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1
Payne, Richard. "Gene discovery in Catharanthus roseus using virus induced gene silencing". Thesis, University of East Anglia, 2015. https://ueaeprints.uea.ac.uk/59379/.
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This thesis presents the use of Virus Induced Gene Silencing (VIGS) for the discovery of enzymes and transporters involved in monoterpene indole alkaloid (MIA) metabolism in the medicinal plant Catharanthus roseus. C. roseus is the source of a number of MIAs that are used as chemotherapeutic agents in the treatment of a variety of cancers, however the complete biosynthetic pathway for these metabolites remains to be elucidated. Additionally, this metabolic pathway is subcellulary compartmented with the key branch point enzyme, strictosidine synthase, localised to the plant vacuole. There is therefore a need for the import of the substrates for strictosidine biosynthesis; secologanin and tryptamine, across the vacuolar membrane, and export of the product, strictosidine, for synthesis of the downstream alkaloids. This thesis presents the identification of two proteins that act as trans-tonoplastic transporters in MIA metabolism. The multidrug and toxic compound extrusion (MATE) protein, CrMATE1952, was localised to the vacuolar membrane and silencing its expression in planta resulted in the accumulation of a secologanin derivative. This implicates CrMATE1952 in the transport of secologanin into the vacuole and highlights the importance of the spatial organisation of the pathway in preventing secologanin derivatisation. Secondly silencing the expression of a tonoplast localised nitrate/peptide (NPF) transporter, CrNPF2.9, resulted in the 20-fold accumulation of strictosidine, suggesting this transporter is the exporter of strictosidine from the vacuole. Furthermore, VIGS also allowed the identification of a reticuline oxidase like protein, CrRO, which resulted in the accumulation of two new MIAs in leaf tissue upon silencing. This thesis highlights a reverse genetics strategy for gene identification in metabolic pathways and is the first time the MATE and NPF transporters, and the reticuline oxidase like enzymes, have been shown to be involved in MIA metabolism in C. roseus.
2
George, Gavin M. (Gavin Mager). "Virus induced gene silencing for the study of starch metabolism". Thesis, Stellenbosch : University of Stellenbosch, 2010. http://hdl.handle.net/10019.1/4024.
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Thesis (PhD (Plant Biotechnology))--University of Stellenbosch, 2010.ENGLISH ABSTRACT: Virus Induced Gene Silencing (VIGS) was optimized to allow for the study of starch metabolism. The plastidial inorganic pyrophosphatase gene, for which a mutant has never been identified, was studied using VIGS and it was found to have a broad role in this subcellular compartment. The accumulation of inorganic pyrophosphate limited the production of starch, carotenoids, chlorophyll, and increased the plants susceptibility to drought stress. These effects highlight the importance of this enzyme in maintaining a low intraplastidial concentration of PPi providing an environment which facilitates these anabolic processes. Several genes involved in starch synthesis and degradation were also targeted with the aim of establishing a system of multiple gene silencing for the study of metabolic pathways. One, two and three genes were successfully silenced using this system which was validated based on previously published data. Interestingly, simultaneous silencing of the two isoforms of disproportionating enzyme led to a novel phenotype as a large reduction in starch instead of the expected increase was observed.
No Afrikaans abstract available
3
Starkus, Laura. "Virus-induced gene silencing of putative Diuraphis noxia (Kurdjumov) resistance genes in wheat". Thesis, Manhattan, Kan. : Kansas State University, 2010. http://hdl.handle.net/2097/4193.
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4
Jaggard, Daniel Andrew William. "The structure and function of RPW8.1 and RPW8.2, powdery mildew disease resistance proteins from Arabidopsis thaliana (L.) Heyhn". Thesis, University of East Anglia, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.251898.
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5
Kawai, Takashi. "Virus-induced gene silencing in Prunus fruit and nut tree species by Apple latent spherical virus vector". Kyoto University, 2017. http://hdl.handle.net/2433/217995.
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Kyoto University (京都大学)0048
新制・論文博士
博士(農学)
乙第13073号
論農博第2843号
新制||農||1046(附属図書館)
学位論文||H29||N5029(農学部図書室)
33224
京都大学大学院農学研究科農学専攻
(主査)教授 北島 宣, 教授 土井 元章, 教授 田尾 龍太郎
学位規則第4条第2項該当
6
Peart, Jack Robert. "The use of virus-induced gene silencing to identify genes required for N-mediated resistance against tobacco mosaic virus". Thesis, University of East Anglia, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.247101.
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Demircan, Turan. "Application Of Virus Induced Gene Silencing Of Brachypodium Distachyon, A Model Organism For Crops". Master's thesis, METU, 2009. http://etd.lib.metu.edu.tr/upload/12610649/index.pdf.
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Grass family is most important family in plant kingdom due to intensive usage of crops in agriculture. To date, molecular biology researches on grass family have had limitations because of inappropriate characteristics of barley and wheat to conduct experiments on them. Brachypodium distachyon that belongs to grass family has recently emerged as a model organism for crops. It shares common characteristics for a model plant due to its small genome, small physical plant size, a short lifecycle, and less demanding growth requirementsas other model organisms
Arabidopsis thaliana, Oryza sativa, and Zea mays (Draper et al. 2001). Especially after appreciating, the genetic distance of O. sativa to grasses (Garvin et al. 2008), it become a key organism to understand complicated genomic organization of agriculturally valuable grasses. Virus-induced gene silencing (VIGS) is one of the revolutionary methods allowing a rapid and effective loss of a gene function through RNA interference (Holzberg et al. 2002
Liu et al. 2008). Barley stripe mosaic virus (BSMV) is still the most effective vector used in monocot gene silencing. It has a tripartite RNA genome having a wide range of infection ability for monocots including barley, oat, wheat, and maize as host (Holzberg et al. 2002
Scofield 2005). In this thesis, Phytoene desaturase (PDS) gene of Brachypodium distachyon was silenced via BSMV mediated VIGS. Additionally, with Green fluorescence protein (GFP) bearing BSMV transcripts, GFP expression was observed under fluorescent microscope. To our knowledge, this is the first report demonstrating a VIGS via BSMV in Brachypodium distachyon. The success of virus induced gene silencing method in Brachypodium distachyon, will be a new convenient tool for evaluating functions of crop genes in this model organism.
8
Lu, Rui. "High throughput virus induced gene silencing for the analysis of disease resistance in plants". Thesis, University of East Anglia, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.398556.
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9
Lee, Jack Alexander. "The use of virus induced gene silencing to investigate Septoria leaf blotch in wheat". Thesis, Durham University, 2016. http://etheses.dur.ac.uk/11465/.
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Septoria leaf blotch, caused by the fungal pathogen Zymoseptoria tritici, is one of the most damaging diseases of wheat (Triticum aestivum), a crop plant of significant worldwide importance. Using the system of Virus-Induced Gene Silencing, to create transient knockdowns of target genes, a novel wheat gene, TaR1, was identified as playing a key role in the host response to this pathogen. Silencing this gene leads to the earlier onset of disease symptoms, but reduced reproduction of the causal pathogen. Sequence analysis, confocal microscopy and protein-protein interaction assays were used to determine that the protein TaR1 localises the nucleus, where its function involves the binding of histones. Precisely, TaR1 is able to bind the Histone 3 subunit, specifically methylated on Lysine 4. Through this action, the host defence response is delayed, and successful pathogen colonisation is promoted. It is hypothesised that this is an example of the pathogen ‘hi-jacking’ TaR1 from its original function, in order to complete its lifecycle.
10
Bozhanaj, Kreshnik. "The Effect Of Virus Induced Gene Silencing Of Fas Associated Factor1 In Blumeria Graminis Infected Barley". Master's thesis, METU, 2009. http://etd.lib.metu.edu.tr/upload/12611139/index.pdf.
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Cereal loss due to fungal pathogens is an ongoing setback in agriculture. Elucidating plant&rsquos resistance and susceptibility mechanisms against these cereal killers, promises progress in agriculture. In the way of understanding barley resistance against fungus Blumeria Graminis we silenced FAS-Associated Factor 1 (FAF1) gene in its mRNA level with Virus Induced Gene Silencing (VIGS) technique. Previous research in our lab had shown an augmentation in mRNA levels of FAF1 gene in fungus infected wheat, suggesting a role of this gene in the resistance mechanism. We hypothesized that the apoptotic role of FAF1 protein in metazoan is conserved in plants by including FAF1 as a factor in hypersensitive response. Barley lines Pallas01 and Pallas03 which are respectively resistant and susceptible against fungus Blumeria graminis hordei 103 (Bgh103) were used for fungal inoculations after FAF1 silencing, to test if the hypersensitive response against fungus Bgh103 was prevented. In this aspect the formation of death lesions on the Pallas01 leaf due to fungal resistance was not prevented demonstrating that FAF1 silencing with VIGS in the resistant Pallas01 line of barley is not sufficient to stop apoptosis. On the other hand the FAF1-silenced barley susceptible line Pallas03 became more sensitive to fungal stress based on conidia (body part of the fungus) counting after trypan blue staining of the infected leaves. In the C-terminus of FAF1 an ubiquitin like domain-X (UBX) is found, which is the cause of stress sensitivity based on the reported data obtained about this domain&rsquo
s loss of function in other proteins. These results suggest that FAF1 is a catalyst in the hypersensitive response and its loss of function makes barley more susceptible to fungal stress. On the other hand a short mRNA homology was found among FAF1 and many pathogen disease related proteins making this homology a possible target site for VIGS of FAF1 generated siRNAs, which might cause some other protein to be responsible for the barley susceptibility against the fungus.
11
Kim, Joonseog. "EFFECTS OF SILENCING CYC2-LIKE GENES ON FLORAL DEVELOPMENT IN SOLANUM LYCOPERSICUM L. AND NICOTIANA OBTUSIFOLIA M. MARTENS & GALEOTTI (SOLANACEAE)". VCU Scholars Compass, 2017. http://scholarscompass.vcu.edu/etd/5014.
