Virus Induced Gene Silencing Optimization in Plants, Things to be Considered (original) (raw)
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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.
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.
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 ...
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...
Virus-Induced Gene Silencing for Functional Characterization of Genes in Petunia
Petunia, 2009
Although functional analysis of genes can be readily carried out in Petunia using standard transformation/regeneration techniques, this process is timeand labor-consuming. High throughput analysis of gene knockouts has been made possible by the use of virus-induced gene silencing (VIGS): fragments of target plant genes are included in the genome of a viral vector, the plant silences them as part of its viral defense mechanism, and the consequences of gene inactivation can be readily analyzed. In Petunia, we use a modified tobacco rattle virus (TRV) vector for VIGS. Infection typically results in chimeric plants, and it is therefore desirable to have a reporter that can show where target genes have been silenced. Inserting a fragment of the gene encoding PHYTOENE DESATURASE (PDS) results in silencing-induced photobleaching of leaves; inserting a fragment of the gene encoding CHALCONE SYNTHASE (CHS) allows us to visualize silencing in floral tissues of purple-flowered Petunia cultivars as white patches, sectors or even entire corollas. We have shown that the VIGS system can silence as many as five independent genes at one time. We describe here the methods that we have found to be efficient and effective for VIGS in Petunia, and describe some results obtained by silencing a range of genes, including some transcription factors.
Agrobacterium and Viral Vectors-Mediated in vivo Gene Silencing Assays in Plants
Open Access Journal of Science
Over the last years, the explosive growth of plant genomic resources and the development of advanced vectors enhanced the emergence of rapid systems to dissect plant gene function. Although transgenic approach has been applied to generate stable transgenic lines, this procedure is time-consuming and labor-intensive. Transient expression systems are continuously emerging during these years as an alternative tool for functional genomics studies in plants. Among these techniques, Agrobacterium-infiltration and infectious clones-derived plant viruses have been developed and successfully applied for versatile transient assays. Their effectiveness over a wide range of plant species is often validated. Here, the green fluorescent protein (GFP) gene was investigated as reporter gene for proof-of-concept to induce gene silencing in GFP-transgenic tobacco and grapevine plants either by inoculation of Arabis mosaic virus infectious clones or through in planta vacuum infiltration of Agrobacterium suspension. Leaves derived from greenhouse-grown grapevine plants were vacuum infiltrated with agrobacteria suspension containing GFP gene to induce silencing. Nicotiana benthamiana plants were agro-inoculated with Arabis mosaic virus infectious clones RNA 1 and RNA 2 along with a modified RNA satellite harboring a GFP construct. Systemic GFP-silencing was monitored during days and weeks after inoculation and data showed that silencing was achieved after 10-12 days post inoculation for tobacco, while it takes 6 weeks to be completely reached in grapevine leaves. The transient expression systems established here were successfully applied for inducing a knockout of endogenous gene. These techniques provide a fast and reliable tool to carry out gene function analysis in plants and open a future outlook with several downstream applications towards pathogen resistance or abiotic stress tolerance.
Annals of botany, 2007
Eschscholzia californica (California poppy) is an emerging model plant for 'evo-devo' studies from the basal eudicot clade of Papaveraceae. California poppy has a relatively small genome, a short life cycle and, most importantly, it is amenable for transformation. However, since this transformation protocol is time consuming, virus-induced gene silencing (VIGS) was evaluated as a fast method to obtain functional data for California poppy genes. Commercially available California poppy plants were infiltrated with Agrobacterium tumefaciens carrying the tobacco rattle virus plasmids pTRV1 and pTRV2. pTRV2 contained part of the eschscholzia Phytoene Desaturase (EcPDS) gene whose loss of function results in photobleaching of the green parts of the plant and in a lack of floral coloration. The degree and duration of these symptoms was evaluated for vegetative rosettes and plants in flower. It is shown that VIGS is able to effectively down-regulate the EcPDS gene in eschscholzia. V...
Virus-induced gene silencing and its applications
CAB Reviews: Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources, 2010
Virus-induced gene silencing (VIGS) is an efficient tool for plant functional genomics. Our current understanding about the phenomenon of post-transcriptional gene silencing (PTGS) in plant defence against viruses has enabled us to modify virus genomes for VIGS in different plant species. In this review, we provide a comprehensive update on the application of VIGS. We have attempted to cover the following topics in this review: different VIGS vectors available to date; VIGS as a tool to identify novel genes involved in various aspects of plant biology; VIGS in different plant species; utility of VIGS in different plant organs; VIGS for deciphering cellular functions of known genes; and future application of VIGS in modern plant biology.
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...
Current Journal of Applied Science and Technology, 2019
Virus-induced gene silencing (VIGS) is a powerful reverse genetics technology used to unravel the functions of genes. It uses viruses as vectors to carry targeted plant genes. The virus vector is used to induce RNA-mediated silencing of a gene or genes in the host plant. The process of silencing is triggered by dsRNA molecules, the mechanism is explained in this chapter. Over the years a large number of viruses have been modified for use as VIGS vectors and a list of these vectors is also included. As the name suggests, virus-induced gene silencing uses the host plant's natural defense mechanisms against viral infection to silence plant genes. VIGS is methodologically simple and is widely used to determine gene functions, including disease resistance, abiotic stress, biosynthesis of secondary metabolites and signal transduction pathways. Here, we made an attempt to describe the basic underlying molecular mechanism of VIGS, the methodology and various experimental requirements, as well as its advantages and disadvantages.