqüències virals. Els nostres resultats suggereixen la possibilitat de interaccions funcionals entre microRNAs (miRNAs) i seqüències complementàries al genoma del virus, tot i que la seua rellevància com a mecanisme de control de la proliferació viral no ha estat tractat en aquest treball. La manera en la que TRV interacciona amb les rutes de silenciament es troba condicionada per l'efecte supressor de la proteïna 16K. Aquesta proteïna impedeix, al menys parcialment, l'acoblament dels complexos efectors del silenciament i pot llavors comprometre l'efecte del silenciament antiviral depenent de sRNAs, tant virals (vsiRNAs) com endògens. L'efecte supressor de TRV no sembla pertorbar globalment el contingut, composició relativa i activitat dels miRNAs, tot i que no es pot descartar que induïsca alteracions en el metabolisme d'espècies concretes.
Martínez Priego, L. (2016). Actividad antiviral de pequeños RNAs endógenos y supresión de silenciamiento génico por la proteína 16K del virus del cascabeleo del tabaco (TRV) [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/61464
TESIS
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Hoffmann, Laurent. "Etude du métabolisme des phénylpropanoïdes; analyse de l'interaction de la caféoyl-coenzyme A 3-O-méthyltransférase (CCoAOMT) avec son substrat et caractérisation fonctionnelle d'une nouvelle acyltransférase, l'HydroxyCinnamoyl-CoA : shikimate/quinate hydroxycinnamoyl Transférase (HCT)." Phd thesis, Université Louis Pasteur - Strasbourg I, 2003. http://tel.archives-ouvertes.fr/tel-00003598.
Full textAbstract:
Le métabolisme des phénylpropanoïdes est un métabolisme secondaire spécifique au règne végétal. Il conduit, à partir de la phénylalanine, à la synthèse d'une grande variété de substances telles que les anthocyanes, les isoflavonoïdes, les stilbènes, des esters d'acides hydroxycinnamiques, ou encore à la lignine. Ces métabolites secondaires interviennent dans la pigmentation florale ou encore la protection des tissus végétaux contre divers stress biotiques et abiotiques. Quant à la lignine, elle assure rigidité aux parois cellulaires végétales et imperméabilité aux tissus conducteurs. La lignine est un polymère tridimensionnel constitué de trois unités monomériques qui possèdent le même squelette carboné phénylpropane mais diffèrent par leur degré de méthoxylation et d'hydroxylation. Une partie de mon travail de thèse a consisté à étudier la relation structure/fonction de la caféoyl-coenzyme A O-méthyltransférase (CCoAOMT) de N. tabacum, responsable de l'introduction de la première des deux fonctions méthyles. Des études bioinformatiques couplées à des approches de biochimie et de mutagenèse dirigée, nous ont permis de modéliser l'interaction de la CCoAOMT avec son substrat, le caféoyl-CoA. Trois acides aminés du site actif ont notamment été identifiés comme intervenant dans la reconnaissance spécifique de la chaîne latérale de CoA. J'ai également caractérisé, chez N. tabacum, une nouvelle acyltransférase à activité HydroxyCinnamoyl-CoA : shikimate/quinate hydroxycinnamoyl Transférase (HCT) impliquée dans le métabolisme des phénylpropanoïdes. Nous avons montré que l'enzyme HCT recombinante synthétisait, in vitro, les substrats de l'hydroxylation en position 3 du noyau aromatique. De plus, la répression de l'expression du gène HCT par le «VIGS» conduit à un ralentissement de la croissance des plantes, à une perturbation importante du pool d'acide chlorogénique, ainsi qu'à une diminution de la quantité et à une modification de la composition de la lignine synthétisée.
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Busto, Jennifer Lee. "Transcriptional changes in Nicotiana benthamiana induced by tobamoviral transfection." Thesis, 2005. http://proquest.umi.com/pqdweb?index=1&did=913527421&SrchMode=1&sid=1&Fmt=2&VInst=PROD&VType=PQD&RQT=309&VName=PQD&TS=1235524057&clientId=23440.
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Huang, Wei-Pin, and 黃薇頻. "Tracing Bamboo Mosaic Virus RNA Molecules in Nicotiana benthamiana Protoplasts." Thesis, 2008. http://ndltd.ncl.edu.tw/handle/14346358446864419971.
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碩士國立中興大學
生物科技學研究所
96
Bamboo mosaic virus (BaMV), a potexvirus, is a single-strand, positive-sense RNA virus which contains five open reading frames (ORFs) which encode proteins for replication, movement and structure and a caped 5''UTR and a 3''UTR with a poly A tail. There is good amount of information on the replication of potexviruses but information on the sub-cellular localization of the viral RNA and the site of replication is lacking. Though there are different methods to detect RNA localization by optical microscopy, most of them are limited to fixed samples. We employ a technique which exploits the ability of phage MS2 coat protein to bind specifically to only its cognate RNA. In this strategy, the MS2 RNA sequence is inserted into target RNA (BaMV genomic RNA) and MS2 coat protein is fused with green fluorescent protein (GFP) which contains a nuclear localization signal (NLS). When the modified target RNA and the GFP-NLS-CPMS2 construct are co-transformed into protoplasts the GFP-NLS-CPMS2 fusion protein binds to the target RNA and the green fluorescence signal indicates the localization of the target RNA. The unbound GFP-NLS-CPMS2 is retained in the nucleus due to the NLS thereby reducing noise. This method can be used for four dimensional study of RNA localization in live cells. First, the MS2 RNA sequence had to be inserted into BaMV RNA genome. According previous study, the PVX 3''UTR insertion construct and only subgnomic RNAs could accumulate normally. Simultaneously, the CPMS2-GFP fusion protein was constructed in pBI 121 vector. As BaMV.S.(MS2)6 was transfected in GFP-NLS-CPMS2 fusion transiently expressed protoplasts1 dpi, observation of GFP localization indicated the BaMV RNA subcellular localization. GFP signal was co-localized and moved with mobile mitochondria stained with Mitotracker OrangeCMTMRos. Employing this novel method we observed BaMV RNA localizing at the mitochondria and that a large proportion of the RNA would most probably be the subgenomic RNAs which also contains the MS2-hps since the mutant RNAs are defective in genomic RNA accumulation.