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CYCLOIDEA (CYC) and DICHOTOMA (DICH) of the CYC2 clade of the TCP gene family have been shown to play a significant role in regulating the identity of the dorsal petals and abortion of the single dorsal stamen in Antirrhinum majus. It is believed that CYC2-like genes are responsible for the convergent evolution of floral zygomorphy, but their role in the development of actinomorphic flowers is still unknown. In Solanaceae, previous analysis has identified two paralogs of CYC2-like genes, CYC2A and CYC2B, resulting from a gene duplication that predates the origin the family. Virus-induced gene silencing (VIGS) is a technique to study the gene function by silencing specific target genes of interest, which is shown to be useful in diverse plant species. Here, we report on the role of CYC2-like genes during floral development in Solanaceae based on the results of VIGS using tobacco rattle virus (TRV)-based vector in Solanum lycopersicum having completely actinomorphic flowers and Nicotiana obtusifolia having slightly zygomorphic flowers. Our VIGS experiments in So. lycopersicum show that downregulation of both CYC2A and CYC2B leads to misshaped petals, the unequal growth of the petals, and most frequently increased number of petals, stamens and sepals, while the carpel and ovule morphology remain the same as the wild type. On the contrary, downregulation of CYC2A and CYC2B in N. obtusifolia results in reduced number of flower organs in sepals, stamens, and petals, however carpels remained the same. For both solanaceous species, silencing CYC2A and CYC2B changes the property of cytoplasm and retards the rate of pollen germination. Our findings suggest that the CYC2-like genes are likely involved in the floral development, mainly regulating the number of floral organs and pollen development in Solanaceae.
12
Lim, Gaik Wee Heidi Clinical School St Vincent's Hospital Faculty of Medicine UNSW. "Promoter targeted siRNAs induce transcriptional gene silencing (TGS) in Simian Immunodeficiency Virus (SIV)". Awarded By:University of New South Wales. Clinical School - St Vincent's Hospital, 2009. http://handle.unsw.edu.au/1959.4/44889.
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RNA interference is a phenomenon by which double-stranded RNA is processed into small interfering RNAs (siRNAs) that can cause gene silencing in plants, yeast, Drosophila (fruit fly) and human cells. Small interfering double stranded RNAs (siRNAs) can induce gene silencing via two pathways: post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS). PTGS involves siRNA triggering of sequence- specific degradation of mRNA takes place in the cell's cytoplasm. In contrast, siRNAs, which induce TGS have sequence complementary to regions within a gene's promoter and the action happens in the nucleus and is associated with de novo methylation of CpG sites and histone methylation within the promoter region. Suziki et al. utilised siRNAs to target regions within 5'-long-terminal repeat (5'LTR) promoter region of human immunodeficiency virus-1 (HIV-1) and subsequently induce silencing of HIV-1 in HeLa cells. Here I recapitulate the previous findings in HIV-1 by showing that certain promoter-targeted siRNAs can also induce silencing of simian immunodeficiency virus (SIV) replication by inducing CpG methylation and epigenetic changes. A similar silencing effect was observed by using shRNA and miRNA mimics based on the most effective siRNAs. The shRNAs and miRNA mimics after conversion gained a PTGS effect in addition of TGS effect. The dual effect appeared to enhance the silencing efficiency. Here I show that the co-localisation of Ago1 and Ago2 with a TGS inducing siRNA (si2A) in nucleus and at the rim of the nucleus respectively in SIV infected cells but not in non-infected cells. The P-body protein, GW182 complexed with si2A was found in both the nucleus and nuclear envelope and was consistent with the location Ago1/si2A and Ago2/si2A complexes. This indicated a role for this protein in TGS together with Ago proteins. Using SIV 5'-LTR luciferase reporter cells, complexes containing Ago1/Ago2/si2A were showed to work in combination and provide the optimal suppression.
13
Mohammad, Hamza [Verfasser]. "Construction of a virus-induced gene silencing system based on Beet necrotic yellow vein virus (BNYVV) and Beet soil-borne mosaic virus (BSBMV) / Hamza Mohammad". Hannover : Technische Informationsbibliothek (TIB), 2018. http://d-nb.info/1152965557/34.
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14
Du, Preez Jacques. "The development and characterisation of grapevine virus-based expression vectors". Thesis, Stellenbosch : University of Stellenbosch, 2010. http://hdl.handle.net/10019.1/4003.
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Thesis (PhD (Genetics))--University of Stellenbosch, 2010.ENGLISH ABSTRACT: Grapevine (Vitis vinifera L.) is a very important agricultural commodity that needs to be protected. To achieve this several in vivo tools are needed for the study of this crop and the pathogens that infect it. Recently the grapevine genome has been sequenced and the next important step will be gene annotation and function using these in vivo tools. In this study the use of Grapevine virus A (GVA), genus Vitivirus, family Flexiviridae, as transient expression and VIGS vector for heterologous protein expression and functional genomics in Nicotiana benthamiana and V. vinifera were evaluated. Full-length genomic sequences of three South African variants of the virus (GTR1-1, GTG11-1 and GTR1-2) were generated and used in a molecular sequence comparison study. Results confirmed the separation of GVA variants into three groups, with group III (mild variants) being the most distantly related. It showed the high molecular heterogeneity of the virus and that ORF 2 was the most diverse. The GVA variants GTG11-1, GTR1-2 and GTR1-1 were placed in molecular groups I, II and III respectively. A collaboration study investigating the molecular divergence of GVA variants linked to Shiraz disease (SD), described two interesting GVA variants of group II, namely GTR1-2 and P163-M5 (Goszczynski et al., 2008). The group II variants were found to be closely linked to the expression of SD. GTR1-2 was isolated from a susceptible grapevine plant that never showed SD symptoms (Goszczynski 2007). The P163-M5 variant that resulted in exceedingly severe symptoms in N. benthamiana and is that used as SD positive control by the grapevine industry, was found to contain a 119 nt insert within the native ORF2. Comparative analysis performed on the complete nt and aa sequences of group II GVA variants suggested that the components in the GVA genome that cause pathogenicity in V. vinifera are more complex (or different) to those that cause pathogenicity in N. benthamiana. The three South African variants (GTR1-1, GTG11-1 and GTR1-2) were assembled into fulllength cDNA clones under control of CaMV 35S promoters. After several strategies were attempted, including a population cloning strategy for GTR1-2, none of the clones generated were able to replicate in N. benthamiana plants. A single amino acid substitution at position 13 (Tyr/Y Cys/C) in ORF 5 of the GTR1-2 cDNA clone was shown to abolish or reduce replication of the virus to below a detectable level. Two infectious clones of Israeli variants of GVA (T7-GVA-GR5 and T7-GVA118, obtained from M. Mawassi) were brought under control of a CaMV 35S promoter (35S-GVA-GR5 and 35S-GVA118). Both clones were infectious, able to replicate, move systemically and induce typical GVA symptoms after agroinfiltration in N. benthamiana. These Israeli clones served as backbone for further experiments in characterisation of transient expression and VIGS vectors. The use of GVA as gene insertion vector (35S-GVA118) and gene exchange vector (35S-GVA-GR5- ORF2+sgMP) in N. benthamiana and V. vinifera was compared. The gene insertion vector, 35S-GVA118 was based on the full-length GVA genome. The gene exchange vector, 35SGVA- GR5- ORF2+sgMP, was constructed in this study by elimination of ORF 2 and insertion of a sgMP and unique restriction sites to facilitate transgene insertion. In N. benthamiana both vectors showed similar GUS expression levels and photobleaching symptoms upon virus-induced NbPDS silencing. In V. vinifera limited GUS expression levels and VIGS photobleaching symptoms were observed for the gene insertion vector, 35SGVA118. No GUS expression was observed for the gene exchange vector 35S-GVA-GR5- ORF2+sgMP in this host. As for silencing, one plant, agroinfiltrated with 35S-GVA-GR5- ORF2-VvPDS+sgMP, developed photobleaching symptoms in 3 systemic infected leaves after 4 months. This study showed that GVA can be used as gene insertion and gene exchange vector for expression and VIGS in N. benthamiana, but in grapevine its use is limited to expression and silencing of genes in the phloem tissue. It is also the first report that ORF 2 of GVA is not needed for long distance movement in grapevine. To investigate the possible role of the P163-M5 119 nt insertion and the GVA ORF 2 (of unknown function), in expression of symptoms in plants, ORF 2 of a 35S-GVA-GR5 cDNA clone was removed and subsequently substituted by the corresponding ORFs of four South African GVA variants. Upon agro-infiltration into N. benthamiana leaves, all chimaeric GVA constructs were able to move systemically through the plant. At this stage no correlation could be found between severity of symptoms, the presence of the P163-M5 insert and the specific GVA ORF 2 present in the chimaeras, indicating that other factors in the viral genome or the host plant probably play a crucial role. This study contributed to the pool of available in vivo tools for study and improvement of the valuable grapevine crop. It also opened several exciting research avenues to pursue in the near future.
