Establishment of gene expression and silencing systems in Echinacea purpurea

Echinacea purpurea is a perennial herbaceous horticultural plant belonging to the Asteraceae family. It is easy to cultivate and well-known for its medicinal ingredients (e.g., chicoric acid) and high ornamental value. However, studies on the synthesis and regulatory mechanisms of its secondary metabolites are limited. Therefore, to advance research on E. purpurea, this study aimed 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. After optimization, we found that injecting the fourth newly emerged leaf with the EHA105 strain and culturing 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 double-stranded RNA targeting EpHTT, we successfully silenced 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 reporter gene, with the maximal silencing effect observed in 15-d-old seedlings. By silencing genes involved in the chicoric acid biosynthesis pathway, such as EpHCT, EpHTT, and EpCAS, we successfully reduced chicoric acid accumulation. In summary, this study successfully established various gene expression and silencing systems for E. purpurea, providing a valuable toolkit for further functional studies.