Plant viruses can be engineered to carry sequences that direct silencing of target host genes, expression of heterologous proteins, or editing of host genes. A set of foxtail mosaic virus (FoMV) vectors was developed that can be used for transient gene expression and single guide RNA delivery for Cas9‐mediated gene editing in maize, Setaria viridis, and Nicotiana benthamiana. This was accomplished by duplicating the FoMV capsid protein subgenomic promoter, abolishing the unnecessary open reading frame 5A, and inserting a cloning site containing unique restriction endonuclease cleavage sites immediately after the duplicated promoter. The modified FoMV vectors transiently expressed green fluorescent protein (GFP) and bialaphos resistance (BAR) protein in leaves of systemically infected maize seedlings. GFP was detected in epidermal and mesophyll cells by epifluorescence microscopy, and expression was confirmed by Western blot analyses. Plants infected with FoMV carrying the bar gene were temporarily protected from a glufosinate herbicide, and expression was confirmed using a rapid antibody‐based BAR strip test. Expression of these proteins was stabilized by nucleotide substitutions in the sequence of the duplicated promoter region. Single guide RNAs expressed from the duplicated promoter mediated edits in the N. benthamiana Phytoene desaturase gene, the S. viridis Carbonic anhydrase 2 gene, and the maize HKT1 gene encoding a potassium transporter. The efficiency of editing was enhanced in the presence of synergistic viruses and a viral silencing suppressor. This work expands the utility of FoMV for virus‐induced gene silencing (VIGS), virus‐mediated overexpression (VOX), and virus‐enabled gene editing (VEdGE) in monocots.

中文翻译：

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使用狐尾花叶病毒载体在单子叶植物中进行蛋白质表达和基因编辑。

植物病毒可以被设计为携带指导靶宿主基因沉默、异源蛋白质表达或宿主基因编辑的序列。开发了一套狐尾花叶病毒 (FoMV) 载体，可用于玉米、狗尾草和本塞姆氏烟草中 Cas9 介导的基因编辑的瞬时基因表达和单引导RNA递送。这是通过复制 FoMV 衣壳蛋白亚基因组启动子、消除不必要的开放阅读框 5A 并在复制的启动子后立即插入包含独特限制性内切核酸酶切割位点的克隆位点来实现的。修饰的 FoMV 载体在系统感染的玉米幼苗的叶子中瞬时表达绿色荧光蛋白（GFP）和双丙磷抗性（BAR）蛋白。通过落射荧光显微镜检测表皮和叶肉细胞中的 GFP，并通过蛋白质印迹分析证实表达。感染带有bar基因的 FoMV 的植物暂时受到草铵膦除草剂的保护，并使用基于快速抗体的 BAR 条带测试证实了表达。这些蛋白质的表达通过重复启动子区域序列中的核苷酸取代而稳定。由重复的启动子表达的单指导RNA介导了N.benthamiana八氢番茄红素去饱和酶基因、S.viridis碳酸酐酶2基因和编码钾转运蛋白的玉米HKT1基因的编辑。在协同病毒和病毒沉默抑制子存在的情况下，编辑效率得到提高。这项工作扩展了 FoMV 在单子叶植物中病毒诱导的基因沉默 (VIGS)、病毒介导的过度表达 (VOX) 和病毒驱动的基因编辑 (VEdGE) 中的效用。