1,721,198 research outputs found
Development of a novel rep-inducible tomato leaf curl virus expression system
No full textPathogen-derived resistance (PDR) strategies, particularly those based on post-transcriptional gene silencing, have been used with great success for the generation of transgenic plants with resistance to RNA viruses. In contrast, a suitable strategy for transgenic resistance to ssDNA plant viruses, including those viruses belonging to the Geminiviridae, has remained elusive. Further, there is no convincing evidence that either post-transcriptional gene silencing, or pathogen-derived resistance in general, would be broadly applicable to ssDNA plant viruses. Researchers at QUT have been developing a novel resistance strategy against ssDNA viruses based on virus-activated expression of a stably integrated suicide gene. The strategy, based on InPAct (In Plant Activation) technology, relies on a "split" suicide gene cassette being arranged in such a way that expression of a lethal ribonuclease (barnase) is dependent on the virus-encoded replication-associated protein (Rep). Upon infection, Rep mediates the release of the construct resulting in the reconstitution of a transcribable and translatable episomal suicide gene expression cassette. The research for this PhD describes the development of an InPAct vector designed to confer resistance to Tomato leaf curl begomovirus (ToLCV), a major cause of disease in Solanaceous crops in the tropics and subtropics.\ud
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ToLCV-based InPAct vectors were constructed based upon two ToLCV isolates from Australia and North Vietnam. Prior to the generation of InPAct cassettes, the entire ToLCV-[Au] and ToLCV-Vie intergenic regions (IRs) were embedded within the castorbean catalase intron of a β-glucuronidase expression vector to determine the effect of the IR upon transcript processing. Using transient reporter gene assays in tobacco NT-1 cells, it was demonstrated that the ToLCV IRs both contained cryptic intron splice sites which interfered with efficient transcript processing and GUS expression. A series of truncations to the IRs were subsequently made to identify the potential cryptic intron splice sites and/or interfering sequences in both the ToLCV-[Au] and ToLCV-Vie IRs. The final truncated IRs, which were used in the construction the InPAct cassettes, comprised approximately 100 bp and appeared to contain all the necessary cis-acting elements required for efficient rolling circle replication (RCR). Using histochemical GUS assays and Southern analyses, the InPAct cassettes were shown to be activated and replicated only in the presence of the cognate viral Rep. GUS expression levels were shown to be further enhanced in the presence of the ToLCV replication-enhancer protein (REn) and by the addition of the Tobacco yellow dwarf mastrevirus origin of second strand synthesis into the cassette. Under these conditions, Rep-activated GUS expression from the InPAct vectors was found to reach levels similar to that of the benchmark CaMV 35S promoter. \ud
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Fifteen independent transgenic lines containing the ToLCV-[Au] and -Vie InPAct-GUS cassettes were generated by Agrobacterium-mediated transformation of tobacco leaf discs. Using agroinfiltration and histochemical assays, Rep-mediated activation of the InPAct cassettes and subsequent GUS expression was demonstrated in 11 out of the 15 lines tested; six of which showed expression levels equivalent to, or higher than, that obtained using a CaMV 35S promoter control. Evidence for activation of the integrated InPAct cassettes at the molecular level was provided by Southern analyses, with showed both linear and open circular forms of the replicating InPAct episome in genomic DNA extracted from infiltrated leaf tissue. \ud
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Following the demonstration of Rep-activatable reporter gene expression and episomal replication of the ToLCV-based InPAct-GUS vectors using transient and stable tobacco transformation assays, new ToLCV-based InPAct vectors were designed to express the lethal RNase, barnase, in an attempt to generate virus resistant plants. Although transient assays in NT-1 cells demonstrated some "leaky" expression of barnase from the InPAct vectors, the level of barnase-mediated cell death from the InPAct vectors was found to be significantly increased in the presence of the cognate Rep and REn. Thirteen independently transformed tobacco lines containing the ToLCV-[Au] InPAct-barnase cassette were generated by Agrobacterium-mediated transformation of tobacco leaf discs. However, agroinfiltration of these plants with ToLCV Rep and REn failed to activate a barnase response. Subsequent molecular analyses on two transgenic lines revealed that both contained mutations in the barnase-coding gene in a region known to encode the active site. These mutations were presumed to result from the leaky barnase expression during initial stages of the Agrobacterium transformation which would favour the selection of barnase mutant InPAct plants. \ud
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To overcome the problems associated with leaky expression of barnase, a barstar-expression cassette was included in the ToLCV-[Au] InPAct-barnase cassette. Transient assays in non-transgenic tobacco leaves demonstrated that the basal levels of barstar expressed from the modified InPAct vector were sufficient to negate the effects of leaky barnase expression. Importantly, however, the level of barnase expression in the presence of Rep and REn was shown to be sufficient to overcome the basal levels of barstar. Seventeen independently transformed lines were generated with the ToLCV-[Au] InPAct-barnase/barstar cassette, and analysis of one line revealed the presence of an uncorrupted barnase-coding region. Using transient agroinfiltration assays, seven of the transgenic lines showed varying levels of cognate Rep and REn-activated, barnase-induced cell death.\ud
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Fifteen transgenic lines were challenged with ToLCV-[Au] by injection of recombinant Agrobacteria containing an infectious ToLCV clone. Unfortunately, all lines displayed typical ToLCV symptoms and tested positive for virus by PCR at 28 days post-inoculation. The inability of the InPAct cassette to confer resistance to ToLCV may have been due to one or a combination of factors, including (i) a delay in barnase-induced cell death, (ii) homology-dependent silencing of the integrated cassette, (iii) generally low-level, Rep-activated barnase expression or (iv) excessive virus load due to the artifical method of inoculation.\ud
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This study details the first report of a ToLCV-based InPAct system for Rep-induced transgene expression in planta. Despite failing to generate ToLCV-resistant plants, the research findings will provide a solid foundation to develop a more effective InPAct vector and ultimately assist in the generation of transgenic plants with resistance to ToLCV and potentially other ssDNA plant viruses, particularly the begomoviruses
Back from the dead
No full textResurrection plants can survive for years in an air-dry state before growing at full capacity when the rain comes. How do they do it, and can this trait be transferred to improve the tolerance of crops to drought, heat, salinity and infection
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
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