Silencing of multiple genes in wheat using barley stripe mosaic virus

Virus-induced gene silencing (VIGS) is a powerful reverse genetics tool wherein the expression of a targeted gene is reduced by a viral vector in a sequence homology-dependent manner. A VIGS system using a modified Barley stripe mosaic virus (BSMV) has been developed and successfully employed in the functional characterization of genes in monocots. There are several inherent limitations to the BSMV-VIGS system including silencing variability and silencing transiency. These limitations make it critical to know where and when the targeted gene is silenced for efficient downstream functional analysis. One effective resolution to these limitations is the utilization of a silencing-reporter gene. In wheat the BSMV-VIGS of phytoene desaturase (PDS) results in a visible photobleaching silencing phenotype. Similarly, BSMV-VIGS of the leaf rust resistance gene, Lr21 results in a leaf rust resistance-breaking silencing phenotype. Here we used BSMV-VIGS to simultaneously silence PDS and Lr21 to test the utility of concurrently silencing two genes. When both PDS and Lr21 were silenced in the same tissue at the same time, there was no compromise in viral infection efficiency, silencing efficiency, or silencing efficacy. The results discussed here suggest that PDS can be used as an endogenous silencing-reporter of a co-targeted gene.