Tuo Decai, Yao Yuan, Yan Pu, Chen Xin, Qu Feihong, Xue Weiqian, Liu Jinping, Kong Hua, Guo Jianchun, Cui Hongguang, Dai Zhaoji, Shen Wentao
National Key Laboratory for Tropical Crops Breeding, Key Laboratory of Biology and Genetic Resources of Tropical Crops (Ministry of Agriculture and Rural Affairs), Hainan Key Laboratory for Protection and Utilization of Tropical Bioresources, Institute of Tropical Bioscience and Biotechnology, Sanya Research Institute, Hainan Institute for Tropical Agricultural Resources, Chinese Academy of Tropical Agricultural Sciences, Haikou & Sanya, Hainan, China.
School of Tropical Agriculture and Forestry, Sanya Nanfan Research Institute, Hainan University, Haikou & Sanya, Hainan, China.
Plant Methods. 2023 Aug 3;19(1):78. doi: 10.1186/s13007-023-01055-5.
Plant virus vectors designed for virus-mediated protein overexpression (VOX), virus-induced gene silencing (VIGS), and genome editing (VIGE) provide rapid and cost-effective tools for functional genomics studies, biotechnology applications and genome modification in plants. We previously reported that a cassava common mosaic virus (CsCMV, genus Potexvirus)-based VIGS vector was used for rapid gene function analysis in cassava. However, there are no VOX and VIGE vectors available in cassava.
In this study, we developed an efficient VOX vector (CsCMV2-NC) for cassava by modifying the CsCMV-based VIGS vector. Specifically, the length of the duplicated putative subgenomic promoter (SGP1) of the CsCMV CP gene was increased to improve heterologous protein expression in cassava plants. The modified CsCMV2-NC-based VOX vector was engineered to express genes encoding green fluorescent protein (GFP), bacterial phytoene synthase (crtB), and Xanthomonas axonopodis pv. manihotis (Xam) type III effector XopAO1 for viral infection tracking, carotenoid biofortification and Xam virulence effector identification in cassava. In addition, we used CsCMV2-NC to deliver single guide RNAs (gMePDS1/2) targeting two loci of the cassava phytoene desaturase gene (MePDS) in Cas9-overexpressing transgenic cassava lines. The CsCMV-gMePDS1/2 efficiently induced deletion mutations of the targeted MePDS with the albino phenotypes in systemically infected cassava leaves.
Our results provide a useful tool for rapid and efficient heterologous protein expression and guide RNA delivery in cassava. This expands the potential applications of CsCMV-based vector in gene function studies, biotechnology research, and precision breeding for cassava.
为病毒介导的蛋白质过表达（VOX）、病毒诱导的基因沉默（VIGS）和基因组编辑（VIGE）设计的植物病毒载体为植物功能基因组学研究、生物技术应用和基因组修饰提供了快速且经济高效的工具。我们之前报道过一种基于木薯普通花叶病毒（CsCMV，马铃薯X病毒属）的VIGS载体被用于木薯基因功能的快速分析。然而，木薯中尚无VOX和VIGE载体。
在本研究中，我们通过对基于CsCMV的VIGS载体进行改造，开发了一种用于木薯的高效VOX载体（CsCMV2-NC）。具体而言，增加了CsCMV外壳蛋白（CP）基因重复的假定亚基因组启动子（SGP1）的长度，以提高木薯植株中异源蛋白的表达。改造后的基于CsCMV2-NC的VOX载体经设计可表达编码绿色荧光蛋白（GFP）、细菌八氢番茄红素合酶（crtB）以及野油菜黄单胞菌木薯致病变种（Xam）III型效应蛋白XopAO1的基因，用于木薯中的病毒感染追踪、类胡萝卜素生物强化以及Xam致病效应蛋白鉴定。此外，我们利用CsCMV2-NC在过表达Cas9的转基因木薯品系中递送靶向木薯八氢番茄红素去饱和酶基因（MePDS）两个位点的单向导RNA（gMePDS1/2）。CsCMV-gMePDS1/2在系统感染的木薯叶片中高效诱导了靶向MePDS的缺失突变，并出现白化表型。
我们的结果为木薯中快速高效的异源蛋白表达和向导RNA递送提供了一个有用的工具。这扩展了基于CsCMV的载体在木薯基因功能研究、生物技术研究和精准育种中的潜在应用。
