Knocking down wheat meiotic genes through Virus-induced gene-silencing (VIGS)

Virus-induced gene-silencing (VIGS) is a reverse genetic technology that can be used to assess gene function in model and crop species. The technique exploits a natural viral defence response in plants, whereby invading viral RNAs are detected, and this triggers a targeted post-transcriptional gene silencing (PTGS) mechanism. In VIGS, a small fragment of a plant gene of interest is inserted into a viral vector to generate a recombinant virus that, upon infection, leads to silencing of both the foreign viral RNA and the endogenous target gene; creating knockdown lines. We have applied barley stripe mosaic virus (BSMV)-mediated VIGS to the study of recombination in hexaploid wheat, and have optimised the targeting of meiotic genes at peak expression. Following VIGS, successful down-regulation of gene expression can be assessed by qRT-PCR, and meiotic phenotypes analysed cytologically. An initial proof-of-concept study was conducted by silencing the recombinase TaDMC1, which resulted in a severe meiotic phenotype with the majority of homologous chromosomes remaining unpaired, visible as univalents, and we are currently expanding this approach to incorporate additional targets. VIGS is proving to be a rapid and cost-effective way of assessing the function of meiotic genes in wheat, enabling us to efficiently screen a broad range of candidate genes, so that further analyses using TILLING or CRISPR/Cas may be employed on promising targets.