Improved virus-induced gene silencing allows discovery of a serpentine synthase gene in Catharanthus roseus

Abstract

Specialized metabolites are chemically complex small molecules with a myriad of biological functions. To investigate plant specialized metabolite biosynthesis more effectively, we developed an improved method for virus-induced gene silencing (VIGS). We designed a plasmid that incorporates fragments of both the target gene and knockdown marker gene (phytoene desaturase, PDS), which identifies tissues that have been successfully silenced in planta. To demonstrate the utility of this method, we used the terpenoid indole alkaloid (TIA) pathway in Madagascar periwinkle (Catharanthus roseus) as a model system. C. roseus is a medicinal plant well known for producing many bioactive compounds such as vinblastine and vincristine. Our VIGS method enabled the discovery of a previously unknown biosynthetic enzyme, serpentine synthase. This enzyme is a cytochrome P450 that produces the β-carboline alkaloids serpentine and alstonine, compounds with strong blue autofluorescence and potential pharmacological activity. The discovery of this enzyme highlights the complexity of TIA biosynthesis and demonstrates the utility of this improved VIGS method for discovering unidentified metabolic enzymes in plants.

Development and application of a virus-induced gene silencing protocol for the study of gene function in narrow-leafed lupin

“…The fact that we did observe the same chemotype suggests that silencing of LaLDC occurred at an earlier time point and then subsided. Indeed, due to their transient nature, VIGS systems in plants are often reported to be variable concerning the timing and extent of silencing [ 24 , 41 , 42 ]. Therefore, we decided to test a ‘PDS co-silencing approach’ where we silenced our visual marker LaPDS alongside the target gene LaLDC using a single construct.…”

“…We developed the PDS co-silencing approach to improve reproducibility and decrease the time, effort, and costs associated with the VIGS experiments. In general, the effectiveness of VIGS systems is often reported as variable with the timing and levels of silencing varying between batches of plants, individual plants, or even patches of cells from a single tissue type [ 24 , 41 , 42 ]. This makes it difficult to determine the exact tissue and time point at which silencing of the target gene is occurring when using the traditional protocols.…”

“…This is particularly relevant for viruses like ALSV, which do not cause visible symptoms in the host plant. A similar PDS co-silencing approach has been employed very recently in two other VIGS systems in Antirrhinum majus [ 46 ] and Catharanthus roseus [ 41 ]. In the case of C. roseus , the PDS co-silencing strategy was key to discovering a gene that resisted identification using the traditional, single-target VIGS method [ 41 ].…”

Terpenoid indole alkaloid biosynthesis in Catharanthus roseus: effects and prospects of environmental factors in metabolic engineering

“…Virus-induced gene silencing (VIGS) has emerged as an effective tool to study the regulation of TIA biosynthesis in C. roseus leaves and flowers (Liscombe and O’Connor 2011 ; Sung et al 2014 ; Liu et al 2019 ; Patra et al 2018 , 2021 ). Recently, an improved C. roseus VIGS method has been developed, in which the target gene and the visual marker gene have been incorporated in the same plasmid to successfully identify the silenced tissues in planta (Yamamoto et al 2021 ). Furthermore, the generation of stable transgenic C. roseus plants have also been reported (Sharma et al 2018b ; Pan et al 2012 ; Wang et al 2012 ).…”

Reconstitution of monoterpene indole alkaloid biosynthesis in genome engineered Nicotiana benthamiana

“…Mass cultivation of C. roseus cells is feasible, but a high producing cell line has yet to be reported (Saiman et al 2018). Though methods for transient expression (Yamamoto et al 2021) and stable genetic transformation (Q. Wang et al 2012) of C. roseus plants have been reported, genetically engineering yield increases within the native plant host remains technically difficult.…”