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Metabolic engineering of plants using a disarmed potyvirus vectorMajer, Eszter 01 September 2016 (has links)
[EN] Plant viruses are obligate intracellular parasites which were used to develop recombinant plant virus vectors to express heterologous proteins and to modify endogenous metabolic pathways of natural products in plants. The main limitation of many plant virus-based systems is the difficulty to co-express various heterologous proteins in the same cell with proper subcellular localization, which is a crucial question in metabolic engineering. This work provides a solution to overcome this problem by using a potyvirus-based vector system. Potyviruses (genus Potyvirus, family Potyviridae) are plus-strand single-stranded RNA viruses, which have a genome expression strategy that allows the equimolar production of most viral proteins. On the basis of an infectious clone of Tobacco etch virus (TEV), Bedoya et al. (2010) developed an expression system in which the RNA-dependent RNA polymerase (NIb) gene was replaced by an expression cassette, harboring several heterologous proteins. This viral vector was able to express three fluorescent proteins with nucleocytoplasmic localization in equimolar amounts in transgenic tobacco plants in which NIb was supplemented in trans. Despite of the apparent simplicity of potyvirus genome expression strategy, foreign cDNA insertion is a complicated task. Thus, our first goal was to analyze the effect of gene insertion on TEV genome stability. As a result of this work, a novel insertion position was discovered at the amino-terminal end of the potyvirus polyprotein, which opened the possibility to explore new questions of recombinant protein expression. Since metabolic pathways are highly compartmentalized, proper subcellular targeting of enzymes is an essential task. Thus, our second objective centralized on the subcellular targeting of expressed proteins from the TEV-based viral vector. cDNAs coding for the green fluorescent protein (GFP) fused to chloroplast, nucleus and mitochondria targeting signal sequences were inserted into the newly described amino-terminal insertion position or into an internal site, replacing the NIb cistron. Our results showed that for protein delivery to chloroplasts and mitochondria, foreign genes have to be inserted at the amino-terminal site of the viral vector, but for nuclear delivery, both insertion positions are suitable. The last objective of this work was to investigate whether the potyvirus-based vector was able to express an entire heterologous multistep biosynthetic pathway in plant cells. For this aim we purposed to produce lycopene, a plant pigment with health promoting properties. To do so, we inserted cDNAs coding for the enzymes of a three-step metabolic pathway of bacterial origin into the potyvirus-based vector. Infected tobacco plants developed orange symptoms indicating lycopene accumulation, which was confirmed by high-performance liquid chromatography analysis and microscopy observations. Our results also illustrated that the sole expression of Pantoea ananatis phytoene synthase, crtB, is enough to induce carotenoid accumulation, conferring yellow coloration to the infected tissue and serves as reporter system to visually track viral infection in several plant species. / [ES] Los virus de plantas son parásitos intracelulares obligados que han sido utilizados para desarrollar vectores virales y expresar proteínas heterólogas y modificar rutas metabólicas endógenas de productos naturales. La principal limitación de muchos sistemas basados en virus de plantas es la dificultad de coexpresar diversas proteínas heterólogas en la misma célula con la localización subcelular apropiada, lo cual es una cuestión crucial en ingeniería metabólica. Este trabajo presenta una solución para superar este problema mediante el uso de un vector viral basado en un potyvirus. Los potyvirus (género Potyvirus, familia Potyviridae) son virus de RNA de cadena positiva simple que tienen una estrategia de expresión génica que permite la producción de la mayoría de las proteínas virales en cantidades equimolares. Basado en un clon infeccioso del virus del grabado del tabaco (Tobacco etch virus, TEV) Bedoya et al. (2010) desarrollaron un sistema de expresión en el que el gen de la RNA polimerasa dependiente de RNA (NIb) fue sustituido por un casete de expresión, que albergaba varias proteínas heterólogas. Este vector viral fue capaz de expresar tres proteínas fluorescentes con localización nucleocitoplásmica en cantidades equimolares en plantas de tabaco transgénicas que complementaban el cistron NIb en trans. A pesar de la aparente simplicidad de la estrategia de expresión génica de los potyvirus, la inserción de un cDNA foráneo es una tarea complicada. Por lo tanto, nuestro primer objetivo fue analizar el efecto de la inserción en la estabilidad del genoma de TEV. Como resultado de este trabajo, descubrimos una nueva posición de inserción en el extremo amino-terminal de la poliproteína viral que nos permitió explorar otras cuestiones sobre la expresión de proteínas recombinantes. Dado que las vías metabólicas son muy compartimentalizadas, la adecuada localización subcelular de enzimas es una tarea esencial en ingeniería metabólica. Por eso, nuestro segundo objetivo se centró en la distribución de las proteínas heterológas expresadas con el vector viral a diferentes