Virus-Induced Gene Silencing as a Scalable Tool to Study Drought Tolerance in Plants

Abstract

Here we describe the methodology of using virus-induced gene silencing (VIGS) as a powerful and scalable tool to screen the function of genes that participate in adaptation to drought. Silencing of endogenous gene expression in Nicotiana benthamiana is achieved by systemic infection of the aerial parts of the plant with a virus engineered to contain homologous fragments of the target gene(s) of interest. Silenced plant material can be consistently produced with little optimization in less than 1 month without specialized equipment, using only simple cloning and transformation techniques. Although maximal silencing is localized to only a few leaves, when whole plants are subjected to water stress, the tissue from these silenced leaves can be characterized for physiological, biochemical, and transcriptional responses to determine the role of the candidate genes in drought tolerance.

Vacuum and Co-cultivation Agroinfiltration of (Germinated) Seeds Results in Tobacco Rattle Virus (TRV) Mediated Whole-Plant Virus-Induced Gene Silencing (VIGS) in Wheat and Maize

“…Vacuum agroinfiltration has been used in VIGS systems in various previous studies (Tague and Mantis, 2006; George et al, 2010, 2012, 2015; Kirigia et al, 2014; Tian et al, 2014). In the experiments reported here it was applied using 20 mL syringes and 10 mL medical glass bottles ( Figures 1B , 2B and Supplementary Figure S3).…”

“…Vacuum-aided agroinfiltration has proven ability to increase infection efficiency in genetic transformation and VIGS procedures, especially for plants that have high resistance to infiltration (Tague and Mantis, 2006; George et al, 2010, 2012, 2015; King et al, 2014; Tian et al, 2014). Thus, we employed vacuum-aided agroinfiltration and found that applying it for 30 and 60 s resulted in the highest silencing efficiencies for wheat and maize, respectively.…”

“…Moreover, it can silence genes in a broad range of plants, such as tomato, tobacco, Arabidopsis , rose, cotton, apple, and the parasitic plant Striga hermonthica (Liu et al, 2002a; Kirigia et al, 2014; Tian et al, 2014; Mustafa et al, 2016). TRV-VIGS in tobacco can be accomplished within 4 weeks, and is reportedly faster and easier than other available gene silencing techniques (George et al, 2012, 2015; Senthil-Kumar and Mysore, 2014). However, it is rarely reported that TRV-VIGS was used in monocot plant species, especially in wheat and maize.…”

Analysis of the Role of the Drought-Induced Gene DRI15 and Salinity-Induced Gene SI1 in Alternanthera philoxeroides Plasticity Using a Virus-Based Gene Silencing Tool

“…Virus-induced gene silencing is a valuable tool that can effectively silence individual genes or families of genes critical for plant development or resistance to biotic and abiotic stresses ( George et al, 2015 ). This tool is a simple and rapid method for assessing gene function ( Burch-Smith et al, 2004 ; Robertson, 2004 ; Becker and Lange, 2010 ; Cakir et al, 2010 ).…”