College of Plant Protection, China Agricultural University, Beijing 100193, China.
Beijing Key Laboratory of Seed Disease Testing and Control, China Agricultural University, Beijing 100193, China.
Int J Mol Sci. 2021 Nov 12;22(22):12237. doi: 10.3390/ijms222212237.
Cucumber green mottle mosaic virus (CGMMV), as a typical seed-borne virus, causes costly and devastating diseases in the vegetable trade worldwide. Genetic sources for resistance to CGMMV in cucurbits are limited, and environmentally safe approaches for curbing the accumulation and spread of seed-transmitted viruses and cultivating completely resistant plants are needed. Here, we describe the design and application of RNA interference-based technologies, containing artificial microRNA (amiRNA) and synthetic -acting small interfering RNA (syn-tasiRNA), against conserved regions of different strains of the CGMMV genome. We used a rapid transient sensor system to identify effective anti-CGMMV amiRNAs. A virus seed transmission assay was developed, showing that the externally added polycistronic amiRNA and syn-tasiRNA can successfully block the accumulation of CGMMV in cucumber, but different virulent strains exhibited distinct influences on the expression of amiRNA due to the activity of the RNA-silencing suppressor. We also established stable transgenic cucumber plants expressing polycistronic amiRNA, which conferred disease resistance against CGMMV, and no sequence mutation was observed in CGMMV. This study demonstrates that RNA interference-based technologies can effectively prevent the occurrence and accumulation of CGMMV. The results provide a basis to establish and fine-tune approaches to prevent and treat seed-based transmission viral infections.
黄瓜绿斑驳花叶病毒（CGMMV）是一种典型的种传病毒，在全球蔬菜贸易中造成了代价高昂且毁灭性的疾病。葫芦科植物对 CGMMV 的遗传抗性资源有限，因此需要采取环境安全的方法来抑制种传病毒的积累和传播，并培育完全抗性的植物。在这里，我们描述了基于 RNA 干扰的技术的设计和应用，包括人工 microRNA（amiRNA）和合成作用小干扰 RNA（syn-tasiRNA），针对 CGMMV 基因组不同株系的保守区域。我们使用快速瞬时传感器系统来鉴定有效的抗 CGMMV amiRNA。开发了病毒种传试验，表明外加的多顺反子 amiRNA 和 syn-tasiRNA 可以成功阻止 CGMMV 在黄瓜中的积累，但不同的毒力株系由于 RNA 沉默抑制子的活性对 amiRNA 的表达表现出不同的影响。我们还建立了稳定表达多顺反子 amiRNA 的转基因黄瓜植株，这些植株对 CGMMV 表现出抗性，并且在 CGMMV 中没有观察到序列突变。这项研究表明，基于 RNA 干扰的技术可以有效地防止 CGMMV 的发生和积累。研究结果为建立和微调预防和治疗基于种子的病毒感染的方法提供了依据。