orgánulos subcelulares. cDNAs que codificaban la proteína fluorescente verde (green fluorescent protein, GFP) fusionada a péptidos señal se insertaron en la nueva posición amino-terminal y en un sitio interno, sustituyendo el cistrón NIb, para enviarla al cloroplasto, núcleo y a la mitocondria. Nuestros resultados mostraron que para la distribución de proteínas al cloroplasto y mitocondria, los genes foráneos deben ser insertados en el sitio amino-terminal del vector viral, pero para la distribución nuclear, ambas posiciones son adecuadas. El último objetivo de este trabajo fue estudiar si el vector viral basado en potyvirus es capaz de expresar una ruta biosíntética de múltiples pasos en células vegetales. Para ello nos propusimos producir licopeno, un pigmento vegetal con propiedades beneficiosas para la salud humana. Para ello, insertamos un cDNA que codificaba las enzimas de una ruta metabólica de tres pasos de origen bacteriano en el vector viral. Las plantas de tabaco infectadas con el vector viral desarrollaron síntomas de color naranja indicando la acumulación de licopeno, que fue confirmado por análisis de cromatografía líquida de alta eficacia y observaciones de microscopía. Nuestros resultados también ilustraron que la sola expresión de la fitoeno sintasa de Pantonea ananatis, crtB, es suficiente para inducir la acumulación de carotenoides que confieren una coloración amarilla al tejido infectado y sirve como sistema reportero visual en varias especies de plantas. / [CAT] Els virus de plantes són paràsits intracel·lulars obligats que han estat utilitzats per a desenvolupar vectors virals i expressar proteïnes heteròlogues y modificar rutes metabòliques endògenes de productes naturals silenciant certs gens o expressant factors de transcripció i enzims metabòlics. La principal limitació de molts sistemes basats en virus de plantes és la dificultat de coexpressar diverses proteïnes heteròlogues en la mateixa cèl·lula amb la localització subcel·lular apropiada, cosa que és una qüestió crucial en enginyeria metabòlica. Aquest treball presenta una solució per a superar aquest problema mitjançant l'ús d'un vector viral basat en un potyvirus. Els potyvirus (gènere Potyvirus, família Potyviridae) són virus d'RNA de cadena positiva simple que tenen una estratègia d'expressió gènica que permet la producció de la majoria de les proteïnes virals en quantitats equimolars. Basat en un clon infecciós del virus del gravat del tabac (Tobacco etch virus, TEV) Bedoya et al. (2010) van desenvolupar un sistema d'expressió en el qual el gen de l'RNA polimerasa depenent d'RNA (NIb) va ser substituït per un casset d'expressió, que albergava diverses proteïnes heteròlogues. Aquest vector viral va ser capaç d'expressar tres proteïnes fluorescents amb localització nucleocitoplàsmica en quantitats equimolars en plantes de tabac transgèniques que complementaven el cistró NIb en trans. Malgrat l'aparent simplicitat de l'estratègia d'expressió gènica dels potyvirus, la inserció d'un cDNA forà és una tasca complicada. Per tant, el nostre primer objectiu va ser analitzar l'efecte de la inserció en l'estabilitat del genoma de TEV. Com a resultat d'aquest treball, hem descobert una nova posició d'inserció en l'extrem amino terminal de la poliproteïna viral que ens va permetre explorar altres qüestions sobre l'expressió de proteïnes recombinants. Atès que les vies metabòliques són molt compartimentalitzades, l'adequada localització subcel·lular d'enzims és una tasca essencial en enginyeria metabòlica. Per açò, el nostre segon objectiu es va centrar en la distribució de les proteïnes heteròlogues expressades amb el vector viral a diferents orgànuls subcelul·lars. cDNAs que codificaven la proteïna fluorescent verda (green fluorescent protein, GFP) fusionada a pèptids senyal es van inserir en la nova posició amino terminal i en un lloc intern, substituint el cistró NIb, per a enviar-la al cloroplast, nucli i al mitocondri. Els nostres resultats van mostrar que per a la distribució de proteïnes al cloroplast i mitocondri, els gens forans han de ser inserits en el lloc amino terminal del vector viral, però per a la distribució nuclear, ambdues posicions són adequades. El lloc amino terminal va resultar ser més adequat per a produir quantitats més grans de proteïnes recombinants, però el lloc d'inserció intern va demostrar ser més estable. Sobre la base d'aquests resultats, hem sigut capaços de distribuir dues proteïnes fluorescents diferents als cloroplasts i nuclis des d'un únic vector viral. L'últim objectiu d'aquest treball va ser estudiar si el vector viral basat en potyvirus és capaç d'expressar una ruta biosintètica de múltiples passos en cèl·lules vegetals. Per açò ens vam proposar produir licopè, un pigment vegetal amb propietats beneficioses per a la salut humana. Per això inserírem un cDNA que codificaba els tres enzims de una ruta metabòlica de tres passos d'origen bacterià en el vector viral. Les plantes de tabac infectades amb el vector viral van desenvolupar símptomes de color taronja indicant l'acumulació de licopè, que va ser confirmat per anàlisi de cromatografia líquida d'alta eficàcia i observacions de microscòpia. Els nostres resultats també van il·lustrar que la sola expressió de fitoè sintasa de Pantonea ananatis, crtB, és suficient per a induir l'acumulació de carotenoides que confereixen una colora / Majer, E. (2016). Metabolic engineering of plants using a disarmed potyvirus vector [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/68477 / TESIS
