Quantitative Analysis of Efficient Endogenous Gene Silencing in Nicotiana benthamiana Plants Using Tomato bushy stunt virus Vectors That Retain the Capsid Protein Gene (original) (raw)
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Suppression of gene silencing: A general strategy used by diverse DNA and RNA viruses of plants
Proceedings of the National Academy of Sciences, 1999
In transgenic and nontransgenic plants, viruses are both initiators and targets of a defense mechanism that is similar to posttranscriptional gene silencing (PTGS). Recently, it was found that potyviruses and cucumoviruses encode pathogenicity determinants that suppress this defense mechanism. Here, we test diverse virus types for the ability to suppress PTGS. Nicotiana benthamiana exhibiting PTGS of a green fluorescent protein transgene were infected with a range of unrelated viruses and various potato virus X vectors producing viral pathogenicity factors. Upon infection, suppression of PTGS was assessed in planta through reactivation of green fluorescence and confirmed by molecular analysis. These experiments led to the identification of three suppressors of PTGS and showed that suppression of PTGS is widely used as a counter-defense strategy by DNA and RNA viruses. However, the spatial pattern and degree of suppression varied extensively between viruses. At one extreme, there are...
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.
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 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.
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.
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.
African Journal of Biotechnology, 2018
Cucurbit yellow stunting disorder virus (CYSDV) and Watermelon chlorotic stunt virus (WmCSV) are the most widespread and damaging viruses to cucurbits in the Middle East. CYSDV and WmCSV are cucurbit-infecting bipartite whitefly-transmitted viruses. Post-transcriptional gene silencing (PTGS) is a universal mechanism by which plants are able to systemically switch off the expression of targeted genes via the reduction of steady-state levels of specific RNAs. PTGS was used in this study to control the two viruses. In this study, the efficiency of the dsRNA for the ability to trigger resistance against the CYSDV and WmCSV was investigated. Three regions of three genes of CYSDV genome were selected; the coat protein gene (CP), heat shock gene (Hsp70) and ORF3, while the two regions of two genes of WmCSV genome were selected; CP gene and rep gene. Bioassay, dot-blot hybridization and polymerase chain reaction (PCR) methods were capable to evaluate the resistance against viruses. Clear symptoms on tobacco plants took two to three weeks to appear and all non-infiltrating tobacco plants (positive control) showed viral symptoms after inoculation. Most of the agro-infiltrating sense/antisense constructs did not yield symptoms of the viruses. Dot-blot hybridization, showed that negative hybridization was obtained with infiltrating tobacco plants with prepared constructs compared to those non-infiltrating tobacco plants used as the control. Only one out of five gave positive signals with the construct pasCYSDV-Hsp70. Using PCR, positive reactions of the expected size of 500 bp fragment with WmCSV and 800 bp with CYSDV were obtained with the infiltrating tobacco plants with sense constructs, which pointed out the existence of viral genome in challenging tobacco plants. Infiltrating tobacco plants with sense/antisense constructs gave negative PCR pointed out the lack of the viral genome.
Efficient Virus-Induced Gene Silencing in Arabidopsis
Plant Physiology, 2006
Virus-induced gene silencing (VIGS) is a plant RNA-silencing technique that uses viral vectors carrying a fragment of a gene of interest to generate double-stranded RNA, which initiates the silencing of the target gene. Several viral vectors have been developed for VIGS and they have been successfully used in reverse genetics studies of a variety of processes occurring in plants. This approach has not been widely adopted for the model dicotyledonous species Arabidopsis (Arabidopsis thaliana), possibly because, until now, there has been no easy protocol for effective VIGS in this species. Here, we show that a widely used tobacco rattle virus-based VIGS vector can be used for silencing genes in Arabidopsis ecotype Columbia-0. The protocol involves agroinfiltration of VIGS vectors carrying fragments of genes of interest into seedlings at the two- to three-leaf stage and requires minimal modification of existing protocols for VIGS with tobacco rattle virus vectors in other species like ...
Transient gene expression is a fast, flexible and reproducible approach to high-level expression of useful proteins. In plants, recombinant strains of Agrobacterium tumefaciens can be used for transient expression of genes that have been inserted into the T-DNA region of the bacterial Ti plasmid. A bacterial culture is vacuum-infiltrated into leaves, and upon T-DNA transfer, there is ectopic expression of the gene of interest in the plant cells. However, the utility of the system is limited because the ectopic protein expression ceases after 2-3 days. Here, we show that post-transcriptional gene silencing (PTGS) is a major cause for this lack of efficiency. We describe a system based on co-expression of a viral-encoded suppressor of gene silencing, the p19 protein of tomato bushy stunt virus (TBSV), that prevents the onset of PTGS in the infiltrated tissues and allows high level of transient expression. Expression of a range of proteins was enhanced 50folds or more in the presence of p19 so that protein purification could be achieved from as little as 100 mg of infiltrated leaf material. The effect of p19 was not saturated in cells that had received up to four individual T-DNAs and persisted until leaf senescence. Because of its simplicity and rapidity, we anticipate that the p19-enhanced expression system will have value in industrial production as well as a research tool for isolation and biochemical characterisation of a broad range of proteins without the need for the timeconsuming regeneration of stably transformed plants.
Plant Biotechnology Journal, 2011
Virus-induced gene silencing (VIGS) is one of the commonly used RNA silencing methods in plant functional genomics. It is widely known that VIGS can occur for about 3 weeks. A few reports show that duration of VIGS can be prolonged for up to 3 months. Increasing the duration of endogenous gene silencing and developing a method for nonintegration-based persistent VIGS in progeny seedlings will widen the application of VIGS. We used three marker genes that provoke visible phenotypes in plants upon silencing to study persistence and transmittance of VIGS to progeny in two plant species, Nicotiana benthamiana and tomato. We used a Tobacco rattle virus (TRV)-based VIGS vector and showed that the duration of gene silencing by VIGS can occur for more than 2 years and that TRV is necessary for longer duration VIGS. Also, inoculation of TRV-VIGS constructs by both Agrodrench and leaf infiltration greatly increased the effectiveness and duration of VIGS. Our results also showed transmittance of VIGS to progeny seedlings via seeds. A longer silencing period will facilitate detailed study of target genes in plant development and stress tolerance. Further, the transmittance of VIGS to progeny will be useful in studying the effect of gene silencing in young seedlings. Our results provide a new dimension for the application of VIGS in plants.