AFRIKAANSE OPSOMMING: Wingerd (Vitis vinifera L.) is ‘n baie belangrike landboukundige gewas wat beskerm moet word. Om die rede word verskeie in vivo gereedskap vir die bestudering van die wingerdplant, en die patogene wat dit infekteer benodig. Die wingerd genoom se volgorde is bepaal en dus is die volgende logiese stap om die gene te annoteer en funksie daaraan toe te skryf. In hierdie studie is die gebruik van Grapevine virus A (GVA), genus Vitivirus, familie Flexiviridae, as tydelike uitdrukking- en virus-geinduseerde geenuitdowingsvektor vir heteroloë proteïen uitdrukking en funksionele genoomstudies in Nicotiana benthamiana en V. Vinifera getoets. Vollengte genoomvolgordes van drie Suid-Afrikaanse variante van die virus (GTR1-1, GTG11-1 en GTR1-2) is gegenereer en in ‘n molekulêre volgorde vergelyking studie gebruik. Resultate het die verdeling van GVA variante in drie groepe, waar groep III die verste verwant is, bevestig. Dit het ook gewys dat die virus ‘n baie hoë molekulêre heterogeniteit het en dat oopleesraam 2 (ORF 2) die mees divers is. ‘n Samewerking studie waar die molekulêre diversiteit van GVA variante, gekoppel aan Shiraz siekte (SD), ondersoek is, is twee interessante variante van groep II beskryf, naamlik GTR1-2 en P163-M5 (Goszczynski et al., 2008). Groep II variante is vooraf gevind om nou verwant te wees aan die ontwikkeling van SD in wingerd. Die GTR1-2 variant is uit ’n vatbare wingerd plant, wat nooit SD-simptome vertoon het nie, geïsoleer (Goszczynski et al., 2007). In die ORF 2 van die P163-M5 variant, wat simptome van die ergste graad in N. benthamiana geïnduseer het, en ook deur die industrie as betroubare SD-positiewe kontrole gebruik word, is ’n 119 nt invoeging gevind. Die vergelykende analise wat uitgevoer is, het daarop gedui dat die determinante van patogenisiteit in die GVA genoom moontlik meer kompleks kan wees in V. vinifera as in N. benthamiana. Die drie Suid-Afrikaanse variante (GTR1-1, GTG11-1 en GTR1-2) is in afsonderlike vollengte cDNA klone, onder beheer van CaMV 35S promotors, aanmekaargesit. Nadat verskeie kloneringstrategieë, insluitend ’n populasie kloneringstrategie vir die GTR1-2 kloon, gebruik is, het geen een van die cDNA klone die vermoë besit om in N. benthamiana te repliseer nie. ’n Enkele aminosuur substitusie in posisie 13 (Tyr/Y Cys/C) in ORF 5 van die GTR1-2 kloon, het die replisering van die virus tot laer as ’n opspoorbare vlak verlaag. Twee infektiewe klone van Israeliese GVA variante (T7-GVAGR5 en T7-GVA118, verkry van M. Mawassi) is onder beheer van ‘n CaMV 35S promotor geplaas (35S-GVA-GR5 and 35S-GVA118). Beide klone het na agro-infiltrasie in N. benthamiana plante gerepliseer, sistemies beweeg en tipiese GVA simptome geinduseer. Hierdie twee klone het as raamwerk gedien vir verdere eksperimente in karakterisering van tydelike uitdrukkings- en VIGS vektore. Die gebruik van GVA as geen-insvoegingsvektor (35S-GVA118) en geen-vervangingsvektor (35S-GVA-GR5- ORF2+sgMP) is in N. benthamiana en V. vinifera vergelyk. Die geen-invoegingsvektor 35S-GVA118, was op die vollengte GVA genoom gebasseer. Die geen-vervangingsvektor 35S-GVA-GR5- ORF2+sgMP, was in hierdie studie gekonstrueer. Dit is gemaak eerstens deur eliminasie van ORF 2 in die 35S-GVA-GR5 kloon, en tweedens deur die invoeging van ’n subgenomiese promotor van die beweginsproteïen (sgMP) en unieke beperkings-ensiemsetels om klonering van transgene te fasiliteer. Beide vektore het in N. benthamiana vergelykbare GUS uitdrukkingsvlakke en fotobleikende simptome getoon na virus-geinduseerde NbPDS uitdowing. In V. Vinifera is beperkte GUS uitdrukkingsvlakke en VIGS fotobleikende simptome opgemerk met die geen-invoegingsvektor, 35S-GVA118. Geen GUS uitdrukking is in hierdie gasheerplant met die geen-vervangingsvektor opgemerk nie. Slegs een wingerdplant het fotobleikende simptome, na 4 maande in 3 sistemies geïnfekteerde blare gewys, na agroinfiltrasie van die 35S-GVA-GR5- ORF2-VvPDS+sgMP konstruk. Hierdie studie het bevestig dat GVA as geen-invoeging en geen-vervangingsvektor, vir heteroloë proteïenuitdrukking en VIGS, in N. benthamiana gebruik kan word, maar dit blyk of die gebruik daarvan in wingerd meer tot die floeëm weefsel beperk is. Hierdie studie wys vir die eerste keer dat ORF 2 nie nodig is vir langafstand beweging van die virus in wingerd nie. Om die moontlike rol van die P163-M5 119 nt invoeging en die GVA ORF 2 (met onbekende funksie), in die uitdrukking van simptome in plante te ondersoek, is ORF 2 van die 35SGVA- GR5 cDNA kloon verwyder en daaropvolgens vervang met die ooreenstemmende ORFs van vier Suid-Afrikaanse GVA variante. Na agro-infiltrasie in N. benthamiana blare, het al die chimeras die vermoë gehad om te repliseer, sistemies te beweeg en simptome te induseer. Geen korrelasie kon gevind word tussen die graad van simptome, die teenwoordigheid van die P163-M5 insersie en die spesifieke GVA ORF 2 teenwoordig in die chimeras nie, wat dus daarop dui dat ander faktore in die virusgenoom of die gasheerplant `n moontlike belangrike rol kan speel. Hierdie studie het bygedrae tot die beskikbare poel van in vivo gereedskap vir die bestudering en verbetering van die kosbare wingerdgewas. Dit het ook talle interessante navorsingsgeleenthede oopgemaak om in die nabye toekoms te betree.
15
Dagdas, Yasin Fatih. "Investigating The Roles Of Micrornas In Biotic Stress Responses And Functional Characterization Of A Novel Ztl-type F-box Protein Via Virus Induced Gene Silencing". Master's thesis, METU, 2009. http://etd.lib.metu.edu.tr/upload/12610641/index.pdf.
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Barley and wheat are the two most important crop species in Turkey. Molecular studies for increasing crop yield of these species are very important for the economic benefits of Turkey. Powdery mildew and yellow rust are the two main pathogens, infecting barley and wheat, respectively in our country and causing a great amount of yield loss each year. Till now, classical genetics studies were performed in order to develop resistant barley and wheat cultivars, but these studies have not been succesful. Therefore, molecular plant-pathogen interactions studies are starting to become the new tool to fight against pathogens. In this thesis, two important aspects of plant microbe interactions were investigated.
In the first part, the role of microRNAs (miRNAs) in powdery mildew-barley pathosysytem, and yellow rust-wheat pathosystem were studied. The expression levels of miRNAs and their putative targets were investigated via miRNA microarray analysis and qRT-PCR, respectively, in response to virulent and avirulent pathogen infections. These data were used to establish a new model for powdery mildew-barley and yellow rust-wheat pathosystems.
In the second part, functional analysis of a novel F-box gene, which was a ZTL-type F-box, was performed by using Barley Stripe Mosaic Virus mediated Virus Induced Gene Silencing. This F-box gene (HvDRF) (Hordeum vulgare Disease Related F-box) was induced upon yellow rust infection and we studied its role in powdery mildew infection. The results confirmed HvDRF as a positive regulator of race specific immunity and enlarged the roles of ZTL-type F-box proteins to biotic stress responses.
16
Ozturk, Ibrahim Kutay. "Elucidation Of The Role Of Gcn2 Gene In Response To Powdery Mildew Infection". Master's thesis, METU, 2012. http://etd.lib.metu.edu.tr/upload/12614563/index.pdf.
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Plant immune system is entirely based on the immunities of the individual cells in which systemic signals originate from the infection sites. Powdery mildew disease is one of the agents causing these infection sites, resulting in significant yield losses, if disease develops. Understanding the molecular basis of plant-pathogen interactions is the new trend for fighting against plant pathogens, since classical methods used in selection of resistant plants are becoming less and less efficient nowadays. Thus, finding out the genes which are responsible in plant&rsquos resistance is becoming very important. In this thesis, effect of &lsquo
General Control Nondepressible-2&rsquo
(GCN2) homolog protein in barley defense mechanism was aimed to be studied. The GCN2 of yeast was v previously identified in our laboratory as an interacting protein when the yeast cDNA library was screened with a putative yellow rust R gene (Yr10) fragment. There are reports available in the literature for the function of GCN2 protein, which makes it a good candidate for a role in disease resistance. Thus, the barley homologue of GCN2 might have a role in the R protein mediated early disease response of which may be proceeding via Programmed Cell Death (PCD). In order to observe such function of HvGCN2 in barley, silencing of its expression via Virus Induced Gene Silencing (VIGS) was investigated. Therefore, the GCN2 homologue was found to function as dampening the severity of the disease. The silencing with triple technical replicates was observed in 5 of the 6 samples, at an average of 43.2% by qRT-PCR analysis. The pathogen growth levels at different time points were analyzed under light microscope on the silenced and the control samples by measuring the primary and secondary hyphae lengths. The total of 24 seedlings and 292 individual spores were analyzed, and then the level of disease formation was quantitated with 603 primary hyphae and 106 secondary hyphae measurements. Up to 25% hyphae growth rate differences between the control and silenced groups were observed with a probability value less than 0.05 on t-test.
17
Krenz, Björn. "Gene Silencing und das Abutilon Mosaik Virus". [S.l. : s.n.], 2007. http://nbn-resolving.de/urn:nbn:de:bsz:93-opus-32318.
Texto completo
18
Thakare, Dhiraj, Jianwei Zhang, Rod A. Wing, Peter J. Cotty y Monica A. Schmidt. "Aflatoxin-free transgenic maize using host-induced gene silencing". AMER ASSOC ADVANCEMENT SCIENCE, 2017. http://hdl.handle.net/10150/623199.
Texto completoResumen
Aflatoxins, toxic secondary metabolites produced by some Aspergillus species, are a universal agricultural economic problem and a critical health issue. Despite decades of control efforts, aflatoxin contamination is responsible for a global loss of millions of tons of crops each year. We show that host-induced gene silencing is an effective method for eliminating this toxin in transgenic maize. We transformed maize plants with a kernel-specific RNA interference (RNAi) gene cassette targeting the aflC gene, which encodes an enzyme in the Aspergillus aflatoxin biosynthetic pathway. After pathogen infection, aflatoxin could not be detected in kernels from these RNAi transgenic maize plants, while toxin loads reached thousands of parts per billion in nontransgenic control kernels. A comparison of transcripts in developing aflatoxin-free transgenic kernels with those from nontransgenic kernels showed no significant differences between these two groups. These results demonstrate that small interfering RNA molecules can be used to silence aflatoxin biosynthesis in maize, providing an attractive and precise engineering strategy that could also be extended to other crops to improve food security.
19
Hunter, B. C. "Genetic modifiers of hairpin-induced gene silencing in Arabidopsis thaliana". Thesis, University of Cambridge, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.604807.
