Virus vector for gene silencing in wheat.

Wheat, rice, and maize account for more than 85% of the world’s cereal production (apps.fao.org/default.jsp), and the increasing global demand for wheat heightens the need for new varieties with improved disease resistance, tolerance to abiotic stresses, and grain quality. Incorporation of such desirable traits via biotechnological approaches first requires thorough characterization of genes and pathways, and high-throughput screening is an essential part of this process (1). Virusinduced gene silencing (VIGS) uses a viral-expressed transgene to trigger RNA silencing against an endogenous gene and can serve as a powerful tool for functional genomics by facilitating high-throughput screening processes (2). However, most VIGS vectors are only available for dicot plants. For example, potato virus X (PVX) and tobacco rattle virus (TRV) have been commonly used for solanaceous species (3,4) and pea early browning virus (PEBV) for legume species (5). Currently, the only published example of the successful use of a VIGS vector in monocots is the use of barley stripe mosaic virus (BSMV) in barley (6,7). The objective of the present study was to determine whether BSMV could serve as a useful VIGS vector in wheat (Triticum aestivum). Although predominantly a barley pathogen, BSMV occurs naturally in wheat as well. The genome of BSMV strain ND18 consists of three RNAs, designated α, β, and γ, and has been described previously (8). We used modified β and γ components of the ND18 strain to facilitate insertion of a PCR product and to increase efficiency of VIGS (Figure 1). The BSMV RNA β mutant “B7” was derived from wildtype RNA β, in which the expression of coat protein was disrupted by a mutation in the start codon (9), thereby enhancing gene silencing (7). The start codon of the γb open-reading frame was modified to create a BamHI site for insertion of cloned DNA fragments (γ-γbBamHI in Figure 1, designated as “B2” in Reference 10), while also blocking expression of the VIGS suppressor γb (8). This modification of γb is the major difference between this vector and the version used previously in barley (6,7). To verify its application, we tested VIGS with three marker genes