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"Expression of Recombinant Zika Virus-Like Particles in Nicotiana benthamiana." Master's thesis, 2018. http://hdl.handle.net/2286/R.I.50512.
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abstract: Zika virus (ZIKV) outbreaks have been linked to several neurological pathologies in the developing fetus, which can progress to spontaneous abortion and microcephaly in newborns whose mothers were infected with the virus during pregnancy. ZIKV has also been correlated with neurological complications in adults such as Guillain-Barré Syndrome (GBS). ZIKV outbreaks often occur in low income areas with limited access to healthcare. Therefore, there is a need to create a low-cost preventative vaccine against the virus. Mature ZIKV particles contain a lipid bilayer, a positive sense single stranded RNA genome and three structural proteins: the envelope (E), membrane (M) and capsid (C) proteins. Congruently, to other members of the Flaviviridae family, ZIKV proteins are synthesized as a polyprotein precursor which needs to be processed to release the mature structural and non-structural viral proteins. Past studies have determined the ZIKV precursor protein is cleaved by a host furin protease which separates the Pr peptide and the M protein, while the host signal peptidase separates the M and E protein. Processing is important for correct folding of the E protein. In turn, the most important neutralizing antibodies upon infection are directed against epitopes of the E protein. In this work, we used a Bean Yellow Dwarf Viral vector system to transiently express, in Nicotiana benthamiana plants, a portion of the ZIKV polyprotein encoding the Pr, M and E proteins. I further demonstrate that plants can proteolytically process the polyprotein to yield the two integral membrane proteins M and E. These proteins can be shown to co-partition into a soluble membrane-particulate fraction, consistent with formation of enveloped virus-like particles (VLPs). This work provides the first step in creating a low-cost sustainable plant-based production system of ZIKV VLPs that can be explored as a potential component 0f a low-cost prophylactic vaccine against ZIKV.Dissertation/Thesis
Masters Thesis Molecular and Cellular Biology 2018
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Guo, Shang-Ming, and 郭尚明. "Interactions between Cymbidium mosaic virus and Odontoglossum ringspot virus in Nicotiana benthamiana." Thesis, 2017. http://ndltd.ncl.edu.tw/handle/e3x9c3.
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碩士國立臺灣大學
植物病理與微生物學研究所
105
Mixed infection of plant viruses usually leads to intrahost virus-virus interactions. Odontoglossum ringspot virus (ORSV) and Cymbidium mosaic virus (CymMV) commonly co-infect orchid plants and cause more severe symptoms, which is defined as synergistic effect. Recently, we found that the synergistic effect between ORSV and CymMV did exist on Nicotiana benthamiana protoplasts. This interaction seems to be regulated by the silencing suppression activity of ORSV p126. In this study, we continued to explore the interactions between ORSV and CymMV on N. benthamiana. In addition to p126, transiently expressed ORSV capsid protein (CP) facilitated CymMV accumulation on the inoculated leaves of N. benthamiana, but ORSV movement protein did not. The mechanism under this phenomenon remains unknown. Individual domains of ORSV p126 were proved without RNA silencing suppression ability and could not improve CymMV accumulation. In this study, we constructed five different domain combination of p126 and found that all four domains are necessary for RNA silencing suppression. Surprisingly, viral RNA and CP accumulation of both ORSV and CymMV had no significant difference between singly and doubly inoculated leaves of N. benthamiana plants through agroinoculation. However, by means of sap inoculation, more severe symptoms on both inoculated and systemic leaves of doubly infected plants were observed compared to singly infected ones. Next, we detected the viruses in systemic leaves of ORSV and CymMV doubly infected plants by indirect-ELISA, and found that the systemic movement-deficient CymMV could systemically infect N. benthamiana. These results suggested that although mixed infection of ORSV and CymMV did not exhibit synergistic interaction on inoculated leaves, ORSV still facilitated CymMV in other mechanism, probably on movement. Interestingly, facilitation on CymMV systemic movement disappeared when CymMV was co-inoculated with systemic movement-deficient ORSV (ORSVE100), which suggested the systemic movement of CymMV may rely on ORSV CP or ORSV infection processes. To understand the specificity of ORSV-CymMV synergism, we co-expressed CymMV with some well-known RNA silencing suppressors (RSSs), e.g. Turnip mosaic virus (TuMV) HCPro, Cucumber mosaic virus 2b, Potato virus X (PVX) p25 and Tomato bushy stunt virus p19. Except for PVX p25, all RSSs could significantly increase the accumulation of CymMV, which indicated that p126-mediated enhancement of CymMV accumulation probably can be replaced by other RSSs. Furthermore, we were curious about whether CymMV can systemically infect N. benthamiana with the aid of other ORSV-related or ORSV-unrelated viruses. For mixed infection of TuMV+CymMV, and PVX+CymMV, about 57% and 50% infected plants showed systemic CymMV infection. Co-infection of CymMV and Tomato mild green mosaic virus (TMGMV), a tobamovirus, facilitated systemic movement of CymMV on all tested plants. Finally, we constructed three eGFP-expressing CymMV clones and used one of them to confirm the experimental results.
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McLachlan, Juanita. "A Study of Nicotiana Benthamiana Protein Interactions with Tomato Bushy Stunt Virus." Thesis, 2013. http://hdl.handle.net/1969.1/149490.
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Two Tomato bushy stunt virus (TBSV) proteins, P19 and P22, have been found to interact with the Nicotiana benthamiana host proteins Hin19 and HFi22 in yeast two,hybrid assays. To determine functional roles of these interacting host proteins, viral induced gene silencing (VIGS) was employed to knock,down their expression. TBSV has been demonstrated to activate a virus,specific antiviral response pathway in N. benthamiana. To characterize this pathway, the antiviral RNAi induced silencing complex (RISC) was isolated from TBSV-infected plants. Additionally, putative RISC-associated proteins were identified in silico and suggested roles for these have been identified through literature and database searches. A further aim was the identification of proteins that coimmunoprecipitate with the TBSV-induced RISC following RISC isolation.
