Development of cassava common mosaic virus-based vector for protein expression and gene editing in cassava

Decai Tuo; Yuan Yao; Pu Yan; Xin Chen; Feihong Qu; Weiqian Xue; Jinping Liu; Hua Kong; Jianchun Guo; Hongguang Cui; Zhaoji Dai; Wentao Shen

doi:10.1186/s13007-023-01055-5

Development of cassava common mosaic virus-based vector for protein expression and gene editing in cassava

Plant Methods. 2023 Aug 3;19(1):78. doi: 10.1186/s13007-023-01055-5.

Authors

Decai Tuo^#¹, Yuan Yao^#¹, Pu Yan¹, Xin Chen¹, Feihong Qu², Weiqian Xue², Jinping Liu², Hua Kong¹, Jianchun Guo¹, Hongguang Cui², Zhaoji Dai², Wentao Shen³

Affiliations

¹ 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.
² School of Tropical Agriculture and Forestry, Sanya Nanfan Research Institute, Hainan University, Haikou & Sanya, Hainan, China.
³ 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. shenwentao@itbb.org.cn.

^# Contributed equally.

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Keywords: Cassava; Cassava common mosaic virus; Genome editing; Protein overexpression.