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<p>Extracellular vesicles (EVs) are emerging as biocompatible carriers for RNA-based delivery in both plant and non-plant systems. In this study, we present the first demonstration that EVs derived from plants can be engineered to deliver artificial microRNAs (amiRNAs) and induce gene silencing <i>in planta</i>. Molecularly modified <i>Nicotiana tabacum</i> plants were generated to express an amiRNA which was designed to target the enhanced green fluorescent protein (EGFP) gene. EVs isolated from tobacco plants expressing amiR-GFP were 100–200&#xa0;nm in diameter and exhibited selective enrichment of the amiRNA at levels comparable to endogenous miR167a and miR168a. Functional assays demonstrated that these EVs were efficiently internalized by <i>EGFP</i>-expressing tobacco protoplasts and intact leaves, leading to a reduction in <i>EGFP</i> expression, as confirmed by fluorescence microscopy and RT-qPCR. Suppression of GFP fluorescence was more rapid in protoplasts and became evident within the first hour post-application, whereas in leaves, a significant effect appeared closer to two hours. These findings establish that plant-derived EVs can be programmed to deliver amiRNAs, highlighting their potential for future applications in plant biotechnology and RNA-based delivery platforms.</p> Graphical abstract <p></p>

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Artificial MicroRNA delivery and gene silencing via extracellular vesicles derived from molecularly modified tobacco

  • 期刊论文

  • 发表日期：

    2025年12月10日

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  • Yury Shkryl,
  • Zhargalma Tsydeneshieva,
  • Tatiana Gorpenchenko,
  • Vitaly Kazarin,
  • Olesya Kudinova,
  • Victor Bulgakov,
  • Yulia Yugay

摘要

Extracellular vesicles (EVs) are emerging as biocompatible carriers for RNA-based delivery in both plant and non-plant systems. In this study, we present the first demonstration that EVs derived from plants can be engineered to deliver artificial microRNAs (amiRNAs) and induce gene silencing in planta. Molecularly modified Nicotiana tabacum plants were generated to express an amiRNA which was designed to target the enhanced green fluorescent protein (EGFP) gene. EVs isolated from tobacco plants expressing amiR-GFP were 100–200 nm in diameter and exhibited selective enrichment of the amiRNA at levels comparable to endogenous miR167a and miR168a. Functional assays demonstrated that these EVs were efficiently internalized by EGFP-expressing tobacco protoplasts and intact leaves, leading to a reduction in EGFP expression, as confirmed by fluorescence microscopy and RT-qPCR. Suppression of GFP fluorescence was more rapid in protoplasts and became evident within the first hour post-application, whereas in leaves, a significant effect appeared closer to two hours. These findings establish that plant-derived EVs can be programmed to deliver amiRNAs, highlighting their potential for future applications in plant biotechnology and RNA-based delivery platforms.

Graphical abstract