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First report of Cucurbit Chlorotic Yellows Virus (CCYV) and Tomato Leaf Curl New Delhi Virus (ToLCNDV) in Algeria and Lack of Evidence for Seed Transmission of ToLCNDV in Melon and PumpkinKheireddine, Amina 14 September 2023 (has links)
[ES] En este trabajo, se detectó por primera vez el virus de la clorosis amarilla de las cucurbitáceas (CCYV, cucurbit chlorotic yellows virus) infectando pepino y calabacín en Argelia. El CCYV (género Crinivirus, familia Closteroviridae) forma parte de un complejo de virus transmitidos por la mosca blanca Bemisia tabaci, que causan enfermedades de amarillamiento en las cucurbitáceas. Se obtuvo la secuencia completa del gen que codifica la proteína de la cápside (CP) y secuencias parciales de los genes RdRp y Hsp70 de un aislado argelino de CCYV y se compararon con las secuencias presentes en las bases de datos. ToLCNDV (género Begomovirus, familia Geminiviridae) también se detectó por primera vez
infectando plantas de cucurbitáceas en Argelia. ToLCNDV es un begomovirus bipartito que causa
importantes epidemias en cultivos económicamente valiosos de las familias Solanaceae y
Cucurbitaceae. Se obtuvo la secuencia genómica completa de un aislado de ToLCNDV de pepino
de Argelia (ToLCNDV-Biskra). La alineación de las secuencias de nucleótidos de los segmentos
de ADN-A y ADN-B reveló identidades del 98,7% y 97,6%, respectivamente, con los segmentos
de los aislados de la cuenca del Mediterráneo, mientras que la identidad con los aislados de Asia
fue de aproximadamente del 90% y 81%. Los aislados presentes en la cuenca mediterránea son
monofiléticos y forman un único grupo, mientras que los aislados de la cepa asiática presentan una
mayor variabilidad genética y forman varios grupos. Todos los aislados de Argelia mostraron una
variación de nucleótidos muy baja. Solo se observó una duplicación de 17 nucleótidos en el ADNB de algunos aislados que daría lugar a una proteína de movimiento 53 aminoácidos más larga, aunque la funcionalidad de esta supuesta proteína es desconocida. A pesar de esa duplicación, no
se detectaron eventos de recombinación entre los aislados secuenciados. La baja variación genética
podría dificultar la detección de recombinantes. / [CA] En aquest treball, es va detectar per primera vegada el virus de la clorosi groga de les cucurbitàcies (CCYV, cucurbit chlorotic yellows virus) infectant cogombres i carabassetes a Algèria. El CCYV (gènere Crinivirus, família Closteroviridae) forma part d'un complex de virus transmesos per mosca blanca que causen malalties d'esgrogueïment en les cucurbitàcies. Es va realitzar la determinació de la seqüència completa de la proteïna de la càpsida (CP) i seqüències parcials dels gens RdRp i Hsp70 d'un aïllat algerià de CCYV i es van comparar amb les seqüències publicades en les bases de dades. ToLCNDV (gènere Begomovirus, família Geminiviridae) també va ser detectat per primera
vegada infectant plantes de cucurbitàcies a Algèria. ToLCNDV és un begomovirus bipartit que
causa importants epidèmies en cultius econòmicament valuosos de les famílies Solanaceae i
Cucurbitaceae. Es va obtindre la seqüència genòmica completa d'un aïllat de ToLCNDV de
cogombre d'Algèria (ToLCNDV-Biskra). La alineació de les seqüències de nucleòtids del
segments d'ADN-A i ADN-B van mostrar identitats del 98.7% i 97.6%, respectivament, amb els
segments corresponents dels aïllats de la conca del Mediterrani, mentre que la identitat amb els
aïllats d'Àsia va ser d'aproximadament el 90% i 81%. Els aïllats presents a la conca del Mediterrani
són monofilètics i formen un únic grup, mentre que els aïllats de la soca asiàtica presenten una
major variabilitat genètica i s'agrupen en diversos grups. Tots els aïllats algerians van mostrar una
variació de nucleòtids molt baixa. Només es va observar una duplicació de 17 nucleòtids en d'ADNB d'alguns aïllats que lonaria lloc a una proteïna de moviment 53 aminoàcids més llarga, encara
que la funcionalitat d¿aquesta suposada proteïnaés desconeguda. A més, malgrat la duplicació, no
es van detectar esdeveniments de recombinació entre els aïllats seqüenciats. La baixa variació
genètica podria dificultar la detecció de recombinants. / [EN] In this work, cucurbit chlorotic yellows virus (CCYV) was first detected infecting cucumber and zucchini in Algeria. CCYV (genus Crinivirus, family Closteroviridae) is part of a complex of whitefly-transmitted viruses that cause yellowing disease in cucurbits. Determination of the complete CP, and partial RdRp and Hsp70 sequences of an Algerian CCYV isolate was conducted to unveil the evolutionary relationships with the published isolates in databases. The phylogenetic analysis showed that the Algerian isolate clustered into group I together with the majority of the reported CCYV isolates. ToLCNDV (genus Begomovirus, family Geminiviridae) was also detected for the first time infecting cucurbit plants in Algeria. ToLCNDV is a bipartite begomovirus that causes major epidemics in economically valuable crops. epidemics in economically valuable crops of the Solanaceae and Cucurbitaceae families. Cucurbitaceae. The complete genome sequence of a ToLCNDV isolate from Algerian cucumber (ToLCNDV from Algeria (ToLCNDV-Biskra) was obtained. Alignment of the nucleotide sequences of the A-DNA and B-DNA segments revealed the identity of the DNA-A and