Virus-induced gene silencing (VIGS) for functional characterization of disease resistance genes in barley seedlings

Authors: GUNUPURU, LOKANADHA - University College Dublin; PEROCHON, ALEXANDER - University College Dublin; ALI, SHAHIN - US Department Of Agriculture (USDA); Scofield, Steven - Steve; DOOHAN, FIONA - University College Dublin

Submitted to: Methods in Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/1/2018
Publication Date: 11/21/2018
Citation: Gunupuru, L.R., Perochon, A., Ali, S.S., Scofield, S.R., Doohan, F.M. 2018. Virus-induced gene silencing (VIGS) for functional characterization of disease resistance genes in barley seedlings. Methods in Molecular Biology. 1900:95-114. https://doi.org/10.1007/978-1-4939-8944-7_7.
DOI: https://doi.org/10.1007/978-1-4939-8944-7_7

Interpretive Summary: Virus-induced gene silencing (VIGS) is a useful functional genomics tool for rapidly creating gene knockout phenotypes that can be used to infer gene function. Until recently, VIGS has only been possible in dicotyledonous plants. However, the development of vectors based on barley stripe mosaic virus (BSMV) has now made VIGS possible in barley and wheat. VIGS has particular advantages for functional genomics in barley and wheat, beause their recalcitranceto transformation greatly hinder strategies for functional identification of genes. Here methods are presented for using the Barley stripe mosaic virus VIGS system (BSMV-VIGS) to silence genes in barley.

Technical Abstract: With the recent advances in sequencing technologies, many studies are generating lists of candidate genes associated with specific traits. The major bottleneck in functional genomics is the validation of gene function. This is achieved by analyzing the effect of either gene silencing or overexpression on a specific phenotypic or biochemical trait. This usually requires the generation of stable transgenic plants and this can take considerable time. Therefore, any technique that expedites the validation of gene function is of particular benefit in cereals, including barley. One such technique is Virus-Induced Gene Silencing (VIGS), which evokes a natural antiviral defense mechanism in plants. VIGS can be used to downregulate gene expression in a transient manner, but long enough to determine its effects on a specific phenotype. It is particularly useful for screening candidate genes and selecting those with potential for disease control. VIGS based on Barley Stripe Mosaic Virus (BSMV) is a powerful and efficient tool for the analysis of gene function in cereals. Here we present a BSMV VIGS protocol for simple and robust gene silencing in barley and describe it to evaluate the role of the hormone receptor BRI1 (Brassinosteroid Insensitive 1) in barley leaf resistance to Fusarium infection.