Virus-induced gene silencing as a tool for functional studies in Cleome violacea

Cleomaceae is emerging as a promising family to investigate a wide range of phenomena, such as C₄ photosynthesis and floral diversity. However, functional techniques are lacking for elucidating this diversity. Herein, we establish virus-induced gene silencing (VIGS) as a method of generating functional data for Cleome violacea, bolstering Cleomaceae as a model system.

We leveraged the sister relationship of Cleomaceae and Brassicaceae by using constructs readily available for Arabidopsis thaliana to provide initial information about the feasibility of VIGS in C. violacea. We then developed endogenous constructs to optimize VIGS efficiency and viability for fruit development.

PHYTOENE DESATURASE was successfully downregulated in C. violacea using both heterologous and endogenous constructs. The endogenous construct had the highest degree of downregulation, with many plants displaying strong photobleaching. FRUITFULL-treated plants were also successfully downregulated, with a high rate of survival but less effective silencing; only a small percentage of survivors showed a strong phenotype.

Our optimized VIGS protocol in C. violacea enables functional gene analyses at different developmental stages. Additionally, C. violacea is amenable to heterologous knockdown, which suggests that a first pass using non-endogenous constructs is a possible route to test additional species of Cleomaceae.