Suppression of gene silencing: A general strategy used by diverse DNA and RNA viruses of plants (original) (raw)
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Silencing of a viral RNA silencing suppressor in transgenic plants
The Journal of general virology, 2002
High expression levels of the helper component proteinase (HC(pro)), a known virus suppressor of RNA silencing, were attained in Nicotiana benthamiana transformed with the HC(pro) cistron of Potato virus A (PVA, genus Potyvirus). No spontaneous silencing of the HC(pro) transgene was observed, in contrast to the PVA coat protein (CP)-encoding transgene in other transgenic lines. HC(pro)-transgenic plants were initially susceptible to PVA and were systemically infected by 14 days post-inoculation (p.i.) but, 1 to 2 weeks later, the new expanding leaves at positions +6 and +7 above the inoculated leaf showed a peculiar recovery phenotype. Leaf tips (the oldest part of the leaf) were chlorotic and contained high titres of PVA, whereas the rest of the leaf was symptomless and contained greatly reduced or non-detectable levels of viral RNA, CP and transgene mRNA. The spatial recovery phenotype suggests that RNA silencing is initiated in close proximity to meristematic tissues. Leaves at p...
Virus-induced gene silencing in plants
Methods, 2003
Virus-induced gene silencing (VIGS) is a technology that exploits an RNA-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying inserts derived from host genes the process can be additionally targeted against the corresponding mRNAs. VIGS has been used widely in plants for analysis of gene function and has been adapted for high-throughput functional genomics. Until now most applications of VIGS have been in Nicotiana benthamiana. However, new vector systems and methods are being developed that could be used in other plants, including Arabidopsis. Here we discuss practical and theoretical issues that are specific to VIGS rather than other gene ''knock down'' or ''knockout'' approaches to gene function. We also describe currently used protocols that have allowed us to apply VIGS to the identification of genes required for disease resistance in plants. These methods and the underlying general principles also apply when VIGS is used in the analysis of other aspects of plant biology.
Applications and advantages of virus-induced gene silencing for gene function studies in plants
Plant Journal, 2004
Virus-induced gene silencing (VIGS) is a recently developed gene transcript suppression technique for characterizing the function of plant genes. The approach involves cloning a short sequence of a targeted plant gene into a viral delivery vector. The vector is used to infect a young plant, and in a few weeks natural defense mechanisms of the plant directed at suppressing virus replication also result in specific degradation of mRNAs from the endogenous plant gene that is targeted for silencing. VIGS is rapid (3–4 weeks from infection to silencing), does not require development of stable transformants, allows characterization of phenotypes that might be lethal in stable lines, and offers the potential to silence either individual or multiple members of a gene family. Here we briefly review the discoveries that led to the development of VIGS and what is known about the experimental requirements for effective silencing. We describe the methodology of VIGS and how it can be optimized and used for both forward and reverse genetics studies. Advantages and disadvantages of VIGS compared with other loss-of-function approaches available for plants are discussed, along with how the limitations of VIGS might be overcome. Examples are reviewed where VIGS has been used to provide important new insights into the roles of specific genes in plant development and plant defense responses. Finally, we examine the future prospects for VIGS as a powerful tool for assessing and characterizing the function of plant genes.
Suppression of virus accumulation in transgenic plants exhibiting silencing of nuclear genes
The Plant Cell Online, 1996
ing of nonviral transgenes prevented virus accumulation. This effect was dependent on sequence homology between the virus and the silenced transgene. Analysis of potato virus X derivatives carrying bacterial l3-glucuronidase (GUS) sequences showed that the 3' region of the GUS coding sequence was a target of the silencing mechanism in two independent tobacco lines. Methylation of the silenced GUS transgenes in these lines was also concentrated in the 3' region of the GUS coding sequence. Based on this concurrence, we propose a link between the DNA-based transgene methylation and the RNA-based gene silencing process.