DNA-B segments revealed identities of 98.7% and 97.6%, respectively, with the segments of the isolates from the Algerian segments of the Mediterranean basin isolates, while the identity with the Asian isolates was approximately 90% and 81%, respectively. was approximately 90% and 81%. The isolates present in the Mediterranean Basin are monophyletic and form a single monophyletic and form a single cluster, whereas isolates from the Asian strain have a higher genetic variability and form several clusters. genetic variability and form several clusters. All isolates from Algeria showed very low nucleotide variation. very low nucleotide variation. Only a duplication of 17 nucleotides was observed in the B-DNA of some isolates which would result in a longer 53 amino acid movement protein, although the functionality of this putative protein is unknown. Despite this duplication, no recombination events recombination events were detected among the sequenced isolates. The low genetic variation could hinder the detection of recombinants. / This work was supported by the following grants: Programa de Ayudas de Investigación y
Desarrollo (PAID-11-21) of the Universitat Politècnica de València; PID2021-125787OR-C33
funded by MCIN/AEI/10.13039/501100011033 and by “ERDF A way of making Europe”; and
PROMETEO project for excellence groups 2021/072 Conselleria de Innovación, Universidades,
Ciencia y Sociedad Digital (Generalitat Valenciana). / Kheireddine, A. (2023). First report of Cucurbit Chlorotic Yellows Virus (CCYV) and Tomato Leaf Curl New Delhi Virus (ToLCNDV) in Algeria and Lack of Evidence for Seed Transmission of ToLCNDV in Melon and Pumpkin [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/196840
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Pflanzenviren als chemische Plattform für die NanotechnologieTscheuschner, Georg 15 July 2024 (has links)
Die Proteinhülle von Pflanzenviren besteht aus Protein-Untereinheiten, die das Genom des Virus verkapseln und transportieren. Der zugrunde liegende self-assembly-Mechanismus kann für nanotechnologische Anwendungen genutzt werden, indem Fremdmoleküle in den Viren verkapselt werden. Diese sogenannten Virus-ähnlichen Partikel (VLP) können zum Wirkstofftransport in der Krebstherapie, für Gentherapie oder als Kontrastmittel bei bildgebenden Verfahren verwendet werden. Auch die Oberfläche der Proteinhülle kann chemisch oder molekularbiologisch funktionalisiert werden. Ein großer Vorteil von Pflanzenviren ist, dass sie keine Gefahr für den Menschen darstellen und einfach in natürlichen Wirtspflanzen hergestellt werden können.
In dieser Arbeit wird am Beispiel des Cowpea Chlorotic Mottle Virus (CCMV) gezeigt, wie diese Nanomaterialien effizient hergestellt und nutzbar gemacht werden können.
Im ersten Kapitel wurde eine Affinitätsextraktion basierend auf einem neuen Peptid-Aptamer entwickelt, um das CCMV selektiv aus dem Extrakt der infizierten Pflanze zu isolieren. Das Reinigungsprotokoll wurde validiert und eine finale Reinheit von 98,4% mittels Umkehrphasen-HPLC bestimmt.
Im zweiten Kapitel wurden CCMV-VLPs durch rekombinante Expression und anschließende in vitro Assemblierung der Kapsidproteine hergestellt. Die Monodispersität der VLPs wurde durch Transmissionselektronenmikroskopie (TEM) bestätigt. Zusätzlich wurde eine Mutante der CCMV-VLPs hergestellt, die physiologischen Bedingungen standhält und für den Einsatz in vivo geeignet ist.
Im dritten Kapitel wurden CCMV und dessen VLPs für nanotechnologische Anwendungen genutzt. Gold-Nanopartikel wurden in CCMV-VLPs verkapselt und im TEM sichtbar gemacht. Das CCMV-Kapsid wurde mit einem Modellpeptid funktionalisiert, um das Potenzial dieses Nanomaterials als chemische Plattform zu demonstrieren. / The protein shell of plant viruses consists of protein subunits that encapsulate and transport the viral genome. The underlying self-assembly mechanism can be utilized for nanotechnological applications by encapsulating foreign molecules within the viruses. These so-called virus-like particles (VLPs) can be used for drug delivery in cancer therapy, gene therapy, or as contrast agents in imaging techniques. Additionally, the surface of the protein shell can be functionalized chemically or via molecular biology techniques. A significant advantage of plant viruses is that they pose no threat to humans and can be easily produced in natural host plants.
This work demonstrates how these nanomaterials can be efficiently produced and utilized using the example of Cowpea Chlorotic Mottle Virus (CCMV).
In the first chapter, an affinity extraction based on a novel peptide aptamer was developed to selectively isolate CCMV from the extract of the infected plant. The purification protocol was validated, achieving a final purity of 98.4% using reverse-phase HPLC.
In the second chapter, CCMV-VLPs were produced through recombinant expression and subsequent in vitro assembly of the capsid proteins. The monodispersity of the VLPs was confirmed by transmission electron microscopy (TEM). Additionally, a mutant of the CCMV-VLPs was produced that withstands physiological conditions and is suitable for in vivo applications.
In the third chapter, CCMV and its VLPs were used for nanotechnological applications. Gold nanoparticles were encapsulated within CCMV-VLPs and visualized using TEM. The CCMV capsid was also functionalized with a model peptide to demonstrate the potential of this nanomaterial as a chemical platform.