A primary aim of this investigation was to identify functional roles for host proteins that interact with the two TBSV 3-terminal encoded proteins, P22 and P19. Each of these has functional roles in viral movement and pathogenicity. In yeast two-hybrid assays, P22 has been shown to interact with HFi22 while P19 interacts with Hin19. VIGS was utilized in attempts to silence the expression of these two host proteins in order to determine their functional roles.
VIGS-mediated suppression of the TBSV-interacting proteins Hin19 and HFi22 has not been accomplished. Despite multiple attempts and multiple approaches, these proteins have not been amenable to silencing. In light of this finding, it is proposed that rather than utilizing VIGS to down-regulate protein levels for Hin19 and HFi22, other approaches should be utilized.
To characterize the TBSV-mediated RNAi pathway, functionally active antiviral RISC was purified from TBSV-infected N. benthamiana plants using ion-exchange chromatography. This RISC was found to be active only in the degradation of TBSV transcripts, indicating the specificity expected from a programmed RISC.
Characterization and identification of proteins that copurify with RISC has not yet been accomplished, though in silico analysis has yielded over 150 putative RISC-associated proteins. Of these, a subset has been identified as highly likely candidates based upon function and/or homology to RISC-associated proteins in non-plant organisms, and a model for the TBSV-induced antiviral pathway has been proposed.
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Li, Jin-Guei, and 黎金桂. "Establishment of Transgenic Nicotiana benthamiana Plants Expressing the Bamboo Mosaic Virus Replicons." Thesis, 2001. http://ndltd.ncl.edu.tw/handle/66779453434479418175.
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碩士國立中興大學
農業生物科技學研究所
89
Bamboo mosaic virus (BaMV) is a flexuous rod-shaped plant virus with a positive-sense RNA, about 6366 nucleotides in length (excluding the poly(A) tail). BaMV have five conserved open reading frames (ORF). The three overlapping ORFs (ORF 2, 3, and 4), known as the triple gene block (TGB), which encode proteins of 28, 13, 6 kDa, respectively. ORF 5 encodes 25 kDa viral capsid protein (CP). The TGB proteins and the coat protein are thought to play roles in virus movement between plant cells. Some BaMV isolates contain a satellite RNA (satBaMV) which is 836 nucleotide long (excluding poly(A)) and contains an ORF for a protein of 20 kDa (P20). Whether the satBaMV uses the same movement machinery as BaMV in plant is not known yet. Coinoculation of BaMV defective in TGB or CP with satBaMV into Nicotiana benthamiana protoplasts had found that both TGB- and CP-defective BaMV could support satBaMV replication. The purpose of this study is to produce the transgenic Nicotiana benthamiana that express the replicative BaMV RNA defective in TGB or CP. Complementary DNAs of defective BaMV RNA that has deletion of TGB and CP gene were constructed in a plant expression vector pKyLx7 and transferred to N. benthamiana by Agrobacterium-mediated transformation. Transgenic plants were selected in kanamycin-containing medium and those expressing defective BaMV RNAs were further identified by the PCR, Southern, Northern and Western blot analyses. The stability of transgene maintained in the F1 progenies was screened in kanamycin-containing medium. The ratio of kanamycin resistance genotype of F1 progenies was 3/4, which followed the Mendelian law. We had selected 15 homozygous transgenic N. benthamiana lines expressing the BaMV dT and 3 homozygous transgenic N. benthamiana lines expressing the BaMV dC. After inoculation with satBaMV, we found that transgenic Nicotiana benthamiana plant expressing the defective BaMV RNA that has deletion of TGBps could not support the movement of satBaMV. This result indicates that BaMV movement protein genes are required for satBaMV movement.
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Hsueh, Chia-Hsin, and 薛家欣. "The effect of NbRbohB on Bamboo mosaic virus replication in Nicotiana benthamiana." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/05121950829248358246.
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碩士國立中興大學
生物科技學研究所
104
Respiratory burst oxidase homologs (Rboh), alias NADPH oxidase, plays a critical role in reactive oxygen species (ROS) generation. In Nicotiana benthamiana, there are two kinds of Rboh, NbRbohA and NbRbohB. NbRbohB, induced by specific pathogen signals, participates in mechanisms for plant disease resistance. Bamboo mosaic virus (BaMV), a member of the Potexvirus genus, contains a single-stranded positive-sense RNA genome. A previous proteomic approach identified a potential interaction between NbRbohB and BaMV replicase. To explore the relationship between NbRbohB and BaMV replication, NbRbohB was down-regulated by the Tobacco rattle virus (TRV)-induced gene silencing method and the silencing plant was subsequently inoculated with the recombinant BaMV virion that carries green fluorescent protein gene (GFP) in this study. Western blot analysis, revealed that the accumulations of GFP and BaMV CP were reduced significantly in NbRbohB-silenced plants. Northern blot and RT-PCR also demonstrated that the accumulation of BaMV genomic RNA was decreased when NbRbohB was silenced. The same VIGS method was used to analyze the effects of NbRbohB on Foxtail mosaic virus (FoMV) and Potato virus X (PVX) replication. Silencing NbRbohB reduced the accumulation of FoMV CP but not PVX CP. According to public EST databases, the full-length cDNAs of NbRbohB1 and NbRbohB2 were acquired, and they were inserted into the expression vector pBI221. Overexpression of NbRbohB1 in BaMV-infected protoplasts significantly increased the accumulation of BaMV CP. In conclusion, NbRbohB1 plays a significant role in promoting BaMV and FoMV replication.
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Odokonyero, Denis 1984. "Identification of ARGONAUTES Involved in Antiviral RNA Silencing in Nicotiana benthamiana." Thesis, 2012. http://hdl.handle.net/1969.1/148228.
