Hossain M, Pfafenrot C, Nasfi S, Sede A, Imani J, Šečić E, Galli M, Schäfer P, Bindereif A, Heinlein M, Ladera-Carmona M, Kogel K H
Institute of Phytopathology, Centre for BioSystems, Land Use and Nutrition, Justus Liebig University, Heinrich-Buff-Ring 26, 35392, Giessen, Germany.
Institute of Biochemistry, Justus Liebig University, Heinrich-Buff-Ring 17, 35392, Giessen, Germany.
Plant Cell Rep. 2025 May 22;44(6):128. doi: 10.1007/s00299-025-03512-y.
We demonstrate non-immunogenic circRNA as a tool for targeted gene regulation in plants, where it acts in an isoform- and sequence-specific manner, enabling future agronomic applications. Circular RNAs (circRNAs) are single-stranded RNA molecules characterized by their covalently closed structure and are emerging as key regulators of cellular processes in mammals, including gene expression, protein function and immune responses. Recent evidence suggests that circRNAs also play significant roles in plants, influencing development, nutrition, biotic stress resistance, and abiotic stress tolerance. However, the potential of circRNAs to modulate target protein abundance in plants remains largely unexplored. In this study, we investigated the potential of designer circRNAs to modulate target protein abundance in plants using Arabidopsis protoplasts as a model system. We show that PEG-mediated transfection with a 50-nt circRNA containing a 30-nt GFP-antisense sequence results in a dose- and sequence-dependent reduction of GFP reporter target protein abundance. Notably, a single-stranded open isoform of circRNA had little effect on protein abundance, indicating the importance of the closed circular structure. Additionally, circRNA also reduced GFP abundance in Arabidopsis mutants defective in RNA interference (RNAi), suggesting that circRNA activity is independent of the RNAi pathway. We also show that circRNA, unlike dsRNA, does not induce pattern-triggered immunity (PTI) in plants. Findings of this proof-of-principle study together are crucial first steps in understanding the potential of circRNAs as versatile tools for modulating gene expression and offer exciting prospects for their application in agronomy, particularly for enhancing crop traits through metabolic pathway manipulation.
我们证明了非免疫原性环状RNA可作为植物中靶向基因调控的工具，它以异构体和序列特异性方式发挥作用，为未来的农艺应用提供了可能。环状RNA（circRNA）是单链RNA分子，其特征在于共价闭合结构，并且正在成为哺乳动物细胞过程的关键调节因子，包括基因表达、蛋白质功能和免疫反应。最近的证据表明，circRNA在植物中也发挥着重要作用，影响发育、营养、生物胁迫抗性和非生物胁迫耐受性。然而，circRNA在植物中调节靶蛋白丰度的潜力在很大程度上仍未被探索。在本研究中，我们以拟南芥原生质体为模型系统，研究了设计的circRNA在植物中调节靶蛋白丰度的潜力。我们发现，用含有30 nt GFP反义序列的50 nt circRNA进行聚乙二醇（PEG）介导的转染，会导致GFP报告靶蛋白丰度呈剂量和序列依赖性降低。值得注意的是，circRNA的单链开放异构体对蛋白丰度影响很小，这表明闭合环状结构的重要性。此外，circRNA在RNA干扰（RNAi）缺陷的拟南芥突变体中也降低了GFP丰度，这表明circRNA的活性独立于RNAi途径。我们还表明，与双链RNA不同，circRNA不会在植物中诱导模式触发免疫（PTI）。这项原理验证研究的结果共同构成了理解circRNA作为调节基因表达通用工具潜力的关键第一步，并为其在农艺学中的应用提供了令人兴奋的前景，特别是通过代谢途径操纵来增强作物性状。
