Virus Induced Gene Silencing (VIGS) for Functional Genomics of Crop Plants
Authors: Basavaprabhu L. Patil*, Monika Dalal, Rohini Sreevathsa, Subodh Kumar Sinha

In the past, plant scientists mostly relied upon forward genetics; which involved identification of a mutant and subsequent cloning of the mutated gene to identify the gene responsible for the function/trait under investigation. During the last several years, Arabidopsis, rice, tomato, cassava, pigeon pea, chick pea and other plant genomes have been sequenced and large data base of sequence information has been generated. Such genome and EST sequencing projects have generated a wealth of sequence information for important plant species of which the majority is difficult to subject to functional genomics. Thus alternative approaches to traditional forward genetics need to be employed to identify the genes responsible for a trait or a function. An important alternative approach is reverse genetics, which investigates the function of a gene by altering the expression of the gene of interest and then identifying the mutant phenotype that is produced. Most reverse genetics approaches described in plants to date rely on posttranscriptional gene silencing (PTGS) or RNA interference (RNAi). Knocking out of genes is the most commonly used strategy of reverse genetics to know the gene functions. Two most common examples of insertional mutagenesis approaches, predominantly used in Arabidopsis are: transferred DNA (T-DNA) and transposon tagging. Although these are robust tools for providing mutants, they have some limitations, such as difficulty in studying the function of duplicated genes in the case of multigene families, the difficulty to reach genome saturation, and the multiple insertional nature of these strategies, which frequently lead to simultaneous knock down of multiple genes, thus making it difficult to identify the correct gene.

Virus-induced gene silencing (VIGS) is a technology that exploits an RNA-mediated (RNAi) antiviral defense mechanism and has been shown to be of great potential in plant reverse genetics or the functional genomics. The term "VIGS" was coined by van Kammen in 1997, to describe the phenomenon of recovery from virus infection. However, the term has since been applied almost exclusively to the technique involving recombinant viruses to knock-down expression of endogenous genes. The discovery of PTGS of endogenous genes by recombinant viruses carrying an identical sequence was made in 1995 and immediately the potential of VIGS as a tool for the analysis of gene function was quickly recognized. Circumvention of plant genetic transformation, methodological simplicity, robustness, and speedy results makes VIGS an attractive alternative approach in plant functional genomics. Thus VIGS provides a powerful tool to facilitate gene functional studies for plant species which are recalcitrant for genetic transformation. Recent approaches allow the use of VIGS as a high throughput method that will exploit the potential of genome and transcriptome projects further. Since the discovery of VIGS, it has been widely employed to characterize the plant genes involved in cellular functions, metabolic pathways, plant-microbe, plant-nematode and plant-insect interaction. In addition to the biotic stresses, several abiotic stresses like drought, salinity, temperature, water are major constraints limiting crop production across the globe. VIGS has also been employed to identify genes for nutrient biofortification in crop plants.

Tobacco mosaic virus (TMV) was the first RNA virus to be used as a silencing vector in 1995 and later in 2001, the limitations of host range and meristem exclusion were overcome with development of VIGS vectors based on Tobacco rattle virus (TRV). One of the more interesting developments to improve the VIGS technology is use of bipartite Cabbage leaf curl virus (CbLCV) in Arabidopsis by Turnage et al. (2002). Lack of appropriate VIGS vectors with broad host range is a major limitation and to facilitate this, VIGS vectors with broad host range have been developed by employing Tobacco rattle virus (TRV) and Apple latent spherical virus (ASLV) vectors. VIGS vectors that produce severe symptoms in host plants should be avoided to have proper visualization of the plant phenotype exhibited by gene/s under investigation. Lack of silencing in certain tissues, uneven or localized silencing is another concern of VIGS and for this reporter gene (GFP) expression along with expression of VIGS vectors should be useful for visualizing the silenced tissues. To prevent low silencing efficiency, insert should be in range of 200-350 bp, localized expression of a viral silencing suppressor improves virus multiplication and off-target silencing by VIGS vectors should be taken care off. Over the years, improved methods for VIGS vector delivery into plants, namely agro-drench, toothpick inoculation and viral sap inoculation have been developed and the availability of such methods has facilitated large-scale screening of plant genes. This also has helped in customized application of VIGS to meet the specific requirements of plant biologists. Thus VIGS as a reverse genetics tool for studies on plant functional genomics presents several advantages, promises rapid generation of functional genomics and even proteomics. With the progress in whole genome sequencing of many important crop plants, VIGS approach will be widely and popularly used.

Suggested Reading:
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