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ARGONAUTE proteins (AGOs) are generally accepted as key components of the post transcriptional gene silencing mechanism, also involved in plant antiviral defense. Except for reports on the antiviral roles of AGO1, AGO2 and AGO7 in Arabidopsis, the exact roles played by the individual AGOs in other plant species are largely unknown. This research focused on the identification and characterization of AGOs involved in antiviral RNAi response to various viruses in N. benthamiana. Based on the temporal and spatial distribution of AGO transcripts in 3 and 8-week old plant root, stem and leaf tissues, expressions of NbAGO mRNAs were found to vary with age and tissue specificity. Plant endogenous AGO mRNAs were knocked down through virus induced gene silencing techniques using the Tobacco rattle virus vector system and posteriorly challenged with a GFP-chimeric virus construct deficient of a silencing suppressor. Unlike in control non-silenced plants, the Tomato bushy stunt virus construct deficient of its P19 silencing suppressor was consistently seen to exhibit a strong fluorescence on N. benthamiana plants silenced for NbAGOs 2 and X. Similar results were also obtained upon silencing of NbAGO2 using hairpin vector techniques. Comparable observations were also made when Tobacco mosaic virus GFP constructs were agroinfiltrated on NbAGO2 silenced plants further hinting the antiviral defense roles played by these AGOs. Agroinfiltration of Foxtailmosaic virus, Sunnhemp mosaic virus, and Turnip crinkle virus GFP chimeric constructs on NbAGO2 silenced N. benthamiana plants, however did not result in accumulation of GFP indicating the AGO antiviral defense specificity to TBSV and TMV. The results also hinted at a role for AGO7. Collectively my findings suggest that the expression of AGOs in N. benthamiana is tissue and age dependent, and that unlike in the model plant Arabidopsis where the main antiviral AGO is thought to be AtAGO1; in N. benthamiana, NbAGOs 2 and X seem to be involved in an antiviral defense role against TBSV and TMV with other AGOs perhaps contributing.
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Ai, Wei-Ping, and 艾瑋苹. "The effect of isocitrate dehydrogenase on Bamboo mosaic virus replication in Nicotiana benthamiana." Thesis, 2015. http://ndltd.ncl.edu.tw/handle/04832184388759104405.
Full textAbstract:
碩士國立中興大學
生物科技學研究所
103
Viruses recruit many of the host cell factors to complete infection cycle. Recently, many studies have committed to explore the relationship between the virus and its host factors. Bamboo mosaic virus ( (BaMV)) , belonging to the genus Potexvirus, is a single-stranded 6.4-kb positive sense RNA virus.. In our lab, BaMV relpicase complex was prepared from Nicotiana benthamiana that had been Agroinfiltrated agroinfiltrated with BaMV replicase-expression cassette, followed by partial purification using immunoprecipitation. Several speculative host factors were subsequently identified by LC-MS/MS, including AtClpC, pleiotropic drug resistance like protein, scarecrow-like protein 5, isocitrate dehydrogenase ( (NbICDH)) and Calcium-transporting ATPase 4. It was found that when NbICDH was down-regulated by virus-induced gene silence silencing ( (VIGS)) , the accumulation of BaMV coat protein was increased. NbICDH Overexpression overexpression NbICDH in protoplasts, decreased the accumulation of BaMV coat protein was decreased. The result suggests that NbICDH may have antiviral function in N. benthamiana. The catalytic site residue Y205 of NbICDH were was replaced by alanine by mutagenesis to examine the involvement of the enzyme’s catalytic activity in BaMV replication. Overexpression of the mutant proteins in protoplasts showed the same inhibition effect on the accumulation of BaMV coat protein as the wild-type NbICDH. This result suggests that the catalytic activity of NbICDH is not required for decreasing the viral replication. In the future, the regulation of BaMV replication by NbICDH should be demonstrated.
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Chuang, Chi-Mau, and 莊棨貿. "Effects of a putative methyltransferase from Nicotiana benthamiana on Bamboo mosaic virus replication." Thesis, 2008. http://ndltd.ncl.edu.tw/handle/94415236392530050905.
Full textAbstract:
碩士國立中興大學
生物科技學研究所
96
After entering host cells, virus requires host factors for replication and hosts might generate defensive mechanisms. Bamboo mosaic virus (BaMV) is a flexuous-rod positive-sense RNA virus belonging to the genus Potexvirus of the family Flexiviridae. BaMV genome contains 5 open reading frames (ORFs). ORF1 encodes a 155-kDa viral replicase, which possesses an RNA-dependent RNA polymerase (RdRp) activity at the C terminus. In previous study, a putative Nicotiana benthamiana methyltransferase (NbMts) was found to interact with BaMV RdRp domain in a yeast two-hybrid screening against a leaf cDNA library of N. benthamiana. According to predictions on websites, NbMts has a signal peptide at N terminus for membrane targeting and motifs for AdoMet binding. In this study, we used protoplast transformation to study the relation of NbMts to the viral replication. To express NbMts under the control of 35S promoter, the corresponding cDNA was inserted in pBI221 to become pBI-NbMts. Protoplasts were transformed with BaMV infectious cDNA (pCBG) and pBI221 (or pBI-NbMts). After cultivation for indicated periods of time, accumulations of the viral coat protein were analyzed by Western blot. The results indicate that NbMts overexpression inhibits BaMV replication in protoplasts, and the BaMV-inhibition effect was dosage dependent. The BaMV-inhibition effect was most obvious at 16~24 h post cotransfection of pCBG and pBI-NbMts. Deletion of the signal peptide or mutations at the putative AdoMet-binding motifs abolish the BaMV-inhibition effects. Addition of AdoMet in the incubation medium enhanced the repression. NbMts overexpression also inhibited the replication of Foxtail mosaic virus, a member of Potexvirus, but not Tobacco mosaic virus in protoplasts.
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Wu, Li-Chin, and 吳麗琴. "Proteomic Analysis of Differential Protein Expression of Bamboo mosaic virus Infected Nicotiana benthamiana." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/89582669429601137127.