Texto completoResumen
The Chalcone Synthase (CHS) gene codes for the first step of biosynthesis of the purple pigment anthocyanin. In this study, transgenic Arabidopsis plants with a hairpin construct homologous to part of the CHS coding sequence were used in a genetic screen to identify modifiers of hairpin silencing. Some putative genetic modifiers were identified, but these appear to be the result of transgene:transgene interaction based on shared promoter homology rather than being due to second site suppressors in the Arabidopsis genome. New transgenic Arabidopsis lines were made with hairpin constructs directed to either the promoter or the reading frame of the CHS gene and both types of transgenic showed an anthocyanin-deficient phenotype. For each construct a representative single-insert homozygous transgenic line was selected for subsequent work. Both transgenic lines showed increased methylation of their respective target site and contained short interfering RNA (siRNA) homologous to their target site. A collection of 59 putative modifiers of CHS silencing were tested with both transgenic lines using segregation analysis of the anthocyanin-deficient phenotype in the F2 generation of appropriate crosses. Mutants in AGO4, AGO6, DRD1, DRM2, NRPD2a and NRPE1 act as recessive second site modifiers of the CHS promoter hairpin line phenotype. These mutants are associated with a decrease in DNA methylation at the hairpin target site. Mutant alleles of the DCL4 and HEN1 genes reduce silencing of the CHS coding sequence hairpin. The reduction in silencing is greater in mutants that are hemizygous for the hairpin construct than in those that are homozygous for it. The increase in siRNA in the construct homozygotes, compared to that in the hemizygotes, is perhaps sufficient to overcome any modification of the phenotype by mutants of DCL4 and HEN1.
20
Wang, Huan. "Investigation of RNA-induced gene silencing in the fission yeast Schizosaccharomyces pombe". Thesis, Queen Mary, University of London, 2007. http://qmro.qmul.ac.uk/xmlui/handle/123456789/1597.
Texto completoResumen
RNA interference (RNAi) is a gene regulating system to which can silence genes at both transcriptional and post-transcriptional levels. In this thesis, a ura4-based RNAi selective assay was developed in the fission yeast Schizosaccharomyces pombe: antisense & sense constructs of mutually inverted ura4 DNA fragments were inserted under the regulation of a thiamine-repressible promoter; when induced by omission of thiamine, these transcripts triggered S. pombe ura4 gene silencing at the molecular level with the efficiency of -30%. Although dozens of proteins involved in RNAi pathways has been identified from different organisms, only a few are negative regulators of RNAi. The helF protein from the soil amoeba, Dictyostelium discoideum is one of the best characterized natural RNAi inhibitors. A homologue of Dictyostelium helF gene has also been identified in S. pombe. It is called. the mfhl gene. This thesis also addresses the question: is the mfhl gene a RNAi inhibitor in S. pombe. A haploid S. pombe mfhl deletion mutant was isolated, and the ura4-based RNAi selective assay was used to study RNAi in this mutant - there was no apparent effect on RNAi activity in this strain. Although no paralogue(s) of helF gene was found in Dictyostelium, a paralogue of S. pombe mfhl gene, mfh2 gene was identified -a mfhl & mf h2 double gene deletion mutant was also tested negative with respect to RNAi using the same assay. A phenotypic characterization of the S. pombe mfhl/mfh2 single/double gene deletion mutants compared to the wild type strain revealed that the mutants were more sensitive to ethanol, hydroxyurea and UV, which indicated that the mf hl and mf h2 genes appeared to protect S. pombe from chemical and UV induced DNA damage. The cellular localization of Dictyostelium helF and S. pombe mfhl genes was also attempted to be revealed by heterologous gene expressing studies using GFP-fusion expression plasmids in both Dictyostelium and S. pombe.
21
au, J. Fosu@murdoch edu y John Fosu-Nyarko. "Studies on Subterranean clover mottle virus towards development of a gene silencing vector". Murdoch University, 2005. http://wwwlib.murdoch.edu.au/adt/browse/view/adt-MU20050421.123457.
Texto completoResumen
Subterranean clover mottle virus (SCMoV) is a positive sense, single-stranded RNA virus that
infects subterranean clover (Trifolium subterraneum) and a number of related legume species.
The ultimate aim of this research was to investigate aspects of SCMoV that would support its
use as a gene silencing vector for legume species, since RNA (gene) silencing is now a
potential tool for studylng gene function. The ability of viruses to induce an antiviral defense
system is being explored by virus-induced gene silencing (VIGS), in which engmeered viral
genomes are used as vectors to introduce genes or gene ii-agments to understand the function of
endogenous genes by silencing them. To develop a gene silencing vector, a number of aspects
of SCMoV host range and molecular biology needed to be studied.
A requirement for a useful viral vector is a suitably wide host range. Hence the first part of this
work involved study of the host range and symptom development of SCMoV in a range of
leguminous and non-leguminous plants. The aim of this work was to identify new and most
suitable hosts among economically important crop and model legumes for functional genomic
studies, and also to study symptom development in these hosts for comparison with host
responses to any SCMoV-based viral vectors that might be used in later infection studies. A
total of 61 plant genotypes representing 52 species from 25 different genera belonging to 7
families were examined for their response to SCMoV infection, including established and new
crop legumes, established pasture, and novel pasture and forage legumes, and 12 host indicator
plants belonging to the families Amaranthaceae, Apiaceae, Chenopodiaceae, Cruciferae,
Cucurbitaceae and Solanaceae. Following mechanical inoculation, plants were examined for
symptoms and tested for primary and secondary infection by RT-PCR andlor ELISA after 2-3
weeks and 3-9 weeks, respectively. Thirty-six legume hosts belonging to eight different genera
of legumes were identified as suitable hosts of SCMoV, 22 of them systemic hosts and 15 were
infected locally. Only two non-legumes were infected with SCMoV-P23, one systemically and
one as a local host, so confirming that SCMoV is essentially a legume-infecting virus. This
work considerably expanded knowledge of the host range of SCMoV.
To provide the information needed to modify the SCMoV genome to develop gene vectors,
the virus was characterized in detail. The complete genomes of four isolates, SCMoV-AL,
SCMoV-MB, SCMoV-MJ and SCMoV-pFL, were sequenced using high fidelity RT-PCR and
molecular cloning, and compared to the first sequenced isolate (SCMoV-P23) to give a
complete picture of the genome organisation of the virus. The 4,258 nucleotide (nt) sequence
of SCMoV RNA is not polyadenylated. The 5' non-coding region (NCR) is 68 nt in length and
the 3' NCR is 174 nt. The coding regon contains four overlapping open reading fi-ames
(ORFs). The first, OW1 (nt 68-608), encodes a putative protein containing 179 amino acids
with a calculated molecular mass (Ma,) of 20.3 kDa. It overlaps with the next ORF, ORF2a, by
four bases. ORF2a (nt 605-2347) encodes a putative protein of 580 amino acids with a Ma, of
63.7 kDa and contains a motif characteristic of chymotrypsin-like serine proteases. The ORF2b
is probably translated as part of a polyprotein by -1 ribosomal fiameshifting in ORF2a. The
transfiame product (Ma, = 107.5 kDa) is made up of 966 amino acids. A GDD motif typical of
RNA virus polymerases is present in ORF2b. The 3' terminal ORF3 (nt 3323-4084) encodes
the 27.3 kDa coat protein (CP).
Nucleotide variation between the complete sequences of the isolates was two to three orders of
magnitude larger than base misincoporation rates of the polymerases used in RT-PCR.
Molecular relationship analysis between all five isolates, undertaken with the complete
nucleotide sequences, clearly separated them into three groups. These groups reflect similar
significantly diverse groupings based on the symptoms and their severity in subterranean
clover. Intra-isolate sequence variability is therefore a possible cause of the differences in
symptom severity. The analysis also showed that there were more nucleotide substitutions at
the 5' terminal half of SCMoV than at the 3' end. This observation was confirmed by the
higher value of nucleotide diversities at nonsynonymous versus synonymous sites (dN/ds ratio)
estimated for the ORF1, compared to the near conservation of sequences of the other ORFs.
These results, together with functional and comparative sequence analysis with other
sobemoviruses, implicate the ORFl gene product in pathogenicity of SCMoV, possibly as a
severity determinant or as a viral suppressor of RNA silencing in plants.
Because more information on SCMoV genome function was required, the possible involvement
of the ORFl gene product (PI) and the CP in movement of SCMoV was studied in cells of
grasspea (Lathyrus clymenum L) and chickpea as C-terminal fusion constructs with jellyfish
(Aequorea victoriae) green fluorescent protein (GFP). A transient expression vector, pTEV, for
in planta synthesis of reporter gene constructs was developed. The vector was based on
pGEM-T with 35s RNA transcriptional promoter of Caulzjlower Mosaic virus (CaMV) and
nopaline synthase gene transcription terminator signal (T-Nos) separated by a multiple
subcloning site. A custom-made particle inflow gun was used to introduce the constructs into
plant cells. The bombardment conditions were fxst optimised for efficient delivery of DNAcoated
particles. Transient gene expression of GFP was monitored 24-96 hours after particle
bombardment. Fluorescence from GFP alone, GFP:CP and GFP:Pl constructs was observed in
the nucleus of single cells, cytoplasm and cell periphery of neighbouring cells. There was
limited spread of these fusion proteins from one cell to another 36-48 hours after
transformation. These results indicate that the P 1 and CP cannot move independently from cell
to cell. Other viral/cellular components might be needed to form a complex with these proteins
to transport the viral genome. Putative nuclear export signals in the P1 and CP sequences of
SCMoV were identified by sequence comparison. These could be tested by mutagenesis using
full-length infectious clones.
To determine the possibility of gene expression of vectors based on SCMoV, three forms of a
full-length cDNA clone of SCMoV-pFL were developed: one with no heterologous
transcriptional factors (pFL), a second under the control of only 35s (p35SFL) and a third with
35s and T-Nos (pTEVFL). Fifteen day-old in vitro-cultured chickpea, grasspea and
subterranean clover seedlings were inoculated by particle bombardment using gold particles
coated with plasmid pTEVFL. In vivo-transcribed RNA transcripts were detected by RT-PCR
after two weeks in grasspea but not in subterranean clover and chickpea.