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Emergence et adaptation du Rice yellow mottle virus : relations entre histoire évolutive, contournement de résistance et interactions hôte/pathogène / Emergence and adaptation of Rice yellow mottle virus : relationships between evolutionary history, resistance-breakdown and host/pathogen interactionsPoulicard, Nils 03 December 2010 (has links)
Le Rice yellow mottle virus (RYMV) est un virus émergeant qui constitue actuellement une contrainte majeure à la riziculture sur le continent africain. Quelques rares variétés de riz, issues des espèces cultivées de riz africain et asiatique (respectivement Oryza glaberrima et O. sativa), ont récemment été identifiées comme hautement résistantes au RYMV. Ce phénotype de résistance est dû à un gène récessif RYMV1 codant le facteur d'initiation de la traduction eIF(iso)4G1 du riz.Les objectifs de cette thèse sont (i) d'étudier la durabilité de la résistance élevée du riz contre le RYMV avant son déploiement à large échelle, (ii) de caractériser les mécanismes d'émergence de génotypes contournants et (iii) d'identifier des signatures moléculaires influençant ces processus d'adaptation. Ainsi, le contournement de deux allèles de résistance, identifiés chez les deux espèces de riz cultivés, a été relié à l'émergence de mutations dans la protéine virale VPg qui permettent de rétablir l'interaction avec le facteur eIF(iso)4G1 de l'hôte résistant. Un site sous sélection diversificatrice de la VPg influence directement la capacité de contournement de la résistance élevée en fonction de l'espèce hôte. Ce site, proche des mutations de contournement, est impliqué dans l'adaptation du virus à l'espèce O. glaberrima au cours de son histoire évolutive. La démarche employée au cours de ce travail combine des études d'évolution expérimentale à des analyses fonctionnelles. Les résultats obtenus par cette approche intégrative participeront à la mise en place de stratégies de lutte intégrée à la fois efficaces et durables face à la maladie de la panachure jaune du riz en Afrique. / The Rice yellow mottle virus (RYMV) is an emerging virus currently considered as the major constraint to rice production in Africa. Some varieties of African and Asian cultivated rice (Oryza glaberrima and O. sativa, respectively), have recently been identified as highly resistant to RYMV. This resistance phenotype is caused by a recessive gene RYMV1 encoding the translation initiation factor eIF(iso)4G1 of rice.The objectives of this thesis are (i) to investigate the durability of the high resistance of rice against RYMV before broadly deployment in fields, (ii) to characterize the mechanisms of emergence of resistance-breaking (RB) genotypes and (iii) to identify molecular signatures that influence these processes of adaptation. The resistance-breaking of two resistance alleles, identified in both cultivated rice species, is mainly associated with the emergence of mutations in the viral protein VPg that restore in resistant hosts the interaction with the factor eIF(iso)4G1. A site of VPg under diversifying selection directly affects the ability to overcome the high resistance depending on the host species. This site, near the RB mutations, is involved in the adaptation of the RYMV to O. glaberrima species during its evolutionary history. The approach used during this work combines experimental evolution and functional analyses. The results of this integrative study will participate in the development of effective and sustainable control strategies toward the Rice yellow mottle virus in Africa.
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Genomic Characterization of the Cacao Swollen Shoot Virus Complex and other Theobroma Cacao-Infecting BadnavirusesChingandu, Nomatter, Chingandu, Nomatter January 2016 (has links)
The cacao swollen shoot disease of Theobroma cacao L. (cacao) is caused by Cacao swollen shoot virus (CSSV; genus, Badnavirus, family, Caulimoviridae). The virus is endemic to West Africa, where it poses a serious threat to cocoa production. Despite efforts to control CSSV spread by replacement of infected trees with tolerant cultivars and mealybug vector management, the disease is widespread in West Africa. In Trinidad, leaf mosaic and vein-banding symptoms have been observed in cacao plants in the field since the 1940s, and recently at the International Cocoa Genebank (ICGT), a custodian of cacao germplasm resources. The strains A and B of the suspect Cacao Trinidad virus (CTV) caused the symptoms, and were thought to be related to CSSV, however, viral causality was not demonstrated, until now. To develop molecular detection methods for CSSV in infected plants, polymerase chain reaction (PCR) amplification of eight regions of the CSSV genome was implemented. The PCR results showed variable amplification frequencies of 19 - 42% at each region, for 124 isolates collected in Cote d'Ivoire and Ghana. Pairwise nucleotide (nt) analyses of the eight regions showed 66-99% shared identities, indicating that CSSV isolates exhibit extensive variability with respect to primer design. The results provided preliminary evidence for the existence of a CSSV complex consisting of four divergent species. The full length genome of 14 CSSV isolates from cacao determined using the Illumina HiSeq platform showed 70-99% shared nt identities. The pairwise nt identities placed CSSV sequences into a group of four distinct species, one of which represented a previously undescribed species. Moreover, the full-length genomes grouped phylogenetically with other badnaviruses and revealed two CSSV subclades with three types of genome arrangements; four, five or six open reading frames (ORFs). Predicted functional protein domains were conserved on each ORF. Two distinct, full-length genome sequences were determined using the Illumina HiSeq platform, from DNA isolated from cacao leaves exhibiting distinct symptoms in Trinidad. The sequences were validated by PCR-amplification and sequencing of overlapping viral genome fragments. Pairwise nt analysis indicated that each genome shared 52-62% nt identities with CSSV and other badnaviruses, suggesting that the two are distinct species. Phylogenetic analysis indicated that the two sequences are not strains of the same virus, as supposed, but they represent two previously undescribed species in the genus, Badnavirus, and they have been named Cacao mild mosaic virus (CaMMV) and Cacao yellow-vein-banding virus (CYVBV). Despite sharing the same host and causing similar symptoms in cacao, CSSV, CaMMV, and CYVBV are phylogenetically-distinct species. The discovery of a CSSV species complex and the identification of three new cacao-infecting badnavirus species will support the development of molecular detection tools using the partial and complete genome sequences determined in this study. The ability to develop validated molecular tools for the detection of CSSV and related viruses, CaMMV and CYVBV, in cacao will aid quarantine efforts and safe movement of germplasm from the ICGT in Trinidad to cacao-growing countries, worldwide. Also, molecular diagnostics tools are expected to be useful in efforts underway to develop CSSV-resistant planting material for countries in West Africa, which are currently experiencing continued or new disease outbreaks.