Full textAbstract:
碩士國立中興大學
生物科技學研究所
95
Bamboo mosaic virus (BaMV), is a single-stranded positive-sense RNA virus with flexuous rod-shaped morphology. A number of studies have been devoted to analyze the replication of plus-stranded RNA viruses; several host proteins are known to be involved in assembling the viral RNA replication complex, activating the complex for RNA synthesis, and other steps. However, the host has the defense system to against viral replication and spreading which are also mediated through proteins. In this study, we have tried to identify the differentially expressed proteins between mock and BaMV inoculated N. benthamiana leaves by proteomics approach. Since positive-sense RNA virus replication is usually associated with rearrangements of membranous organelles, we first isolated the membrane fraction (P30) of the N. benthamiana leaves. Proteins associated with P30 fraction were resolved by two-dimensional polyacrylamide gel electrophoresis (2-DE) and identified by MALDI-TOF MS. Comparison of protein patterns from P30 fractions of mock and BaMV-infected N. benthamiana in 2-D gels revealed several differential expressed proteins. The differential expression of ribosomal protein L25, RubisCO small chain, calmodulin-1 and glycoprotein endopeptidase-like protein. Among these proteins, ribosomal protein L25 was only found in P30 fractions sample from BaMV-infected plants. It had been reported that ribosomal protein L25 is homologous to general stress proteins CTC. We utilized TRV- based virus-induced gene silencing (VIGS) system to generate L25-knockdown plants and to investigate the effect of this protein on BaMV accumulation. Real-time PCR results showed that the levels of the L25 mRNA in the various L25 knock-down plant were reduced to about 25 and 40 % to those of the control plants. Western blots and northern blots were used to analyze the accumulation of viral coat protein and viral RNA at seven day post-inoculation of BaMV virions. Results showed that the accumulation of BaMV coat protein and viral RNA in L25-knockdown plant were reduced. But no interference on the accumulation of FoMV coat protein was observed in all L25-knockdown plants. Together, these data suggest that L25 likely plays an important role in the BaMV accumulation. Keywords：Bamboo mosaic virus / host factors / proteomic
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Wang, Ssu-yuan, and 王斯遠. "The effect of NbSCL6 on Bamboo mosaic virus infection cycle in Nicotiana benthamiana." Thesis, 2014. http://ndltd.ncl.edu.tw/handle/67005081453115320570.
Full textAbstract:
碩士國立中興大學
生物科技學研究所
102
Bamboo mosaic virus ( BaMV) is a single-stranded positive-sense RNA virus with a 5￠cap and a 3￠ poly(A) tail. BaMV belongs to the genus Potexvirus and the family Flexiviridae. The gene expression profile in Nicotiana benthamiana may be altered after BaMV infection. To identify the possible host genes involving the infection cycle of BaMV, our lab used cDNA-AFLP technique to screen the differentially expressed genes in BaMV-inoculated N. benthamiana plants. ACTC7-1 is an upregulated gene when BaMV infects N. benthamiana. To characterize the function of ACTC7-1 involving in BaMV infection cycle, I used the virus-induced gene silencing (VIGS) technique to known down the expression of ACTC7-1 leveling N. benthamiana plant and then inoculated BaMV onto the knockdown leaves. The accumulation levels of BaMV coat protein was determined by Western blotting analysis. The accumulation of BaMV was enhanced when after the expression of ACTC7-1 was knocked down. To further analyze the effect of ACTC7-1 on BaMV infection is on viral RNA replication or virus movement, I infected BaMV RNA into the ACTC7-1-knockdown protoplasts which were derived from the knockdown plants. The result of the accumulation of BaMV coat protein in N. benthamiana protoplasts was also increased. Overall of these preliminary results suggest that ACTC7-1 is probably involved in the replication of BaMV. Furthermore, I used RACE technique to clone the full-length of ACTC7-1 and blasted the sequence to WORKBENCH. The identity of ACTC7-1 could be the SCARECROW-LIKE transcriotion factor in GRAS family. The future work of this research will focus on the transient expression the full-length of ACTC7-1 fused with Orange fluorescent protein in N. benthamiana to localize this protein and examine the effect on BaMV accumulation
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Chien, Wan-Chu, and 簡婉竹. "Generation of transgenic Nicotiana benthamiana plants conferring resistance against Cucurbit chlorotic yellows virus." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/eghd44.
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Cheng, Chun-Wei, and 程鈞煒. "The effect of Bamboo mosaic virus accumulation by a putative methyltransferase in Nicotiana benthamiana." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/59291778919213580102.
Full textAbstract:
博士國立中興大學
生物科技學研究所
99
Abstract Bamboo mosaic virus (BaMV), a positive-sense RNA virus with the length of 6.4-kb, contains five ORFs. ORF1 of the BaMV encodes a 155-kDa replicase consisting of mRNA capping enzyme domain, helicase-like domain, and RNA-dependent RNA polymerase (RdRp) domain. In the previous study, the interaction between the BaMV RdRp domain and a host putative methyltransferase (PMtsNb1) was identified from a yeast two-hybrid screening against a leaf cDNA library of Nicotiana benthamiana. Over expression of PMtsNb1 in N. benthamiana protoplasts by CaMV 35S promoter significantly decreased the viral coat protein accumulation by 40% and the viral in vitro RdRp activity also by 50%. To assure that these inhibitions were related to the interaction of PMtsNb1 and RdRp, the PNbMts1 fused with GFP and HA-tagged RdRp were co-expressed in protoplast. In this experiment, the HA tagged RdRp could be recognized in the immunoprecipitation of PNbMts1-GFP fusion protein. In addition, mutations at the putative AdoMet-binding motifs abolished the BaMV-inhibition effect. Furthermore, addition of AdoMet in the incubation medium of protoplast enhanced the inhibition effect of PNbMts1. Besides, the accumulation of viral coat protein was less in PNbMts1-overexpressing N. benthamiana than in the wild type plant. In contrast, knock-down of PNbMts1 by virus-induced gene silencing in N. benthamiana increased the accumulation of the viral coat protein about four to six times. In summary, we have identified a novel virus-resistant protein, PNbMts1, which can inhibit the accumulation of BaMV.