Experiments were undertaken towards developing the SCMoV genome into a VIGS vector.
Three forms each of five major GFP chimeric constructs of pFL (the full length SCMoV cDNA
clone) were generated from which in vitro- and in vivo-transcribed RNA transcripts could be
derived. The rationale used in developing these constructs was gene insertion andlor
replacement with d p , and duplication of the putative subgenomic RNA promoter (sgPro) of
SCMoV. The major constructs were as follows:
pFLCPgfp, pFL with the d p gene fused to the 3' end of the CP gene,
pFLP 1 gfp, pFL with gj27 gene fused to the 3 ' end of the ORF 1,
pFLCPsgprogfp, pFL with a putative sgPro sequence and a translatable & gene cloned
in tandem between the CP gene and the 3' NCR of SCMoV,
pFLCPVsgprogf$, pFL with a putative sgPro sequence and a translatable gfp gene
cloned in tandem between a truncated CP gene and the 3' NCR and
pFLREPsgprog@, pFL with the ORF2b, a putative sgPro sequence and a translatable
&fP gene cloned in tandem between a truncated CP gene and the 3' NCR
These constructs were all made, but a detailed assessment of their vector potential could not be
done because there was a delay of about one year whilst the Office of the Gene Technology
Regulator processed the application for permission for glasshouse testing. Although additional
work needs to be undertaken to complete development of a final RNA silencing vector, this
study has contributed to new knowledge in terms of extending understanding of SCMoV host
range, symptoms, sequence variation and control of gene expression. The constructs made
have also laid the groundwork for development of a legume gene silencing vector based on
SCMoV.
22
Fosu-Nyarko, John. "Studies on Subterranean clover mottle virus towards development of a gene silencing vector". Fosu-Nyarko, John (2005) Studies on Subterranean clover mottle virus towards development of a gene silencing vector. PhD thesis, Murdoch University, 2005. http://researchrepository.murdoch.edu.au/54/.
Texto completoResumen
Subterranean clover mottle virus (SCMoV) is a positive sense, single-stranded RNA virus that infects subterranean clover (Trifolium subterraneum) and a number of related legume species. The ultimate aim of this research was to investigate aspects of SCMoV that would support its use as a gene silencing vector for legume species, since RNA (gene) silencing is now a potential tool for studylng gene function. The ability of viruses to induce an antiviral defense system is being explored by virus-induced gene silencing (VIGS), in which engmeered viral genomes are used as vectors to introduce genes or gene ii-agments to understand the function of endogenous genes by silencing them. To develop a gene silencing vector, a number of aspects of SCMoV host range and molecular biology needed to be studied.
A requirement for a useful viral vector is a suitably wide host range. Hence the first part of this work involved study of the host range and symptom development of SCMoV in a range of leguminous and non-leguminous plants. The aim of this work was to identify new and most suitable hosts among economically important crop and model legumes for functional genomic studies, and also to study symptom development in these hosts for comparison with host responses to any SCMoV-based viral vectors that might be used in later infection studies. A total of 61 plant genotypes representing 52 species from 25 different genera belonging to 7 families were examined for their response to SCMoV infection, including established and new crop legumes, established pasture, and novel pasture and forage legumes, and 12 host indicator plants belonging to the families Amaranthaceae, Apiaceae, Chenopodiaceae, Cruciferae, Cucurbitaceae and Solanaceae. Following mechanical inoculation, plants were examined for symptoms and tested for primary and secondary infection by RT-PCR andlor ELISA after 2-3 weeks and 3-9 weeks, respectively. Thirty-six legume hosts belonging to eight different genera of legumes were identified as suitable hosts of SCMoV, 22 of them systemic hosts and 15 were infected locally. Only two non-legumes were infected with SCMoV-P23, one systemically and one as a local host, so confirming that SCMoV is essentially a legume-infecting virus. This work considerably expanded knowledge of the host range of SCMoV.
To provide the information needed to modify the SCMoV genome to develop gene vectors, the virus was characterized in detail. The complete genomes of four isolates, SCMoV-AL, SCMoV-MB, SCMoV-MJ and SCMoV-pFL, were sequenced using high fidelity RT-PCR and molecular cloning, and compared to the first sequenced isolate (SCMoV-P23) to give a complete picture of the genome organisation of the virus. The 4,258 nucleotide (nt) sequence of SCMoV RNA is not polyadenylated. The 5' non-coding region (NCR) is 68 nt in length and the 3' NCR is 174 nt. The coding regon contains four overlapping open reading fi-ames (ORFs). The first, OW1 (nt 68-608), encodes a putative protein containing 179 amino acids with a calculated molecular mass (Ma,) of 20.3 kDa. It overlaps with the next ORF, ORF2a, by four bases. ORF2a (nt 605-2347) encodes a putative protein of 580 amino acids with a Ma, of 63.7 kDa and contains a motif characteristic of chymotrypsin-like serine proteases. The ORF2b is probably translated as part of a polyprotein by -1 ribosomal fiameshifting in ORF2a. The transfiame product (Ma, = 107.5 kDa) is made up of 966 amino acids. A GDD motif typical of RNA virus polymerases is present in ORF2b. The 3' terminal ORF3 (nt 3323-4084) encodes the 27.3 kDa coat protein (CP).
Nucleotide variation between the complete sequences of the isolates was two to three orders of magnitude larger than base misincoporation rates of the polymerases used in RT-PCR. Molecular relationship analysis between all five isolates, undertaken with the complete nucleotide sequences, clearly separated them into three groups. These groups reflect similar significantly diverse groupings based on the symptoms and their severity in subterranean clover. Intra-isolate sequence variability is therefore a possible cause of the differences in symptom severity. The analysis also showed that there were more nucleotide substitutions at the 5' terminal half of SCMoV than at the 3' end. This observation was confirmed by the higher value of nucleotide diversities at nonsynonymous versus synonymous sites (dN/ds ratio) estimated for the ORF1, compared to the near conservation of sequences of the other ORFs. These results, together with functional and comparative sequence analysis with other sobemoviruses, implicate the ORFl gene product in pathogenicity of SCMoV, possibly as a severity determinant or as a viral suppressor of RNA silencing in plants.
Because more information on SCMoV genome function was required, the possible involvement of the ORFl gene product (PI) and the CP in movement of SCMoV was studied in cells of grasspea (Lathyrus clymenum L) and chickpea as C-terminal fusion constructs with jellyfish (Aequorea victoriae) green fluorescent protein (GFP). A transient expression vector, pTEV, for in planta synthesis of reporter gene constructs was developed. The vector was based on pGEM-T with 35s RNA transcriptional promoter of Caulzjlower Mosaic virus (CaMV) and nopaline synthase gene transcription terminator signal (T-Nos) separated by a multiple subcloning site. A custom-made particle inflow gun was used to introduce the constructs into plant cells. The bombardment conditions were fxst optimised for efficient delivery of DNAcoated particles. Transient gene expression of GFP was monitored 24-96 hours after particle bombardment. Fluorescence from GFP alone, GFP:CP and GFP:Pl constructs was observed in the nucleus of single cells, cytoplasm and cell periphery of neighbouring cells. There was limited spread of these fusion proteins from one cell to another 36-48 hours after transformation. These results indicate that the P 1 and CP cannot move independently from cell to cell. Other viral/cellular components might be needed to form a complex with these proteins to transport the viral genome. Putative nuclear export signals in the P1 and CP sequences of SCMoV were identified by sequence comparison. These could be tested by mutagenesis using full-length infectious clones.
To determine the possibility of gene expression of vectors based on SCMoV, three forms of a full-length cDNA clone of SCMoV-pFL were developed: one with no heterologous transcriptional factors (pFL), a second under the control of only 35s (p35SFL) and a third with 35s and T-Nos (pTEVFL). Fifteen day-old in vitro-cultured chickpea, grasspea and subterranean clover seedlings were inoculated by particle bombardment using gold particles coated with plasmid pTEVFL. In vivo-transcribed RNA transcripts were detected by RT-PCR after two weeks in grasspea but not in subterranean clover and chickpea.
Experiments were undertaken towards developing the SCMoV genome into a VIGS vector. Three forms each of five major GFP chimeric constructs of pFL (the full length SCMoV cDNA clone) were generated from which in vitro- and in vivo-transcribed RNA transcripts could be derived. The rationale used in developing these constructs was gene insertion andlor replacement with d p , and duplication of the putative subgenomic RNA promoter (sgPro) of SCMoV. The major constructs were as follows:
* pFLCPgfp, pFL with the d p gene fused to the 3' end of the CP gene
* pFLP1gfp, pFL with gj27 gene fused to the 3' end of the ORF 1,
* pFLCPsgprogfp, pFL with a putative sgPro sequence and a translatable and gene cloned in tandem between the CP gene and the 3' NCR of SCMoV,
* pFLCPVsgprogfp, pFL with a putative sgPro sequence and a translatable gfp gene cloned in tandem between a truncated CP gene and the 3' NCR and
* pFLREPsgprogfp, pFL with the ORF2b, a putative sgPro sequence and a translatable gfP gene cloned in tandem between a truncated CP gene and the 3' NCR
These constructs were all made, but a detailed assessment of their vector potential could not be done because there was a delay of about one year whilst the Office of the Gene Technology Regulator processed the application for permission for glasshouse testing. Although additional work needs to be undertaken to complete development of a final RNA silencing vector, this study has contributed to new knowledge in terms of extending understanding of SCMoV host range, symptoms, sequence variation and control of gene expression. The constructs made have also laid the groundwork for development of a legume gene silencing vector based on SCMoV.
23
Choy, Yee-wai Elizabeth. "A study of Epstein-Barr virus-encoded small regulatory RNAs". Click to view the E-thesis via HKUTO, 2007. http://sunzi.lib.hku.hk/HKUTO/record/B39557261.
Texto completo
24
蔡依慧 y Yee-wai Elizabeth Choy. "A study of Epstein-Barr virus-encoded small regulatory RNAs". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2007. http://hub.hku.hk/bib/B39557261.