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Molecular Characterization Of Movement Protein Encoded By ORF-1 Of Sesbania Mosaic Virus (SeMV)Chowdhury, Soumya Roy 01 1900 (has links) (PDF)
No description available.
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Identification of viral-based replicating vectors suitable for the development of a sugarcane bioreactorPirlo, Steven Dominic January 2007 (has links)
The circular, single-stranded (ss) DNA genomes of plant viruses in the families Geminiviridae and Nanoviridae are replicated within the nucleus of a host cell by a mechanism called rolling circle replication (RCR). Although this process relies almost exclusively on the replication machinery of the host cell, initiation occurs via the interaction of the viral replication initiation protein (Rep) with regulatory DNA sequences within the viral genome. The use of a virus-based episomal amplification technology as a plant bioreactor platform exploits the process of Rep-mediated RCR for the high-level amplification of virus-based episomes in plants and subsequent expression of heterologous proteins; such an approach offers advantages over existing gene expression technologies. This PhD thesis describes research towards the development of a virus-based episomal amplification system for use in sugarcane. Such a crop is ideally suited for a plant bioreactor system due to the efficient high-level production of plant biomass and the existence of established production, harvesting and processing infrastructure. In order to rapidly assess the potential of a virus-based episomal amplification system in sugarcane, a transient assay system was established. Sugarcane callus was identified as the most suitable cell preparation; providing rapid cell regeneration, uniform experimental samples and upon isolation, total DNA suitable for Southern analysis. This assay system once established, proved effective in rapidly identifying virus-based episomes capable of undergoing RCR within sugarcane host cells. This transient assay system was then used to test the functionality of a virus-based episomal amplification system based on the ssDNA virus, Banana bunchy top virus (BBTV) in sugarcane. BBTV-based episomal amplification vectors were constructed with a reporter gene expression cassette flanked by two copies of the BBTV regulatory DNA sequences. The episomal amplification vectors were bombarded into sugarcane and banana host cells in various combinations and evidence of RCR was assessed through Southern blot analysis. RCR products were identified in banana host cells bombarded with the BBTV-based episomal amplification vectors in combination with vectors encoding BBTV Master-Rep (M Rep). RCR products were not identified within sugarcane cells bombarded with the same construct combinations. Integrated InPAct (In Plant Activation) episomal vectors based on BBTV were then employed to confirm the transient results, in addition, the functionality of an InPAct vector based on an alternate virus, Tobacco yellow dwarf virus (TYDV) was also assessed. InPAct vectors based on BBTV were constructed with an untranslatable expression cassette for integration within the sugarcane genome. Transient experiments were performed to assess the ability of BBTV M-Rep and TYDV Rep to initiate RCR of their respective InPAct vectors. Visual observation of GFP expression indicated that BBTV M-Rep was capable of initiating RCR of the BBTVbased InPAct vectors within banana host cells but no evidence was observed in sugarcane host cells. TYDV Rep was capable of initiating RCR of the TYDV-based InPAct vector within sugarcane host cells with a 100-fold increase in the number of fluorescent foci compared to cells bombarded with the TYDV InPAct vector alone. The BBTV-based InPAct vector was stably integrated within the sugarcane genome and the ability for BBTV M-Rep to initiate episome formation and RCR was assessed by Southern blot analysis. Evidence of BBTV M-Rep mediated RCR was not detected within the transgenic sugarcane bombarded with BBTV M-Rep. Transgenic sugarcane containing the TYDV-based InPAct vectors was assessed for the ability to be activated by TYDV Rep and undergo RCR. Southern blot analysis demonstrated that TYDV Rep was capable of recognising the integrated TYDVbased InPAct vector and RCR was detected within the transgenic sugarcane. The observation that episomal vectors based on TYDV were functional within sugarcane host cells and BBTV-based vectors were not, was unexpected. It had been hypothesised that an episomal vector based on a monocot-infecting virus would replicate in an alternate monocot host, while an episomal vector based on a dicot infecting virus would not. Virus replication is thought to be host-specific however most host range studies have been conducted with full length infectious clones and not deconstructed virus-based episomes. The implication that viral Reps may be functional in plant cells of non-host species was then investigated. The ability for viral Reps to recognise their cognate IR and initiate RCR of virus-based episomes in different host cells was assessed through cross-replication experiments. Four ssDNA plant viruses; BBTV, TYDV, Chloris striate mosaic virus (CSMV) and Tomato leaf curl virus - Australia (ToLCV-Au) were assessed via Southern blot analysis for their ability to initiate both autonomous replication of infectious clones and episomal amplification within three different plant hosts; tobacco, sugarcane and banana. Results from cross replication studies indicated a complex interaction between viral and host replication components. BBTV infectious clones and episomal vectors were restricted to replication within banana host cells providing a