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Valenzuela, Aguila Sofia [Verfasser]. "Transformation of Nicotiana benthamiana with different BWYV (Beet western yellows virus) sequences to test for virus resistance = Transformation von Nicotiana benthamiana mit verschiedenen Sequenzen des BWYV (Beet western yellows virus) zur Virus-Resistenztestung / von Sofia Valenzuela Aguila." 2000. http://d-nb.info/959528695/34.
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Chen, Xiang-Yu, and 陳相伃. "The study of ferredoxin-NADP+ oxidoreductase involved in Bamboo mosaic virus replication in Nicotiana benthamiana." Thesis, 2017. http://ndltd.ncl.edu.tw/handle/75888843185615854959.
Full textAbstract:
碩士國立中興大學
生物科技學研究所
105
Bamboo mosaic virus (BaMV) is a single-stranded positive-sense RNA virus belonging to the genus Potexvirus of the family Alphaflexiviridae. We used cDNA-AFLP technique to isolate the host gene fragment which had differential expression after BaMV infection in tobacco plant (Nicotiana benthamiana). One of downregulated gene fragments, ACAG1, is sharing high identity to the non-specific ferredoxin NADP+ reductase sequence in the database; we then designated this gene NbFNR. It is a flavoenzyme that involved in the process of photosynthesis electron transport chain, catalyzes NADP+ into NADPH reaction. In order to investigate whether NbFNR affects the infection cycle of BaMV, we used virus-induced gene silencing (VIGS) technique to reduce the expression level of NbFNR gene in leaves and protoplasts. After BaMV inoculation, the accumulation of BaMV coat protein and viral RNA was significantly lower than that of the control group. Further experiment of transiently expressed NbFNR fused with T7-tag or Orange fluorescent protein (OFP) in plants that could elevate the accumulation of BaMV coat protein. The localization of NbFNR-OFP was observed at chloroplast as expected by confocal microscopy. Overall of these results suggest that NbFNR localized at the chloroplast plays a positive role in BaMV replication.
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Liu, Hsin-Yi, and 劉欣宜. "The study of gibberellic acid insensitive involved in Bamboo mosaic virus replication in Nicotiana benthamiana." Thesis, 2017. http://ndltd.ncl.edu.tw/handle/88268758104731508985.
Full textAbstract:
碩士國立中興大學
生物科技學研究所
105
Bamboo mosaic virus (BaMV) is a single-stranded positive-sense RNA virus, a member of Potexvirus genus of Alphaflexiviridae family. In a previous study, 90 differentially expressed genes were identified from BaMV-inoculated N. benthamiana plants using the technique cDNA-amplified fragment length polymorphism (cDNA-AFLP). One of the differentially expressed genes ACCT7-1 was upregulated in BaMV-inoculated N. benthamiana plants. Furthermore, the full-length cDNA was cloned using rapid amplification of cDNA ends (RACE) and sequence. The identity of ACCT7-1 was revealed as a homolog of GA-INSENSITIVE (GAI), a member of DELLA family, when compared to the databases of National Center for Biology Information (NCBI). Therefore, ACCT7-1 is then designated as NbGAI. NbGAI was characterized by BaMV inoculated on virus-induced gene silencing (VIGS) leaves and protoplasts. The results showed that the accumulation of BaMV coat protein was decreased significantly in both knockdown leaves and protoplasts compared to the controls. These results can be inferred that NbGAI may play a positive role in assisting the virus replication. Furthermore, the fusion protein NbGAI with Orange fluorescent protein (OFP), NbGAI-OFP is expressed in plants and BaMV is then inoculated. The accumulation of BaMV coat protein is increased significantly compared with the control plants with the expression of OFP only. The results indicate that NbGAI play a role in assisting BaMV accumulation. NbGAI is a member of DELLA family and contains a nuclear localization signal (NLS). In this study, I have used confocal microscopy to confirm the nuclear localization of NbGAI in cell. In addition, the NLS of NbGAI is predicted by screening online software: cNLS Mapper. These results indicated that NbGAI could play an assisting role in BaMV replication.
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Lin, Jhe-Wei, and 林哲緯. "Investigating the relation between NbXRN4 and replication efficiency of Bamboo mosaic virus in Nicotiana benthamiana." Thesis, 2015. http://ndltd.ncl.edu.tw/handle/55638555805311058677.
Full textAbstract:
碩士國立中興大學
生物科技學研究所
103
Bamboo mosaic virus (BaMV) is a 6.4-Kb positive-sense RNA virus. It depends on the host factors for replication and movement. Nicotiana benthamiana infiltrated with Agrobacterium that carries the viral polymerase-coding region was used to produce BaMV replication complex, which was then isolated by Immunoprecipitation using specific antibodies. Twenty-two hypothetical host factors in the complex were identified by LC tandem mass spectrometry. The expression of the potential factors in N. benthamiana were downregulated by the TRV-induced gene silencing method and the silenced plant was subsequently inoculated with the recombinant BaMV virion that carries GFP gene to assess the effects of the factors on BaMV replication. Among the potential factors, silence of NbXRN4, confirmed by the RT-PCR method, significantly reduced the expression of GFP, as evidenced by the green fluorescent spots, on the leaves that had been inoculated with the recombinant BaMV. The expression of GFP in the inoculated leaves was examined by Western blot, and the result confirmed that the accumulation of GFP was decreased in the NbXRN4-silenced plant. Northern blot also indicated that the accumulation of genomic and subgenomic RNAs of BaMV was decreased in NbXRN4-silenced plant. The putative nucleotide sequence of NbXRN4 cDNA was assembled according to the various EST data. In order to obtain the cDNA in full length, specified primers were designed to amplify the gene and put it into pBI221 expression vector. Overexpression of NbXRN4 in N. benthamiana protoplasts significantly increased the viral coat protein accumulation. Next, we expect to make sure the co-localization of the BaMV RdRP and NbXRN4 in plant cell.
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Lin, Yan-Cheng, and 林彥丞. "The study of NbGAI involved in the accumulation of Bamboo mosaic virus in Nicotiana benthamiana." Thesis, 2015. http://ndltd.ncl.edu.tw/handle/11635732051930245340.