Texto completo
25
Lee, Hung-chiu. "Synthetic RNA interference against influenza A virus". Click to view the E-thesis via HKUTO, 2005. http://sunzi.lib.hku.hk/hkuto/record/B35537814.
Texto completo
26
Lee, Hung-chiu y 李洪釗. "Synthetic RNA interference against influenza A virus". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2005. http://hub.hku.hk/bib/B35537814.
Texto completo
27
Gammelgård, Elin. "Interactions of potato virus A with host plants : recombination, gene silencing and non-hypersensitive resistance /". Uppsala : Dept. of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences, 2007. http://epsilon.slu.se/2007111.pdf.
Texto completo
28
Wagner, Laura A. "Silencing mutant Huntingtin by RNA interference for the treatment of Huntington Disease". Thesis, University of British Columbia, 2008. http://hdl.handle.net/2429/937.
Texto completoResumen
Huntington Disease (HD) is a dominantly inherited neurological disease attributed to a CAG expansion within the HD gene. The HD mutation gives rise to a polyglutamine expansion in exon 1 of the protein huntingtin (Htt). Since the discovery of the HD mutation in 1993, various HD gene mouse models have been developed to contain either fragments or full-length copies of the mutant HD gene. The existence of these HD mouse models enables focused therapeutic testing to develop potential treatments for HD. RNA interference (RNAi) therapy is a targeted gene silencing approach whereby synthetic RNA constructs are shuttled into the cell by viral vectors and used by the cell’s endogenous RNAi machinery to silence a gene of interest. RNAi therapy holds promise for mutant huntingtin (muHtt) allele-specific silencing as a treatment for HD. The purpose of this thesis was to develop the tools for pre-clinical testing of RNAi-mediated gene silencing of human muHtt in the YAC128 mouse model of HD. First, AAV serotypes were compared for delivery to striatal neurons, the neurons most affected in HD. From this work AAV serotype 1 was selected as the most effective serotype for construct delivery. Second, synthetic RNAi constructs including short-hairpin RNA (shRNA) and microRNA-based constructs (miR-shRNAs) were compared for silencing of human muHtt expression in vivo. Here, miR-shRNAs were found to have increased gene silencing and improved tolerance in avoiding immune activation compared to shRNAs. Alternatively, the shRNAs induced dramatic immune activation and morbidity in some cases. Ultimately these findings will contribute to a pre-clinical trial in YAC128 mice investigating Htt RNAi-mediated gene silencing in the treatment of HD, which is also discussed in this thesis. This future work provides proof-of-principle for muHtt allele-specific silencing as a treatment of HD.
29
Hayes, Ian MacDonald. "Virus-induced changes in host gene expression in infected cowpeas". Thesis, University of East Anglia, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.277172.
Texto completo
30
Van, Eeden C. (Christiaan). "The construction of gene silencing transformation vectors for the introduction of multiple-virus resistance in grapevines". Thesis, Stellenbosch : Stellenbosch University, 2004. http://hdl.handle.net/10019.1/53764.
Texto completoResumen
Thesis (MSc)--University of Stellenbosch, 2004.ENGLISH ABSTRACT: Viruses are some of the most important pathogens of grapevines. There are no effective chemical treatments, and no grapevine- or other natural resistance genes have been discovered against grapevine infecting viruses. The primary method of grapevine virus control is prevention by biological indexing and molecular- and serological screening of rootstocks and scions before propagation. Due to the spread of grapevine viruses through insect vectors, and in the case of GRSPaV the absence of serological screening, these methods of virus control are not always effective. In the past several methods, from cross-protection to pathogen derived resistance (PDR), have been applied to induce plant virus resistance, but with inconsistent results. In recent years the application of post-transcriptional gene silencing (PTGS), a naturally occurring plant defense mechanism, to induce targeted virus resistance has achieved great success. The Waterhouse research group has designed plant transformation vectors that facilitate specific virus resistance through PTGS. The primary focus of this study was the production of virus specific transformation vectors for the introduction of grapevine virus resistance. The Waterhouse system has been successfully utilised for the construction of three transformation vectors with the pHannibal vector as backbone. Each vector contains homologous virus coat protein (CP) gene segments, cloned in a complementary conformation upstream and downstream of an intron sequence. The primary vector (pHann-SAScon) contains complementary CP gene segments of both GRSPaV and GLRaV-3 and was designed for the introduction of multiple-virus resistance. For the construction of the primary vector the GRSPaV CP gene was isolated from RSP infected grapevines. A clone of the GLRaV-3 CP gene was acquired. The second vector (pHann- LR3CPsas) contains complementary CP gene segments of GLRaV-3. The third vector (pHann-LR2CPsas) contains complementary CP gene segments of GLRaV-2. The cassette containing the complementary CP gene segments of both GRSPaV and GLRaV-3 was cloned into pART27 (pART27-HSAScon), and used to transform N tabacum cv. Petit Havana (SRI), through A. tumefaciens mediated transformation. Unfortunately potential transformants failed to regenerate on rooting media; hence no molecular tests were performed to confirm transformation. Once successful transformants are generated, infection with a recombinant virus vector (consisting of PYX, the GFP gene as screenable marker and the complementary CP gene segments of both GRSPaV and GLRaV-3) will be used to test for the efficacy of the vectors to induce resistance. A secondary aim was added to this project when a need was identified within the South African viticulture industry for GRSPaV specific antibodies to be used in serological screening. To facilitate future serological detection of GRSPaV, the CP gene was isolated and expressed with a bacterial expression system (pETI4b) within the E. coli BL2I(DE3)pLysS cell line. The expressed protein will be used to generate GRSPaV CP specific antibodies.
AFRIKAANSE OPSOMMING: Virusse is van die belangrikste patogene by wingerd. Daar bestaan geen effektiewe chemiese beheer nie, en geen wingerd- of ander natuurlike weerstandsgene teen wingerdvirusse is al ontdek nie. Die primêre metode van beheer t.o.v. wingerdvirusse is voorkoming deur biologiese indeksering, en molekulêre- en serologiese toetsing van onderstokke en entlote voor verspreiding. As gevolg van die verspreiding van wingerdvirusse deur insekvektore, en in die geval van GRSPa V die tekort aan serologiese toetsing, is dié metodes van virusbeheer nie altyd effektief nie. In die verlede is metodes soos kruis-beskerming en patogeen-afgeleide weerstand (PDR) gebruik om virusweerstand te induseer, maar met inkonsekwente resultate. In onlangse jare is post-transkripsionele geenonderdrukking (PTGS), 'n natuurlike plantbeskermingsmeganisme, met groot sukses toegepas om geteikende virusweerstand te induseer. Die Waterhouse-navorsingsgroep het planttransformasievektore ontwerp wat spesifieke virusweerstand induseer d.m.v. PTGS. Die vervaardiging van virus spesifieke tranformasievektore vir die indusering van wingerdvirusweerstand was die primêre doelwit van hierdie studie. Die Waterhouse-sisteem was gebruik vir die konstruksie van drie transformasievektore, met die pHannibal vektor as basis. Elke vektor bevat homoloë virus kapsiedproteïen (CP) geensegmente, gekloneer in 'n komplementêre vorm stroom-op en stroom-af van 'n intronvolgorde. Die primêre vektor (pHann-SAScon) bevat komplementêre CP geensegmente van beide GRSPaV en GLRaV-3, en was ontwerp vir die indusering van veelvoudige-virusweerstand. Die CP-geen van GRSPa V was vanuit RSP-geïnfekteerde wingerd geïsoleer, vir die konstruksie van die primêre vektor. 'n Kloon van die GLRa V-3 CP-geen was verkry. Die tweede vektor (pHann-LR3CPsas) bevat komplementêre CP geensegmente van GLRaV-3. Die derde vektor (pHann-LR2CPsas) bevat komplementêre CP geensegmente van GLRa V-2. Die kasset bestaande uit die komplementêre CP geensegmente van beide GRSPaV en GLRaV-3, was gekloneer in pART27 (pART27-HSAScon), en gebruik om N tabacum cv. Petit Havana (SRI) te transformeer d.m.v. A. tumefaciens bemiddelde transformasie. Ongelukkig het potensiële transformante nie geregenereer op bewortelingsmedia nie; gevolglik was geen molekulêre toetse gedoen om transformasie te bevestig nie. Na suksesvolle transformante gegenereer is, sal infeksie met 'n rekombinante-virusvektor (bestaande uit PYX, die GFP geen as waarneembare merker en die komplementêre CP geensegmente van beide GRSPa V en GLRa V-3) gebruik word om die effektiwiteit van die vektore as weerstandsinduseerders te toets. 'n Sekondêre doelwit is by die projek gevoeg toe 'n behoefte aan GRSPaV spesifieke teenliggame binne die Suid-Afrikaanse wynbedryf geïdentifiseer is, vir gebruik in serologiese toetsing. Om toekomstige serologiese toetsing van GRSPa V te bemiddel, was die CP-geen geïsoleer en in 'n bakteriële uitdrukkingsisteem (PETI4b) uitgedruk, in die E. coli BL21(DE3)pLysS sellyn. Die uitgedrukte proteïne sal gebruik word vir die vervaardiging van GRSPa V CP spesifieke antiliggame.
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Buchmann, Cody. "Reversal of RNA-mediated gene silencing pathways by geminivirus AL2 and L2 proteins". The Ohio State University, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=osu1221847080.
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Howard-Till, Rachel A. "Small RNA pathways and the roles of tudor nucleases in gene silencing and DNA deletion in Tetrahymena thermopila /". Thesis, Connect to this title online; UW restricted, 2006. http://hdl.handle.net/1773/5064.
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Dung, Le Phuong [Verfasser]. "Analysis of sense transgene-induced gene silencing in introgression lines reveals the presence of silencing modulators in Arabidopsis thaliana accession genomes / Le Phuong Dung". Halle, 2017. http://d-nb.info/1132840481/34.
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Deddouche, Safia. "Regulation and function of the virus induced gene Vago in drosophila". Strasbourg, 2009. http://www.theses.fr/2009STRA6263.