clear indication that episomal amplification vectors based on BBTV are restricted to Musa spp. BBTV M-Rep was unable to recognise the viral regulatory DNA sequences of the other three ssDNA viruses. TYDV infectious clones and episomal vectors were capable of replicating within all three host cells tested, indicating that TYDV is capable of undergoing RCR within a broad range of plant hosts. TYDV Rep was also capable of recognising the viral regulatory DNA sequences of both CSMV and BBTV given favourable conditions within specific plant hosts. Replication of the CSMV infectious clone was not detected in any of the three host cells, although fidelity of this clone requires further confirmation. CSMV episomal vectors were functional within banana host cells only, indicating that although closely related to TYDV, episomal amplification vectors based on CSMV have a restricted host range. CSMV Rep could not initiate RCR of episomal amplification vectors containing the viral regulatory DNA regions of the other three viruses in any of the plant host cells. ToLCV-Au infectious clones were capable of replicating within banana and tobacco host cells. Episomal amplification vectors based on ToLCV-Au extended the host range to sugarcane. ToLCV-Au Rep was unable to recognise the viral regulatory DNA sequences of the other three viruses in any of the plant host cells. The ability for a viral Rep to recognise its own cognate regulatory DNA sequences within alternate plant host cells is variable. Episomal amplification vectors based on TYDV and ToLCV-Au appear to be the most suitable for the further development of a virusbased bioreactor system in sugarcane. This study details the initial steps taken towards the development of a virus-based episomal amplification system in sugarcane. In doing so, fundamental knowledge into the mechanisms involved in Rep recognition of viral regulatory DNA sequences has been gathered. These research findings will provide a solid foundation for the further development of a sugarcane-based bioreactor.
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L'infection d'Arabidopsis thaliana par le virus de la mosaique du colza (ORMV) : analyse du transcriptome et étude spécifique de CIPK12 et CDC48 / Oilseed rape mosaic virus infection in Arabinopsis thaliana : RNA profiling and specific analysis of CIPK12 and CDC48Gereige, Dalya 11 May 2012 (has links)
L’infection virale compatible des plantes est une cause majeure des pertes de rendements des récoltes. Nousavons analysé, suite à une infection par les tobamovirus, les réponses de l’hôte au niveau de la régulation del’expression des gènes ainsi qu’au niveau des mécanismes moléculaires déterminant l'infection. Des plantesd'Arabidopsis infectées par le Virus de la mosaïque du colza (ORMV) ont été utilisées pour étudier leschangements d’expression des gènes induits par le virus. Conformément à l'expression d'un suppresseur desilencing par le virus, les forts changements de populations d’ARNs ne corrèlent pas d’une façon significativeavec les changements de taux d’ARNm cibles. En se concentrant sur les altérations des processusphysiologiques, deux gènes potentiellement impliqués dans les réponses à l'infection virale ont étéfonctionnellement caractérisés. Le premier gène code pour CIPK12. L’ARNm issu de ce gène montre uneaccumulation suite à une infection par ORMV. De plus, l'expression transitoire de CIPK12 dans des feuilles deN. benthamiana semble inhiber la propagation de l’ORMV dans cet hôte. La deuxième protéine étudiée dansce travail est CDC48B qui s'accumule suite à une infection tobamovirale et agit dans la maintenance desmembranes du réticulum endoplasmique (RE). De plus, CDC48B interagit avec la MP virale qui s’accumuledans des inclusions associées aux membranes du RE en vue d’améliorer son extraction des membranes et salocalisation au niveau des microtubules et dans le cytosol. Ainsi, deux protéines induites par l’infection viraleavec un potentiel d'interagir avec la MP virale et d'influer sur son activité lors de l'infection, sont décrites danscette thèse. / Infection of crop plants with compatible viruses is a major cause of losses in harvest yield. Here, we analyzedhost responses to tobamovirus infection at the level of gene regulation and expression, and at the level ofmolecular mechanisms determining the outcome of infection. ORMV-infected Arabidopsis plants were used toaddress virus-induced changes in gene expression. Consistent with the expression of a silencing suppressor bythe virus, strong changes in sRNA populations in virus-infected plants are not significantly correlated withcorresponding changes in the levels of the corresponding mRNA targets. Focusing on altered physiologicalprocesses, two genes with potential involvement in responses to virus infection were functionallycharacterized. The first gene encodes CIPK12, a (CBL)-interacting protein kinase, involved in decodingcalcium signatures. The mRNA of this gene accumulates upon ORMV infection and further observationsprovide evidence indicating that the regulation of the CIPK12 mRNA level occurs at the post-transcriptionallevel. Transient expression of CIPK12 in N. benthamiana leaves appears to inhibit the cell-to-cell spread ofORMV in this host. The second protein addressed in this work is CDC48B. CDC48B accumulates upontobamoviral infection and functions in ER-membrane maintenance. Consistent with this function it interactswith virus-accumulated MP in ER-associated inclusions to promote its extraction form the membrane and itslocalization to the cytosol. Thus, in this thesis I describe two virus-induced proteins with potential to interactwith viral MP and to influence the subcellular localization of this protein during infection.