Full textAbstract:
碩士國立中興大學
生物科技學研究所
103
Bamboo mosaic virus (BaMV) is a single-stranded positive-sense RNA virus belonging to the genus Potexvirus of the family Flexiviridae. The RNA genome comprises 6366 nts with a 5 cap and a 3 poly (A) tail. In general, the expression profile of host genes could be altered when infected by viral pathogens. These differentially expressed genes might play positive or negative roles in regulating BaMV infection cycle. In a previous study, our lab isolated 90 differentially expressed genes from BaMV-inoculated N. benthamiana plants using cDNA-amplified fragment length polymorphism (cDNA-AFLP) technique. One of the upregulated genes post BaMV infection ACCT7-1 was found to involve in BaMV replication since the accumulation of BaMV coat protein was reduced when the expression of ACCT7-1 was knocked down by virus-induced gene silencing (VIGS) in the inoculated leaves and protoplasts. Furthermore, the full-length cDNA was cloned using rapid amplification of cDNA ends technique and sequenced. The identity of ACCT7-1 was revealed as a homolog of GA-INSENSITIVE (GAI), a member of DELLA family, when compared to the databases of National Center for Biology Information (NCBI). Therefore, we then designated this gene as NbGAI. To localize NbGAI in cell, I subcloned NbGAI into pEpyon vector with which can produce the fusion protein of NbGAI with Orange fluorescent protein (OFP) when expressed in plant cells. Under the circumstance of the fusion protein expression, the accumulation of BaMV coat protein is increased compared to the control plants with the expression of OFP only. The results indicate that NbGAI plays a positive role in assisting BaMV accumulation. Since NbGAI is one of the DELLA proteins that regulate plant hormones including gibberellin (GA) and Jasmonate (JA), the involvement of regulating in BaMV replication could be through the JA pathway. In applying JA or GA in plants and followed BaMV inoculation revealed that JA has either no effect or negative on the accumulation of BaMV, whereas the GA plays a positive role on that of BaMV. These results are conflict on the effect of NbGAI in BaMV. Therefore, we conclude that NbGAI, although it is a DELLA protein, may not go through the signaling pathway.
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Heish, Cheng-Kai, and 謝丞凱. "The study of NbRTE1 from Nicotiana benthamiana involved in the replication of Bamboo mosaic virus." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/13049716685092917346.
Full textAbstract:
碩士國立中興大學
生物科技學研究所
104
Bamboo mosaic virus (BaMV) is a single-stranded positive-sense RNA virus which belongs to the Potexvirus of alphaflexiviridae. We used cDNA-amplified fragment length polymorphism technique to screen the differentially expressed genes of Nicotiana benthamiana post BaMV inoculation. One of downregulated genes, ACTG7-1, sharing 59% identify to REVERSION-TO-ETHYLENE SENSITIVITY (RTE1) of Arabidopsis was further investigated. We used virus-induced gene silencing (VIGS) technique to knock down the expression of RTE1 and investigate the involvement of NbRTE1 in BaMV infection cycle. The results derived from previous studies revealed that NbRTE1 could negatively regulate the replication of BaMV. My research goals are aiming at the localization of NbRTE1 in cell by using confocal microscopy and clarifying whether the ethylene response pathway is involved in BaMV infection. The RTE1 in Arabidopsis was reported to associate with endoplasmic reticulum (ER), accordingly the localization of NbRTE1 on the ER needs to be confirmed. Because RTE1 is one of the components in the ethylene response pathway and is involved in BaMV replication in N. benthamiana, we would like to examine more components in the pathway and inspect if they are also involved in BaMV infection. NbETR1, the ethylene receptor, specifically interacts with RTE1 to transduce the signaling. The results derived from VIGS indicated that the expression level of ETR1 was decreased to 18% that of the control plants, the accumulation of BaMV coat protein was reduced to 75%. To further inspect if ethylene is involve in the BaMV infection cycle, we have treated the protoplasts with the ethylene inhibitor to block the signaling pathway. The results revealed no significant difference of the BaMV accumulation. Overall, the results suggest that NbRTE1 involved in BaMV replication might not go through the ethylene signaling pathway. The mechanism of NbRTE1 involved in BaMV replication is still need to be clarified
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Chiang, Wen-Shan, and 江雯珊. "The study of NbDXR from Nicotiana benthamiana involved in the replication of Bamboo mosaic virus." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/38369194472158216201.
Full textAbstract:
碩士國立中興大學
生物科技學研究所
104
Bamboo mosaic virus (BaMV) is a single-stranded positive-sense RNA virus, belonging to the Potexvirus genus of Alphaflexiviridae. To reveal the relationship of host genes involved in BaMV infection cycle, our lab used cDNA-amplified fragment length polymorphism (cDNA-AFLP) to screen the differentially expressed genes from Nicotiana benthamiana after infection. One of the upregulate genes is ACGT8-2, was identified as 1-deoxy-ᴅ-xylulose-5-phosphate reductoisomerase (DXR) in our previous study. The full-length of this gene was cloned and designated as NbDXR. The enzyme is involved in the first committed step of the MEP pathway that converted 1-deoxy-ᴅ-xylulose-5-phosphate (DXP or DOXP) to 2-C-methyl-ᴅ-erythritol 4-phosphate (MEP) for isoprenoid biosynthesis. To further characterize the role of NbDXR in BaMV infection cycle, we used the virus induced gene silencing (VIGS) technology to knock down the expression of DXR in N. benthamiana. The accumulation of BaMV coat protein in both NbDXR-knockdown plants and protoplasts were shown lower than those of the control. These results are implied that NbDXR may play a role in assisting the replication step of BaMV infection cycle. We then further transiently expressed the T7-tag fused NbDXR in plant and shown to have 1.8 folds enhancement of the accumulation of BaMV coat protein compared to that of the control (expressed OFP-T7). Furthermore, the OFP-fused NbDXR was transiently expressed in N. benthamiana and revealed chloroplast localization by confocal microscopy. These results indicated that NbDXR could play an assisting role in BaMV replication. The upstream and downstream genes of DXR in the MEP pathway will be characterized in the future.