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Des infections virales continuent à être une cause majeure de morbidité et de mortalité dans le monde entier. En particulier, ces trois dernières décennies ont été témoin de l’apparition d'environ 25 nouvelles maladies virales. De plus, les récentes pandémies, comme celle associé au virus du Chikungunya à la Réunion, démontrent l'énorme problème de santé publique associé aux infections virales transmisses par les arthropodes. À cette étape, notre compréhension de la réponse du vecteur face à l'infection virale est très limitée. Le but de mes études de doctorat était d'utiliser la Drosophile melanogaster comme un modèle pour étudier la réponse hôte d'insectes à l'infection virale. La réponse antivirale de la drosophile, comme chez d'autres invertébrés et les plantes, repose principalement sur l'interférence à l'ARN. En plus de ce mécanisme, l'infection virale déclenche chez la drosophile, l'activation d’une centaine de gènes. Un des gènes induits, Vago, code un polypeptide riche en cystéines de 18 kDa. L’étude génétique de la fonction du produit du gène Vago, nous a permis de mettre en évidence l’importance de Vago dans le contrôle de la charge virale dans le corps gras après l'infection avec le virus C de la drosophile. Mes travaux ont également mis en évidence que l'induction du gène Vago dépend de l’hélicase Dicer-2. Dicer-2 appartient à la même famille de DeXD/H-Box helicase que les récepteurs Retinoic acid Inducible gene-I Like Receptor (RLR) qui sont impliqués dans la détection de l'infection virale et l'induction d’interferon chez des mammifères. Ce travail permis de mettre en lumière un nouveau parallèle entre l'immunité des mammifères et de la drosophileViral infections continue to be a major cause of morbidity and mortality world-wide. In particular, the past three decades have witnessed the onset of some 25 new viral diseases. Moreover recent outbreaks such as Chikungunya fever in La Réunion demonstrate the enormous public health problem associated with arthropod-borne virus infections. At this stage, our understanding of how the vector responds to virus infection is very limited. The goal of my PhD studies was to use Drosophila melanogaster as a model to study the host response of insects to virus infection. In flies like in mammals, viral infection triggers the expression of a large number of genes. I have provided genetic evidence that the inducible gene Vago limits viral replication. This was the first demonstration that an inducible molecule controls viral replication in drosophila. Interestingly, I have shown that Vago induction is dependant of the Dicer-2 molecule. I also note that Dicer-2 belongs to the same DEXD/H-box helicase family as RIG-I like receptors, which sense viral infection and mediate interferon induction in mammals. I posit that this family represents an evolutionary conserved set of sensors, which detect viral nucleic acids and direct antiviral responses. My work points out that the well known RNaseIII enzyme Dicer-2 plays a dual role during infection: (i) a direct role counteracting viral replication by cutting viral RNA and (ii) a sensing role that triggers antiviral gene expression in Drosophila
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Pathi, Krishna [Verfasser]. "Establishment of maize resistance to fungal diseases by host-induced gene silencing and site-directed mutagenesis / Krishna Pathi". Hannover : Gottfried Wilhelm Leibniz Universität, 2021. http://d-nb.info/1235138437/34.
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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.
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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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Elmén, Joacim. "Nucleic acid based therapeutic approaches /". Stockholm, 2005. http://diss.kib.ki.se/2005/91-7140-047-8/.
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Wong, Kam-wai y 黃錦偉. "The molecular mechanism of mitotic arrest induced by a novel diterpenoid pseudolaric acid B and a novel gene encoding RNA-bindingprotein 22". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2006. http://hub.hku.hk/bib/B37679090.
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Chu, Chia-Ying. "Molecular Mechanism of RNA-Mediated Gene Silencing in Human Cells: A Dissertation". eScholarship@UMMS, 2008. https://escholarship.umassmed.edu/gsbs_diss/388.
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Small non-coding RNAs regulate gene expression at posttranscriptional level in eukaryotic cells. Two classes of such small (~21-25 nt) RNAs that have been extensively studied in gene silencing are short interfering RNAs (siRNAs) and microRNAs (miRNAs). RNA interference (RNAi) is process whereby double-stranded RNA induces the sequence-specific degradation of homologous mRNA. The RNAi machinery can also be programmed in human cells by introducing 21-nt siRNA duplexes that are assembled into RNA-induced silencing complexes (RISC). In this dissertation, systematic analysis of siRNAs with deletions at the passenger and/or guide strand reveals that a short RNAi trigger, 16-nt siRNA, induces potent RNAi in human cells. The 16-nt siRNA more effectively knocked down mRNA and protein levels than 19-nt siRNA when targeting the endogenous CDK9 gene. In vitro kinetic analysis of human RISC indicates that 16-nt siRNA has a higher RISC-loading capacity than 19-nt siRNA. These results suggest that 16-nt duplexes can be designed as potent triggers for RNAi.
RISC can be programmed by small interfering RNAs (siRISC) to cleave a perfectly complementary target mRNA, or endogenous microRNAs (miRISC) to inhibit translation by binding imperfectly matched sequences in the 3’-untranslated region (3’-UTR) of target mRNA. Both RISCs contain Argonaute2 (Ago2), which localizes to cytoplasmic mRNA processing P-bodies. This dissertation shows that RCK/p54, a DEAD box helicase, interacts with Ago2, in affinity-purified active siRISC or miRISC, facilitates formation of P-bodies. Depletion of RCK/p54 disrupted P-bodies and dispersed Ago2 throughout the cytoplasm, but did not significantly affect siRNA-mediated RNAi. Depleting RCK/p54 releases general and miRNA-induced translational repression. These findings imply that miRISC-mediated translation repression requires RCK/p54, also suggest that location of miRISC to P-bodies is not required for miRNA function, but is the consequence of translation repression.
To elucidate the function of RCK/p54 in miRNA-mediated gene silencing, analysis of a series of YFP-tagged RCK/p54 mutants reveals the motif required for P-body localization, interaction with Ago2, and/or facilitating the miRNA-mediated translation repression. Additionally, rabbit reticulocyte lysate system was used to recapitulate the miRISC function in a cell-free system and confirmed the requirement of RCK/p54 for miRNA function in vitro. Analysis of Ago2 distribution in the polysome profiling in RCK/p54-depleted cells, compared to that in normal cells, revealed that RCK/p54 facilitates miRISC by trapping it at translation initiation complex. These data suggest that interaction of RCK/p54 with Ago2 is involved in the repression of translation initiation of miRNA function.
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Nauth, Brittany J. "Soybean QTL Mapping and Candidate Gene Identification for Pythium irregulare and Phytophthora sojae Partial Resistance; and Root-Knot Nematode Induced Suppression of Gene Silencing". The Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1406151869.
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Wong, Kam-wai. "The molecular mechanism of mitotic arrest induced by a novel diterpenoid pseudolaric acid B and a novel gene encoding RNA-binding protein 22". Click to view the E-thesis via HKUTO, 2006. http://sunzi.lib.hku.hk/hkuto/record/B37679090.
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Guan, Haoji y 關浩基. "Double-stranded RNA induced gene silencing of neuropeptide genes in sand shrimp, Metapenaeus ensis and development of crustacean primarycell culture". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2006. http://hub.hku.hk/bib/B36893626.
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Guan, Haoji. "Double-stranded RNA induced gene silencing of neuropeptide genes in sand shrimp, Metapenaeus ensis and development of crustacean primary cell culture /". Click to view the E-thesis via HKUTO, 2006. http://sunzi.lib.hku.hk/hkuto/record/B36893626.
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Rupp, Jessica Lynn Shoup. "RNA interference mediated virus resistance in transgenic wheat". Diss., Kansas State University, 2015. http://hdl.handle.net/2097/20387.
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Doctor of PhilosophyPlant Pathology
John P. Fellers
Harold N. Trick
Wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV) are two viruses affecting wheat in the Great Plains region of the United States. Genetic resistance is severely limited, requiring management methods focusing on the deployment of resistant varieties and various cultural practices. Evaluation of resistance is complicated by the lack of a standard rating scale. The objective of this work was to develop new avenues to mitigate these challenges. A standardized virus symptom rating scale was developed using historical Kansas rating scales, and validated using multiple wheat populations. Two independent RNA interference (RNAi) expression vectors targeting portions of viral coat protein (CP) of WSMV and TriMV were previously transformed into wheat. T₂ plants and beyond were evaluated using PCR, reverse transcription-PCR and bioassays in which plants were challenged with their respective virus. These lines were evaluated for resistance through the T₆ generation. Crosses were made with the susceptible winter wheat cultivars, ‘Overley’ and ‘Karl 92.’ Real-time PCR results show viral titer was up to 20-fold lower in the T₆ transgenic lines, the F₁, and the BC₁F₁ compared to control plants. This provides evidence that this RNAi silencing method is stable in wheat over multiple generations. WSMV and TriMV use host eukaryotic initiation factors (eIF) in order to facilitate replication of their genomes. Previously created RNAi expression vectors were derived from the sequences of the wheat genes eIF(iso)4E-2 and eIF4G. Evaluation of these lines began in the T₁ generation. Resistance has been demonstrated in three lines of eIF(iso)4E-2 and four lines of eIF4G, derived by single seed descent. T₆ progeny co-infected with WSMV and TriMV continue to be resistant. Crosses have been performed with the winter wheat ‘Karl 92’ and three Kansas elite lines, KS030887K-6, KS09H19-2-3, and KS10HW78-1-1. RNAi construct effectiveness was evaluated using real-time PCR. Results show up to 18-fold reduction in viral titer in the transgenic lines, the F₁, and the BC₁F₁ in comparison to control plants. This research provides the first evidence that a single host transgene can provide resistance to multiple viruses and has great potential benefits to both breeders and producers.
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Höfle, Lisa [Verfasser]. "Investigations on the mechanism of Host-Induced Gene Silencing of the fungal CYP51 genes in the Fusarium Arabidopsis pathosystem / Lisa Höfle". Gießen : Universitätsbibliothek, 2018. http://d-nb.info/1163945307/34.
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Owor, Betty Elizabeth. "Maize streak virus (MSV) diversity in Uganda and the assessment of gene silencing as a tool for development of resistance to MSV". Doctoral thesis, University of Cape Town, 2008. http://hdl.handle.net/11427/4315.
