Virus-induced gene silencing (VIGS), a posttranscriptional gene silencing method, plays a role as an antivirus defense system in plants [1-3]. When the virus with targeting fragment has successfully entered into host plant cells, it begins to replicate and transcribes its DNA or RNA genome within cells. Double-stranded RNA (dsRNA) intermediates are produced and induce the degradation of target genes. Thus, changes in plant phenotype or physiological indicators can be induced to identify gene function effectively . VIGS has emerged as a widely functional genomics tool for knocking down the transcript level of genes in plants, especially in related genes of plant disease resistance, stress resistance, growth and development, and metabolic regulation [4-8]. Its advantages continue to emerge in related studies, including easy manipulation, wide application scope, yielding of fast results, high effectiveness, independence of genetic transformation, suitableness for largescale analysis of genes, and so on [7, 9-12].
In these years, various viral vectors have been successfully used in VIGS, such as tobacco mosaic virus (TMV) , satellite virus-induced silencing system (SVISS) , potato virus X (PVX) , barley stripe mosaic virus (BSMV) , Cotton leaf crumple virus (CLCrV) , African cassava mosaic virus (ACMV) , tobacco rattle virus (TRV) , cabbage leaf curl virus (CaLCuV) [20-21], and turnip yellow mosaic virus (TYMV) . Among them, TRV has the widest host range and induces mild viral symptoms after infecting plants such as dicotyledons and monocotyledons [22, 27-32]. Each viral vector has a certain host range, and the effect of inducing silencing is different. Several species have been used to establish VIGS systems, and gene function has been successfully verified, e.g., in Arabidopsis, apple, lettuce, eggplant, tobacco, strawberry, papaya, N. benthamiana, etc [22, 27-32]. Moreover, the silencing efficiency of VIGS is influenced by many parameters, including vector targeting fragment (insertion direction and the size of the insert sequence) [18, 32], infection pattern [33-34], culture environment [35-36] and plant growth stage . Therefore, more research and exploration are needed for more efficient VIGS systems.
Cabbage (Brassica oleracea L. var. capitata), an important Brassicaceae crop, is reported to be recalcitrant to transformation in many genotypes. VIGS has received recent attention because its low-cost and rapid high-throughput evaluation of gene function. VIGS systems have been established in some Brassicaceae species and play a critical role in analyzing gene function of these species. In the model plant Arabidopsis thaliana, some VIGS vectors have been successfully established and assist in gene function verification, including TRV, CaLCuV, pTYs (TYMV) [21, 37-39]. Yu et al. (2018) established an improved VIGS system based on the TYMV-derived vector that efficiently silenced the phytoene desaturase (PDS) gene in B. rapa through particle bombardment . Zheng et al. (2010) applied the VIGS vector TRV to knock down endogenous PDS expression in three plant species (A. thaliana, B. nigra and N. benthamiana) . These VIGS studies set foundations for VIGS studies in cabbage and other Brassicaceae family crops. However, there has been no report of VIGS of cabbage and it is unknown whether any VIGS vectors can be applied to unravel gene functions in cabbage. Therefore, it is necessary to explore and develop the VIGS system in cabbage.
In this study, we tested TRV, pTYs and CaLCuV to develop VIGS systems in cabbage, using PDS gene as an efficient control for VIGS. Finally, we efficiently down-regulate the PDS in cabbage by pTYs and CaLCuV, and this latter system could also be applied to other Brassicaceae. The VIGS system will pave an important way for analyzing gene function in Brassicaceae.