Optimisation of tobacco rattle virus-induced gene silencing in Arabidopsis
Changchun Wang A C , Xinzhong Cai A D , Xuemin Wang B and Zhong Zheng AA Institute of Biotechnology, and Department of Plant Protection, College of Agriculture and Biotechnology, Zhejiang University, 268 Kai Xuan Road, Hangzhou 310029, People’s Republic of China.
B State Key Laboratory of Plant Physiology and Biochemistry, College of Life Science, Zhejiang University, 268 Kai Xuan Road, Hangzhou 310029, People’s Republic of China.
C College of Life and Environmental Science, Zhejiang Normal University, Jinhua 321004, People’s Republic of China.
D Corresponding author. Email: xzhcai@zju.edu.cn
Functional Plant Biology 33(4) 347-355 https://doi.org/10.1071/FP05096
Submitted: 26 April 2005 Accepted: 4 January 2006 Published: 3 April 2006
Abstract
Arabidopsis thaliana (L.) Heynh. is a model plant species in which to study plant gene functions. Recently developed virus-induced gene silencing (VIGS) offers a rapid and high-throughput technique platform for gene function analysis. In this paper we report optimisation of tobacco rattle virus (TRV)-induced gene silencing in Arabidopsis. The parameters potentially affecting the efficiency of VIGS in Arabidopsis were investigated. These included the concentration and pre-incubation of Agrobacterium inocula (agro-inocula), the concentration of acetosyringone included in agro-inocula, the Agrobacterium inoculation (agro-inoculation) method, the ecotypes and the growth stages of Arabidopsis plants for agro-inoculation, and the growth temperature of agro-inoculated plants. The optimised VIGS procedure involves preparing the agro-inocula with OD600 of 2.0, pre-incubating for 2 h in infiltration buffer containing 200 μm acetosyringone, agro-inoculating by vacuum infiltration, and growth of agro-inoculated plants at 22 −24°C. Following this procedure consistent and highly efficient VIGS was achieved for the genes encoding phytoene desaturase (PDS) and actin in Arabidopsis. The silencing phenotype lasts for at least 6 weeks, and is applicable in at least seven ecotypes, including Col-0, Cvi-0, Sd, Nd-1, Ws-0, Bay-0 and Ler. TRV-induced VIGS was expressed not only in leaves, but also in stems, inflorescences and siliques. However, VIGS was not transmissible through seed to the subsequent generation. The optimised procedure of the TRV-induced gene silencing should facilitate high-throughput functional analysis of genes in Arabidopsis.
Keywords: Agrobacterium, Arabidopsis thaliana, optimisation, tobacco rattle virus, vacuum infiltration, virus-induced gene silencing (VIGS).
Acknowledgments
Dr He Zuhua and Ms Wang Muyang are acknowledged for providing the Arabidopsis seeds. We thank the members of Cai Xinzhong’s laboratory for their help with agro-infiltration. This work was financially supported by the National Natural Science Foundation of China (grant nos. 30070492 and 30200179) and the Fok Ying Tong Education Foundation.
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