Pfotenhauer Alexander C, Jones Samantha M, Clark Mikayla, Concha Bryn L, Goldstein Elliot B, Harbison Stacee, Martin Lana H, Reuter D Nikki, Reed Andrew C, Stewart C Neal, Lenaghan Scott C
Center for Agricultural Synthetic Biology, The University of Tennessee, Knoxville, Tennessee 37996, United States.
Department of Plant Sciences, The University of Tennessee, Knoxville, Tennessee 37996,United States.
ACS Synth Biol. 2025 Jun 20;14(6):2230-2240. doi: 10.1021/acssynbio.5c00153. Epub 2025 May 29.
Tobacco rattle virus (TRV) is a bipartite single-stranded RNA virus that encodes a replicase, movement protein, and silencing suppressor on TRV1 and a capsid protein on TRV2. Researchers typically insert target silencing sequences into TRV2 and coexpress this with TRV1 to achieve virus-induced gene silencing (VIGS). However, TRV1 does not require TRV2 for mobility or replication within a plant host. With this knowledge, we engineer TRV1 alone as a self-replicating RNA (srRNA) that moves systemically throughout plants for targeted gene repression of up to 89%. As TRV1 is encapsidated in trans by the capsid protein encoded on TRV2, we demonstrate the ability to encapsidate our TRV1 srRNAs for application to target plants by coexpression of the capsid protein off a nonviral expression vector. The subsequent encapsidated TRV1 srRNA can then be harvested and applied to new plants by using a simple spray-on application. Since the RNA for the capsid does not accompany the TRV1 srRNA, our srRNAs are incapable of spreading from plant to plant after the initial application. Minimal to no phenotypic penalties were observed when we used our spray-on srRNA approach. To our knowledge, this is the first demonstration of engineering a sprayable TRV1-based srRNA that is highly capable of repressing target genes. As TRV has a broad host range and our encapsidated srRNAs are unlikely to persist in the environment, we envision that this platform can be used for targeted gene silencing in agriculture.
烟草脆裂病毒（TRV）是一种双链单链RNA病毒，在TRV1上编码一种复制酶、运动蛋白和沉默抑制子，在TRV2上编码一种衣壳蛋白。研究人员通常将目标沉默序列插入TRV2，并将其与TRV1共表达，以实现病毒诱导的基因沉默（VIGS）。然而，TRV1在植物宿主内移动或复制并不需要TRV2。基于这一认识，我们将TRV1单独设计成一种自我复制RNA（srRNA），它能在植物体内系统移动，实现高达89%的靶向基因抑制。由于TRV1由TRV2上编码的衣壳蛋白反式包装，我们通过在非病毒表达载体上共表达衣壳蛋白，展示了将我们的TRV1 srRNAs包装用于目标植物的能力。随后，包装好的TRV1 srRNA可以通过简单的喷雾应用收获并应用于新植物。由于衣壳RNA不伴随TRV1 srRNA，我们的srRNAs在初次应用后无法在植物间传播。当我们使用喷雾srRNA方法时，观察到最小到没有表型惩罚。据我们所知，这是首次证明设计出一种基于TRV1的可喷雾srRNA，它具有高度抑制靶基因的能力。由于TRV具有广泛的宿主范围，且我们包装的srRNAs不太可能在环境中持续存在，我们设想这个平台可用于农业中的靶向基因沉默。