The Plant Cell, 1993
Transgenic tobacco plants expressing either a full-length form of the tobacco etch virus (TEV) coat protein or a form truncated at the N terminus of the TEV coat protein were initially susceptible to TEV infection, and typlcal systemic symptoms developed. However, 3 to 5 weeks after a TEV infection was established, transgenlc plants "recovered" from the TEV infection, and new stem and leaf tissue emerged symptom and virus free. A TEV-resistant state was induced ln the recovered tissue. The resistance was virus speclflc. Recovered plant tissue could not be lnfected wlth TEV, but was susceptlble to the closely related virus, potato virus Y. The resistance phenotype was functional at the slngle-cell leve1 because protoplasts from recovered transgenic tissue did not support TEV repllcation. Surprlslngly, steady state transgene mRNA levels in recovered tissue were 12-to 22-fold less than transgene mRNA levels in uninoculated transgenic tissue of the same developmental stage. However, nuclear run-off studles suggested that transgene transcription rates in recovered and uninoculated plants were similar. We propose that the resistant state and reduced steady state levels of transgene transcript accumulation are mediated at the cellular leve1 by a cytoplasmic actlvity that targets speclfic RNA sequences for inactlvation.
Transcriptional and Posttranscriptional Plant Gene Silencing in Response to a Pathogen
Science, 1998
Plants are able to respond to pathogen attack to restrain development of a systemic infection. The response of Brassica napus (oilseed rape) to systemic infection with the DNA virus cauliflower mosaic virus was shown to result in enhancement and subsequent suppression of viral gene expression in parallel with changes in symptom expression. Transgenes with homology to viral sequences were also affected. This phenomenon, which was shown to be mediated by both transcriptional and posttranscriptional mechanisms, might be related to regulation of highly expressed genetic elements.
Antiviral strategies in plants based on RNA silencing
Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, 2011
One of the challenges being faced in the twenty-first century is the biological control of 19 plant viral infections. Among the different strategies to combat virus infections, those 20 based on pathogen-derived resistance (PDR) are probably the most powerful approaches 21 to confer virus resistance in plants. The application of the PDR concept not only 22 revealed the existence of a previously unknown sequence-specific RNA-degradation 23 mechanism in plants, but has also helped to design antiviral strategies to engineer viral 24 resistant plants in the last 25 years. In this article, we review the different platforms 25 related to RNA silencing that have been developed during this time to obtain plants 26 resistant to viruses and illustrate examples of current applications of RNA silencing to 27 protect crop plants against viral diseases of agronomic relevance. 28 29 1. Introduction 30 Plant viruses represent important threats to modern agriculture. Although accurate 31 figures for crop losses due to viruses are not available, it is generally accepted that 32 among the different plant pathogens, the economic relevance of viruses comes second to 33 fungi. Until the emergence of genetic engineering technologies, plant viruses have been 34 partially controlled using conventional cultivation techniques such as crop rotation, 35 early detection and eradication of the diseased plants, cross protection, breeding for 36 resistance, or chemical control of their vectors [1]. In the 1980s, the successful transfer 37 of foreign DNA into the nuclear genome using Agrobacterium as a vector prompted the 38 introduction of genetic engineering for crop improvement and the development of virus-39 resistant plants [2, 3]. Today, different antiviral strategies are being undertaken, either 40 by exploiting natural plant defence mechanisms, or designing new tools, which in most 41 cases are ultimately also based on natural defence mechanisms. 42 Most of the achievements obtained in plant biotechnology in the area of plant virus 43 resistance are based on the principle of pathogen-derived resistance (PDR) [4]. The 44 concept of PDR was proposed by Sanford and Johnston [5] twenty-five years ago using 45 the bacteriophage Qß as a model, and considers that expression of pathogen genetic 46 elements outside the context of infection may lead to resistance. This approach opened 47 an interesting possibility for the practical control of diseases. For plant viruses, the 48 concept of PDR was first validated with its use in tobacco plants transformed with the 49 tobamovirus Tobacco mosaic virus (TMV) coat protein (CP) gene [6]. Soon this 50 observation was validated using other viral CPs and other viral sequences that code for 51 proteins such as replicases, proteinases and movement proteins [for review, see 7-11]. 