Development of a Broad bean wilt virus 2-based expression vector for gentian

- A novel viral expression vector was developed using Broad bean wilt virus 2 (BBWV-2).
- The first example of a viral vector from the genus Fabavirus.
- GFP was successfully expressed by BBWV-2 vector in various organs of gentian.
- GFP expression via BBWV-2 vector was stable at least up to 162 days in gentian.
- BBWV-2 vector can be used in petal color modification of gentian flower.

Japanese gentian is one of the most important ornamental flowers in Japan. Its most attractive trait is the vivid blue flower, whose color results from polyacylated anthocyanins. Other important traits in gentian breeding include flower shape, flowering time, and overwintering characteristics. To show the molecular mechanism by which these traits are regulated, transgenic gentian plants are often generated by Agrobacterium-mediated transformation. However, this method is not suitable for all gentian cultivars because of the difficulties in regeneration and/or transformation. Also, it takes a long time to obtain transgenic plants and analyze their phenotypes. Therefore, it is important to develop a more efficient method to analyze gene function in gentian. In the present study, we developed a novel viral expression vector for efficient gene functional analysis in gentian using Broad bean wilt virus 2 (BBWV-2) infectious cDNA. First, we expressed two reporter genes, GFP and GUS, in gentian using the BBWV-2 vector. Fluorescence from GFP was observed in various organs including the leaf, flower, root, and overwintering bud. β-Glucuronidase (GUS) activity was detected in the inoculated leaf, but not in upper uninoculated leaves because the GUS insert was unstable in the BBWV-2 vector. Next, we inoculated the BBWV-2 vector carrying GtMYB3, a transcription factor regulating anthocyanin biosynthesis, fused with SRDX, which encodes the EAR transcriptional repression domain. Expression of GtMYB3-SRDX via the BBWV-2 vector partly changed the gentian petal color from blue to white. The expression levels of genes encoding anthocyanin biosynthetic enzymes (flavanone 3-hydroxylase, dihydroflavonol-4-reductase, anthocyanidin synthase, flavonoid 3′,5′-hydroxylase, and anthocyanin 5,3′-aromatic acyltransferase) were markedly lower in the white parts of petals than in the blue parts. These results clearly indicated that GtMYB3-SRDX expressed via the BBWV-2 vector was functional in gentian flowers. This BBWV-2-based gene expression system enabled us to analyze a phenotypic change in gentian flower color within a few months.