Resources for virus-induced gene silencing in the grasses.

Virus-induced gene silencing (VIGS) is a very useful research tool for rapid creation of gene knockdown phenotypes that can be used to assess plant gene function (Kumagai et al., 1995; Ratcliff et al., 1997; Baulcombe, 1999). VIGS exploits the fact that infection by many RNA viruses activates a conserved, RNAbased plant antiviral defense response, which targets the RNA produced by infecting viruses for sequencespecific degradation (Ratcliff et al., 1997). By inserting a fragment of sequence into the viral vector from a plant gene under study, transcripts of the gene also become targets for degradation, thus causing the gene of interest to be significantly down-regulated or knocked down. Several aspects of VIGS make it a particularly useful tool for plant functional genomics studies. First, it is a rapid experimental procedure. In most instances, the knockdown phenotype of a gene of interest can be generatedwithin 1 to 2months of identifying the target sequence. This is far quicker than what is possible through the production and analysis of knockout mutants or stably transformed RNAi plants (Burch-Smith et al., 2004). Second, VIGS does not require full-length cDNA sequences to function, so experiments can be initiated without complete gene sequence information. Third, silencing is initiated by infecting plants with the VIGS construct, so silencing occurs transiently and the VIGS phenotype affects only a portion of the plant. This is unlike what occurs in stable RNAi or mutant plants where the loss-of-function phenotype occurs throughout the plant, thereby increasing the occurrence of lethal phenotypes, which can limit gene function evaluations. Related to this, VIGS can be performed on species that are difficult to transform for stable RNAi studies. Fourth, VIGS can be particularly useful for research in polyploid plants because gene silencing occurs through homology-dependent RNA-mediated gene silencing, and therefore any genes sharing at least 85% sequence identity are likely to be down-regulated (Kumagai et al., 1995; Holzberg et al., 2002). In this way, knockdown phenotypes can be observed because the closely related homeologous genes present in polyploids are likely to be silenced as well. However, one major limitation to the widespread adoption of VIGS has been the lack of suitable VIGS vectors for different plant species. Initially, VIGS was almost exclusively performed in Nicotiana benthamiana using vectors derived fromTobaccomosaic virus (Kumagai et al., 1995), Potato virus X (Ratcliff et al., 1997), and Tobacco rattle virus (Ratcliff et al., 2001; Liu et al., 2002b). In recent years, new protocols and vectors have expanded the list of dicotyledonous plants inwhichVIGS can be employed (e.g. tomato [Solanum lycopersicum; Liu et al., 2002a], Arabidopsis [Arabidopsis thaliana; Burch-Smith et al., 2006; Pflieger et al., 2008]), and potato [Solanum tuberosum; Brigneti et al., 2004; FaivreRampant et al., 2004]), but it was not until the report of silencing inbarley (Hordeumvulgare) usingbarley stripe mosaic virus (BSMV)-based vectors that VIGS became an option for functional genomics research in monocotyledonous plants and, more specifically, the grass species (Holzberg et al., 2002). This article will describe the VIGS systems currently in use in grass species and discuss what has been learned about their capabilities and limitations as functional genomics research tools.