52 CP is the most successful and widely applied viral protein for PDR. However, the 53 protection conferred by CP-mediated resistance varies significantly from strong 54 interference with virus multiplication to delay or attenuation of symptoms. The PDR 55 based on the expression of viral proteins, with either the wild type or the mutated one, 56 in transgenic plants has several general characteristics: i) it is not very specific, and 57 protects against a broad range of viral strains; ii) it shows a positive correlation between 58 the levels of accumulation of the viral product and the effectiveness in resistance; iii) it 59 is usually overcome by high doses of inoculum. Despite extensive studies, the 60 molecular mechanisms underlying protein-mediated resistance are not fully understood. 61 What appears to be certain is that they are diverse, that they probably affect several 62 steps of the infection process, and that each virus/transgenic plant combination has 63 specific features. Moreover, it soon became apparent that many virus resistances 64 initially envisaged as protein-mediated PDR did not rely on the expression of the 65 corresponding viral proteins and that a majority of PDR phenomena seemed to work 66 through RNA-mediated mechanisms [12]. 67 68 2. RNA silencing and virus resistance 69 In the early nineties, two independent research groups found that the expression of 70 a transgene mRNA with a high sequence similarity to an endogenous mRNA, led to 71 specific degradation of both mRNAs through post-transcriptional gene silencing 72 (PTGS), also known as "cosuppression" [13, 14]. Later, the W. Dougherty research 73 group suggested that a similar mechanism might be involved in the resistance 74 phenomena observed in transgenic plants transformed with viral genes. Some of the 75 transgenic lines showed anomalous phenotypes; unexpectedly and unpredictably the 76 highest level of resistance was observed in the transgenic lines showing very low levels 77 of transgene mRNA accumulation, whereas plant lines expressing the same gene at high 78 levels were fully susceptible. Interestingly, the virus resistant plants had actively 79 transcribed genes but they had low steady-state levels of transgene mRNA. A 80 breakthrough discovery, from transgenic lines included to serve as negative controls, 81 showed that resistance occurred even with non-translatable versions of the viral genes, 82 which demonstrated that the RNA itself was responsible for the virus resistance 83 observed in the transgenic plants [15-17]. All the molecular analysis of these transgenic 84 plants challenged the existing paradigm of genetic regulation and became the first 85
Virus Induced Gene Silencing Optimization in Plants, Things to be Considered
Postdoc Journal, 2014
Study of biological processes is mostly limited to model plant species possessing considerable advantages like small genome size, tractability for genetic studies, ease of use, short generation time, and consequently availability of immense genetic resources. Discoveries from model species are extremely valuable but not enough for improvement of agronomic characteristics of economically important plants mainly due to divergence of mechanisms through evolution. Transient techniques are emerging as powerful tools for functional studies in diverse plant species and for validation of discoveries made in model species. Virus Induced Gene Silencing (VIGS), a transient reverse genetics tool, is extensively being used for performing rapid loss-of-function experiments in plants. Several of the advantages of VIGS including its suitability for high throughput studies will extend functional studies to diverse plant species, contributing to our understanding of unique biological processes. One of the main factors hindering even wider application of VIGS is its requirement for specific conditions with each species. This manuscript reviews the available information in the literature regarding efforts invested in several plant species and points out the key factors to be considered optimizing for achievement of efficient gene knock-down phenotypes in novel plant species.
Molecular Plant-Microbe Interactions, 2007
Tomato bushy stunt virus (TBSV) coat protein (CP) replacement vectors have been used previously to silence transgenes (e.g., the green fluorescent protein gene) but have not been effective for silencing endogenous plant genes. New TBSV vectors which retained the CP gene were developed by engineering an XhoI restriction site in three positions (3f, CEB, and CEA) of the pTBSV-100 infectious clone. Magnesium chelatase (ChlH) and phytoene desaturase (PDS) were chosen as targets for endogenous gene silencing. Initial experiments using CP replacement vectors with a 230-bp sense or antisense ChlH insert gave a silencing phenotype prominent only in the first new leaves above those inoculated. No silencing phenotype was apparent beyond these leaves whereas, for PDS, no silencing phenotype was observed. When plants were inoculated with the XhoI insert vectors containing ChlH and PDS sequences, plants showed a silencing phenotype extensively throughout the challenged plant, indicating an impro...