Tobacco rattle virus-induced VcANS gene silencing in blueberry fruit

Blueberry (Vaccinium corymbosum L.), a woody perennial bush in the genus Vaccinium, is an economically important and popular fruit crop worldwide. Development the superior cultivars, which including excellent fruit traits, not only means higher yielding and economic efficiency, but also produce fruit that to meet the preferences of different consumers. Excavating fruit quality-related genes, studying their functions, and using transgenic or molecular-assisted breeding are beneficial to the development of excellent blueberry varieties. Genetic transformation is an excellent way to study the function of genes in plants, however, it is a labor-intensive and time-consuming process to genetically transform many woody plants, including blueberry. Virus-induced gene silencing (VIGS) provides an efficient approach to knock-down the expression of target genes for functional analysis. In this study, tobacco rattle virus induced genes silencing (TRV-VIGS) was established in blueberry fruits using the VcANS gene as a reporter. The silenced sector of the skin of blueberry fruits injected with pTRV2 (plasmid Tobacco Rattle Virus, TRV-RNA2)::VcANS remained green or white at 25 days after agroinfiltration. In agroinfiltrated materials, the VcANS transcript levels were much lower in fruits with phenotypic changes (delayed color change) than in those infiltrated with the pTRV2 empty vector. Silencing of VcANS also affected the expression of other genes involved in the anthocyanin synthesis pathway. The experimental results support that VcANS can be used as an effective marker gene for VIGS system. In addition, the TRV-VIGS system has been successfully established in blueberry fruits, which provided an effective verification method for functional identification of unknown genes in blueberry fruits.