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Apport des récentes évolutions de la cryo-microscopie électronique et du traitement d’images dans l’étude structurale de virus de plantes / Contribution of latest developments in cryo-electron microscopy and image processing for the structural study of plant virusesLecorre, François 14 December 2016 (has links)
La cryo-microscopie électronique a connu une révolution majeure ces dernières années, liée à des évolutions technologiques importantes, tant au niveau des microscopes, que des caméras ou des logiciels de traitement d’images. Ainsi, avec l’arrivée de microscopes électroniques plus stables mécaniquement et électroniquement, il est possible d’enregistrer plusieurs milliers d’images de façon automatique en quelques jours, et par conséquent d’obtenir un jeu initial d’images conséquent des particules des complexes protéiques étudiés. Sauf que maintenant, il ne s’agit plus d’image au sens strict, mais de film. En effet, les nouvelles caméras, dites à détection directe d’électrons, permettent de décomposer l’image en plusieurs fractions d’images au cours de l’exposition, et ce, avec une sensibilité dix fois supérieure par rapport à celle des anciennes caméras. L’analyse de ces fractions d’images a permis de montrer que les particules protéiques, bien que piégées dans une mince pellicule de glace, bougeaient sous l’effet du faisceau d’électrons. L’alignement des fractions et leur sommation permettent ainsi de corriger ces mouvements, améliorant la qualité du signal contenu dans chaque image. Ainsi, alors que pendant des dizaines d’années, les informations structurales extraites des images des microscopes électroniques étaient limitées à la moyenne résolution, c’est à dire entre 5 et 15 Å de résolution, nous voyons apparaître depuis ces deux-trois dernières années, de très nombreuses cartes de densités électroniques de complexes protéiques issues de la microscopie électronique, à des résolutions inférieures à 4 Å, permettant de construire des modèles atomiques à l’aide d’outils jusqu’alors réservés à la cristallographie aux rayons X. C’est dans ce contexte, que j’ai étudié par cryo-microscopie électronique et traitement d’images, l’organisation structurale de trois virus de plante :- Le virus de la mosaïque de l’arabette (ArMV), un Nepovirus uniquement transmis par le nématode Xiphinema diversicaudatum, qui est responsable de la maladie du court-noué de la vigne. -Le virus de la tâche de la fève (BBSV), un Comovirus transmis par les coléoptères, responsable de la dégénération chez les légumineuses.- Le virus de la mosaïque du chou-fleur (CaMV), un Caulimovirus servant de virus modèle pour l’étude de la transmission des virus non circulants.Les virus sont des parasites endocellulaires obligatoires, dont l’efficacité dépend de leur capacité de réplication au sein de la cellule infectée et de leur transmission vers de nouveaux hôtes. En raison de l’immobilité des plantes, les phytovirus font souvent appel à des vecteurs pour la transmission plante à plante, qui sont principalement des insectes, des nématodes, des champignons ou des acariens. Les phytovirus sont généralement responsables d’une baisse importante de croissance de la plante et des fruits, voire de la mort de l’hôte infecté. Les dégâts ainsi causés engendrent des pertes de rendement dans les cultures partout dans le monde, se traduisant par d’énormes pertes économiques pour les cultivateurs. Ce travail de thèse présente les structures atomiques de l’ArMV et du BBSV obtenues par cryo-microscopie électronique, ainsi que les premiers résultats obtenus sur la structure de la capside du CaMV, et sa protéine de transmission P2. / A revolution has taken over the world of cryo-electron microscopy for the last years, by dint of a major breakthrough both in technology, with the rise of new microscopes and cameras, and in image processing. With the advent of high-end microscopes, mechanically and electronically more stable, one can expect to record an initial data set of thousand images in few days, thanks to automated acquisition. Besides, the new direct electron detectors can not only record images, but also movies with a better sensitivity than the one we used to have. The movie processing revealed the existence of a beam-induced motion occurring during acquisition. The correction of the motion through frame alignment improves significantly the quality of data. Thus, cryo-electron microscopy was only limited to a middle resolution range (5 to 15 Å) until two or three years ago, when several density maps above 4 Å started to appear, allowing the building of atomic model using tools that were only restricted to X-ray crystallography.In this context, I have studied the structural organization of three plant viruses, using cryo-electron microscopy and image processing:- Arabis Mosaic Virus (ArMV), it’s a Nepovirus only transmitted by the nematode Xiphinema diversicaudatum, responsible for disease of vineyards.- Broad Bean Stain Virus (BBSV), it’s a Comovirus transmitted by beetles, responsible for the degeneration of leguminous plants.- Cauliflower Mosaic Virus (CaMV), it’s a Caulimovirus used as model to characterize the transmission of non circulative viruses.Viruses are obligate intracellular parasites, which efficiency is directly related to its replicative capacity inside the infected cell, and its transmission to new hosts. Due to the immobility of plants, plant viruses often use vectors for the transmission plant to plant, which are mainly insects, nematodes, fungi or mites. Plant viruses are generally responsible for a significant decrease in plant and fruit growth, and even the death of the plant. The plant viruses are devasting fields worldwide, causing huge loss in crop yield each year. This study highlights the atomic structures of ArMV and BBSV, as well as the first data about the CaMV capsid and its transmission protein.
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Plant virus identification and virus-vector-host interactionsGaafar, Yahya Zakaria Abdou 08 November 2019 (has links)
No description available.
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