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Includes abstract.Includes bibliographical references (leaves 158-197).
Maize streak virus (MSV: Family Geminiviridae, Genus Mastrevirus) is the causal agent of maize streak disease (MSD) that contributes significantly to low maize yields in Africa, thereby threatening food security of sub-Saharan Africa’s poorest people. In Uganda, MSD has been identified as one of the most important constraints to maize production. In order to have a better understanding of the disease in that country, this thesis set out to establish MSD levels in farmers’ fields; develop a new sampling and virus isolation method; assess the diversity of MSVs throughout Uganda; and, through the cloning of sampled virus genomes, to determine the genetic characteristics of different isolates. In addition, this study also included an assessment of RNA silencing as a resistance strategy against MSV.
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Zhang, Chunquan. "GENETIC DIVERSITY OF BEAN POD MOTTLE VIRUS (BPMV) AND DEVELOPMENT OF BPMV AS A VECTOR FOR GENE EXPRESSION IN SOYBEAN". UKnowledge, 2005. http://uknowledge.uky.edu/gradschool_diss/437.
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Bean pod mottle virus (BPMV), a member of the genus Comovirus in the family Comoviridae, is widespread in the major soybean-growing areas in the United States. The complete nucleotide sequences of the genomic RNAs of the naturally occurring partial diploid strain IL-Cb1 were determined. Intermolecular RNA1 recombinants were isolated from strain IL-Cb1 and characterized at the molecular level. Structurally similar recombinant RNA1 was also generated after four passages in soybean derived from plants previously inoculated with a mixture of infectious RNA1 transcripts from two distinct strains. BPMV was developed as a plant viral vector that is appropriate for gene expression and virus-induced gene silencing (VIGS) in soybean. The foreign gene was inserted between the movement protein (MP) and the large coat protein (L-CP) coding regions. The recombinant BPMV constructs were stable following several serial passages in soybean and relatively high levels of protein expression were attained. Successful expression of several proteins with different biological activities was demonstrated from the BPMV vector. Double infection of soybean by BPMV and SMV triggers a synergistic interaction leading to a serious disease. To investigate the underlying mechanism, helper componentprotease (HC-Pro) genes from several SMV strains and TEV were expressed from BPMV vectors. The recombinant BPMV vectors carrying the HC-Pro genes from SMV strain G7 or TEV induced very severe symptoms on soybean whereas constructs containing the HC-Pro gene from SMV isolate P10, a mild strain with an apparent defect in synergism, induced only very mild symptoms. Transient agroinfiltration assays using GFP-transgenic Nicotiana benthamiana showed that HC-Pro from SMV isolate P10 was not a RNA silencing suppressor, whereas those of SMV strain G7 and TEV exhibited strong suppressor activities. Analysis of chimeric HC-Pro genes and point mutations indicated that a positively charged amino acid at position 144 is critical for the suppressor function of not only SMV HC-Pro but also other potyvirus HC-Pro proteins. Although amino acid substitution at position 144 resulted in changes in small RNA profile, it did not affect HC-Pro stability.
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Plows, David John. "Natural and induced variation in the fusion glycoprotein gene of human respiratory syncytial virus subgroup A". Thesis, University of Warwick, 1994. http://wrap.warwick.ac.uk/104816/.
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The nucleotide sequences of the fusion (F) protein encoding gene of five isolates of subgroup A respiratory syncytial (RS) virus, representing the five lineages associated with current epidemics of RS virus-associated disease, were determined and compared with F gene sequences from laboratory strains. Overall natural variation in the fusion protein gene among subgroup A isolates is low. Amino acid identities of between 97% to 99.5% were deduced. The relationship of the five lineage isolates, based on the sequence of their fusion protein genes, agrees broadly with evolutionary relationships inferred from comparison of their G, SH and N protein genes. There is a region of high amino acid variation at the C-terminal end of the F2- subunit, specifically between residues #101 and #105. None of the apparent amino acid coding changes are located in known epitopes. Using predictive structural models it is suggested that the few amino acid changes observed may alter the fusion protein structure especially in the C-terminal domain of the F2-subunit. The secondary structure of the Fi-subunit is predicted to remain unaltered. It is hypothesised that amino acid variation in the F2-subunit may result in antigenic variation, by altering a potential conformational epitope formed by interaction between the N-terminal region of Fi and the C-terminal region of F2. Induced variation in the fusion protein gene of two candidate vaccine strains was investigated. Temperature-sensitive mutant MAI, derived by intensive mutagenesis from the A2 strain, has two distinctive phenotypes; temperature-sensitivity and a retarded fusion protein mobility in non-reducing gels. Previous analysis of the phenotypic characteristics of tsA1 indicated that the F gene might be the site of the ts mutation. The sequence data derived in this study suggest that the site of ts lesion is located at residue #66 (Glu —> Lys) and that the mobility phenotype is located at residue #102 (Pro —► Ser). Mutant tsA1 exhibits a complex pattern of reversion with two classes of revertant viruses observed; a fully revertant virus which has wild-type growth characteristics but still retains retarded fusion protein mobility; and a partially revertant virus that possesses near wild-type growth characteristics and wild-type mobility. In fully revertant viruses the correction of the ts phenotype has been identified as the reversion of amino acid #66 to the wild-type residue (Lys —> Glu) whilst the coding change at amino acid #102 is retained, resulting in a mobility phenotype similar to that found in mutant tsA1. In partially revertant viruses the coding changes at amino acid #66 and amino acid #102 are retained. In partially revertant viruses the coding change correcting for the mobility phenotype, and partially correcting for the ts phenotype, has been tentatively identified as additional coding changes at residues #103 (Thr —► Ala) and/or #105 (Asn —> Ser). In vitro expression of the fusion gene products of mutant, revertant and wild-type viruses in mammalian cells has confirmed that the mobility phenotype is solely a consequence of changes in the fusion protein gene. Temperature-sensitive mutant MIC is a triple ts mutant derived from the RSS-2 strain. The mutations detected in the fusion gene of mutant MIC are conservative in nature and are not located in known epitopes. Therefore it is unlikely that the coding changes observed in the fusion protein gene account for the ts-phenotype or viral attenuation. It is also thought that the induced mutations have not altered the antigenic properties of the virus.
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Poulin, Louise. "Expression differentielle du produit du gene 'src' dans les tumeurs induites par le virus de sarcome aviaire = Differential expression of the 'src' gene product in tumor cells induced by avian sarcoma virus". Thesis, McGill University, 1987. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=74020.
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Tsao, Theresa Tsun-Hui. "Towards the development of transgenic banana bunchy top virus (BBTV)-resistant banana plants : interference with replication". Queensland University of Technology, 2008. http://eprints.qut.edu.au/17031/.
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Banana bunchy top virus (BBTV) causes one of the most devastating diseases of banana. Transgenic virus resistance is now considered one of the most promising strategies to control BBTV. Pathogen-derived resistance (PDR) strategies have been applied successfully to generate plants that are resistant to numerous different viruses, primarily against those viruses with RNA genomes. BBTV is a circular, single-stranded (css) DNA virus of the family Nanoviridae, which is closely related to the family Geminiviridae. Although there are some successful examples of PDR against geminiviruses, PDR against the nanoviruses has not been reported. Therefore, the aim of this thesis was to investigate the potential of BBTV genes to interfere with virus replication when used as transgenes for engineering banana plants resistance to BBTV. The replication initiation protein (Rep) of nanoviruses is the only viral protein essential for viral replication and represents an ideal target for PDR. Therefore, this thesis focused on the effect of wild-type or mutated Rep genes from BBTV satellite DNAs or the BBTV integral genome on the replication of BBTV in banana embryogenic cell suspensions.
A new Rep-encoding satellite DNA, designated BBTV DNA-S4, was isolated from a Vietnamese BBTV isolate and characterised. When the effect of DNA-S4 on the replication of BBTV was examined, it was found that DNA-S4 enhanced the replication of BBTV. When the replicative capabilities of DNA-S4 and the previously characterised Rep-encoding BBTV satellite, DNA-S1, were compared, it was found that the amount of DNA-S4 accumulated to higher levels than DNA-S1. The interaction between BBTV and DNA-S1 was also examined. It was found that over-expression of the Rep encoded by DNA-S1 using ubi1 maize polyubiquitin promoter enhanced replication of BBTV. However, when the Rep-encoded by DNA-S1 was expressed by the native S1 promoter (in plasmid pBT1.1-S1), it suppressed the replication of BBTV. Based on this result, the use of DNA-S1 as a possible transgene to generate PDR against BBTV was investigated.
The roles of the Rep-encoding and U5 genes of BBTV DNA-R, and the effects of over-expression of these two genes on BBTV replication were also investigated. Three mutants of BBTV DNA-R were constructed; plasmid pUbi-RepOnly-nos contained the ubi1 promoter driving Rep expression from DNA-R, plasmid pUbi-IntOnly-nos contained the ubi1 promoter driving expression of the DNA-R internal gene product (U5), while plasmid pUbi-R.ORF-nos contained the ubi1 promoter driving the expression of both Rep and the internal U5 gene product. The replication of BBTV was found to be significantly suppressed by pUbi-RepOnly-nos, weakly suppressed by pUbi-IntOnly-nos, but strongly enhanced by pUbi-R.ORF-nos.
The effect of mutations in three conserved residues within the BBTV Rep on BBTV replication was also assessed. These mutations were all made in the regions in the ATPase motifs and resulted in changes from hydrophilic to hydrophobic residues (i.e. K187→M, D224→I and N268→L). None of these Rep mutants was able to initiate BBTV replication. However, over-expression of Reps containing the K187→M or N268→L mutations significantly suppressed the replication of BBTV.
In summary, the Rep constructs that significantly suppressed replication of DNA-R and -C in banana embryogenic cell suspensions have the potential to confer resistance against BBTV by interfering with virus replication. It may be concluded that BBTV satellite DNAs are not ideal for conferring PDR because they did not suppress BBTV replication consistently. Wild-type Rep transcripts and mutated (i.e. K187→M and N248→L) Rep proteins of BBTV DNA-R, however, when over-expressed by a strong promoter, are all promising candidates for generating BBTV-resistant banana plants.