Virus-induced gene silencing as a reverse genetics tool to study gene function.

Abstract

Reverse genetics has proven to be a powerful approach to elucidating gene function in plants, particularly in Arabidopsis. Virus-induced gene silencing (VIGS) is one such method and achieves reductions in target gene expression as the vector moves into newly formed tissues of inoculated plants. VIGS is especially useful for plants that are recalcitrant for transformation and for genes that cause embryo lethality. VIGS provides rapid, transient knockdowns as a complement to other reverse genetics tools and can be used to screen sequences for RNAi prior to stable transformation. High-throughput, forward genetic screening is also possible by cloning libraries of short gene fragments directly into a VIGS plasmid DNA vector, inoculating, and then looking for a phenotype of interest. VIGS is especially useful for studying genes in crop species, which currently have few genetic resources. VIGS facilitates a rapid comparison of knockdown phenotypes of the same gene in different breeding lines or mutant backgrounds, as the same vector is easily inoculated into different plants. In this chapter, we briefly discuss how to choose or construct a VIGS vector and then how to design and carry out effective experiments using VIGS.