Establishment of gene expression and silencing assays in Echinacea purpurea

1. Optimized the experimental parameters for the Agrobacterium tumefaciens -mediated transient expression and the virus-induced gene silencing systems in E. purpurea leaves. 2. Established gene expression and silencing systems in E. purpurea based on the Agrobacterium rhizogenes -mediated cut-dip-budding system. 3. Established an efficient protoplast transient expression system in E. purpurea . Echinacea purpurea ( E. purpurea ) is a perennial herb and horticulture plant belonging to the Asteraceae family. It is an easy cultivating plant that is well-known for its medicinal (e.g. chicoric acid) and high ornamental value. However, information based on the synthesis and regulatory mechanisms its secondary metabolites are limited. Therefore, to improve research progress on E. purpurea , this study aims to establish gene expression and silencing systems in E. purpurea . First, a transient gene expression system mediated by Agrobacterium tumefaciens was developed in E. purpurea leaves. Following optimization, it was determined that injecting the fourth newly emerged leaf with the EHA105 strain and maintaining it for 4 d yielded the best expression results. Then a previously reported cut-dip-budding (CDB) system was adapted and improved to establish a gene expression system based on Agrobacterium rhizogenes . By infecting the roots with A. rhizogenes , we detected efficient expression of the target gene after 30 d. Using this system, we achieved expression of EpHTT , a key gene involved in chicoric acid synthesis, and significantly increased the accumulation of chicoric acid. By expressing a double-strand RNA targeting EpHTT , we successfully silenced the EpHTT expression and reduced chicoric acid accumulation. We further investigated the protoplast-based transient gene expression system, studied key parameters, such as enzyme concentration and osmotic pressure, and successfully achieved transient expression of the target gene in protoplasts. Finally, a gene-silencing system in E. purpurea mediated by the tobacco rattle virus (TRV) was established and EpCHLH was identified as the optimal silencing indicator gene, with the best silencing effect observed in 15-day-old seedlings. By silencing genes involved in the chicoric acid synthesis pathway, such as EpHCT, EpHTT , and EpCAS , chicoric acid accumulation was successfully reduced. In summary, this study successfully established various gene expression and silencing systems for E. purpurea and providing a valuable toolkit for further functional studies.