An efficient virus-induced gene silencing (VIGS) system for functional genomics in Brassicas using a cabbage leaf curl virus (CaLCuV)-based vector

Background: Virus-induced gene silencing (VIGS), a posttranscriptional gene silencing method, represents an effective technology for the analysis gene functions in plants. However, no VIGS vectors are available for Brassica oleracea till now. The gene silencing vectors TRV, pTYs and CaLCuV were chose to improve the VIGS system in cabbage, using phytoene desaturase (PDS) gene as an efficient visual indicator for VIGS.Results: We successfully silenced the expression of PDS and observed the photobleaching phenomenon in cabbage by pTYs and CaLCuV, with the latter displaying higher efficiency and lower cost. Then, the factors potentially affecting the silencing efficiency of VIGS by CaLCuV in cabbage, including targeting fragment strategy, inoculation method and incubation temperature, were compared. The optimized CaLCuV-based VIGS system is as follows: approximately 500 bp insert sequence, Agrobacterium OD600=1.0, vacuum osmosis method at the bud stage, and the incubation temperature of 22 °C, through which we achieved a stable silencing efficiency of 65%. To further test the effectiveness of the system, we selected two other genes in cabbage including Mg-chelataseH subunit (ChlH) and lobed-leaf 1 (LL1) to knock down their expression, and we observed the expected yellow or lobed leaves. In addition, we successfully applied the CaLCuV-based VIGS system in two other representative Brassica crops including B. rapa and B. nigra and thus expanded its application scope.Conclusion: These results demonstrate that CaLCuV-based VIGS effectively work in cabbage and can be used for analyzing gene function. Our VIGS system described here will contribute to efficient functional genomics research in Brassica crops.