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Yu, Jong-Ding, and 余中鼎. "The study of Argonautes in Nicotiana benthamiana involved in the infection of Bamboo mosaic virus." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/16142441951239615532.
Full textAbstract:
碩士國立中興大學
生物科技學研究所
104
Bamboo mosaic virus (BaMV) is a single-stranded positive-sense RNA virus belonging to the genus Potexvirus of the family Alphaflexiviridae. The genome of BaMV is about 6.4 kb with a 5?cap and a 3?poly (A) tail. In this study we use Nicotiana benthamiana as a model plant to inspect the relationship between Argonautes (AGOs) and BaMV. RNA interference (RNAi) in Eukaryotes is known to play an antiviral defense role especially the RNA viruses. During the replication of viral RNAs, the double-stranded (ds) RNA accumulates and triggers the host silencing process. DICER-LIKE (DCL) proteins cleave the viral dsRNA into short interfering RNAs (siRNAs) of 21~24 nucleotides in length. These siRNAs are loaded into AGOs to form the RNA induced silencing complex (RISC). The RISC contains the specific siRNAs and targets to the viral RNAs. There are 10 AGOs identified in Arabidopsis thaliana. By contrast, only four AGOs (AGO1, AGO2, AGO4, and AGO5) were sequenced in N. benthamiana. In this thesis, I used Tobacco rattle virus (TRV)-based gene silencing technique to knock down the expression of different AGOs in N. benthamiana to reveal the relationship between AGOs and BaMV. The results indicated that reducing the expression of AGO4 did not have any effect on the accumulation of BaMV. The results also imply that AGO4 may not play a key role against BaMV in N. benthamiana. By contrast, the reduction of the expression of AGO1 could result in an increase of the accumulation of BaMV in plants and protoplasts. Furthermore, but the accumulation of BaMV has no significant change in AGO2-silencing plants. Moreover, the expression of AGO2 is increased in AGO1-knockdown plants. The results suggest that AGO2 might be controlled by AGO1 as that revealed in Arabidopsis. The relationship between AGO1 and AGO2 in N. benthamiana needs to be further explored.
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Wu, Yi-Jhen, and 吳宜臻. "The effect of MAP kinase phosphatase 1 on Bamboo mosaic virus replication in Nicotiana benthamiana." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/41201104240517043841.
Full textAbstract:
碩士國立中興大學
生物科技學研究所
104
MAP kinase phosphatase 1 (MKP1) in Nicotiana benthamiana has 859 amino acid residues. In Arabidopsis thaliana AtMKP1 interacts with MAP kinase 3, 4, and 6 (MPK3, 4, and 6). The interaction compromises the ability of MPK3 and 6 to provoke the pathogen-defending function and salicylic acid production. Recently, NbMKP1 was found to interact with the replication protein of Bamboo mosaic virus (BaMV) through a proteomic approach. In this study, the virus-induced gene silencing (VIGS) method was used to reduce the expression of NbMKP1, and the silencing plant was inoculated with BaMV-GFP virions. The accumulations of BaMV coat protein (CP) and GFP were both enhanced due to the decreased expression of NbMKP1. BaMV genomic RNA was also increased in the same condition. In protoplasts, overexpression of NbMKP1 reduced the accumulation level of BaMV CP. These data suggest that NbMKP1 can inhibit the proliferation of BaMV in N. benthamiana. In addition, the effect of NbMKP1 on accumulations of Foxtail mosaic virus (FoMV) and Potato virus X (PVX) was examined. Decreasing NbMKP1 led to higher expression of FoMV but lower expression of PVX. The inactivated NbMKP1 was expressed in protoplasts to understand if the phosphate-removing activity is critical to the protein’s inhibitory effect on BaMV accumulation. Overexpression of the inactivated NbMKP1 actually accumulated even more BaMV CP than the negative control, suggesting that the catalytic function of NbMKP1 is critical on this matter.
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Chiu, Ling-Ying, and 邱齡瑩. "The study of NbLTP1 from Nicotiana benthamiana involved in the replication of Bamboo mosaic virus." Thesis, 2014. http://ndltd.ncl.edu.tw/handle/65508777785579651265.
Full textAbstract:
碩士國立中興大學
生物科技學研究所
102
Bamboo mosaic virus (BaMV) is a single-stranded positive sense RNA virus belonging to genus Potexvirus of the family Flexiviridae. The objective of this study is to understand the relationship of the host proteins involved in the replication mechanism of BaMV. The results derived from a previous study, a downregulated gene fragment ACGT12 from Nicotiana benthamiana post BaMV infection identified by cDNA-AFLP technique is further characterized. We used the Tobacco rattle virus (TRV)-based silencing system (virus-induced gene silencing; VIGS) to knock down the expression of ACGT12 and infected the BaMV on the knockdown plants. The results indicate that the accumulation levels of BaMV coat protein in the knockdown plants are lower than that in the control plants. Similar results are observed in the knockdown protoplasts that less coat protein accumulated than that in the control protoplasts. These results suggest that ACGT12 may be involved in the replication process rather than in viral movement. The full-length cDNA of ACGT12 is obtained by rapid amplification of cDNA ends (RACE) technique. The sequence of ACGT12 is blasted to the Biology WorkBench database and matches to that of non-specific lipid transfer protein 1 (nsLTP1) and designated as NbLTP1. Furthermore, the cellular localization of NbLTP1-OFP (fused with Orange fluorescence protein) is mainly associated with the extracellular matrix. However, when the signal peptide is removed, the majority of the expressed mutant protein is associated with chloroplasts. The accumulation of BaMV coat protein is enhanced when NbLTP1 is transiently expressed in plants. Overall, the results indicate that the newly identified host protein NbLTP1 is a positive regulator for the replication of BaMV RNA. In addition to the hydrophobic pocket to accommodate the lipid, NbLTP1 has a conserved calmodulin (CaM)-binding site at the very C-terminus. Therefore, the lipid binding and the CaM-binding properties involving in the replication of BaMV is needed to be further characterized.