Abstract
Main conclusion
CaLCuV-based VIGS effectively works in cabbage and contributes to efficient functional genomics research in Brassica crop species.
Abstract
Virus-induced gene silencing (VIGS), a posttranscriptional gene silencing method, is an effective technique for analysing the functions of genes in plants. However, no VIGS vectors have been available for Brassica oleracea until now. Here, tobacco rattle virus (TRV), pTYs and cabbage leaf curl virus (CaLCuV) gene-silencing vectors (PCVA/PCVB) were chosen to improve the VIGS system in cabbage using the phytoene desaturase (PDS) gene as an efficient visual indicator of VIGS. We successfully silenced the expression of PDS and observed photobleaching phenomena in cabbage in response to pTYs and CaLCuV, with the latter being more easy to operate and less expensive. The parameters potentially affecting the silencing efficiency of VIGS by CaLCuV in cabbage, including the targeting fragment strategy, inoculation method and incubation temperature, were then compared. The optimized CaLCuV-based VIGS system involves the following: an approximately 500 bp insert sequence, an Agrobacterium OD600 of 1.0, use of the vacuum osmosis method applied at the bud stage, and an incubation temperature of 22 °C. Using these parameters, we achieved a stable silencing efficiency of 65%. To further test the effectiveness of the system, we selected the Mg-chelatase H subunit (ChlH) gene in cabbage and knocked down its expression, and we observed yellow leaves, as expected. We successfully applied the CaLCuV-based VIGS system to two other representative Brassica crop species, B. rapa and B. nigra, and thus expanded the application scope of this system. Our VIGS system described here will contribute to efficient functional genomics research in Brassica crop species.
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The datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request.
Abbreviations
- CaLCuV:
-
Cabbage leaf curl virus
- ChlH:
-
Chelatase H subunit
- PDS:
-
Phytoene desaturase
- TRV:
-
Tobacco rattle virus
- VIGS:
-
Virus-induced gene silencing
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Acknowledgements
The work was performed at the Key Laboratory of Biology and Genetic Improvement of Horticultural Crops, Ministry of Agriculture, Beijing 100081, China. We thank Su Xiaoming (Shandong Academy of Agricultural Sciences, Jinan, Shandong Province, China) for providing the TRV vectors, Prof Zhang Changwei (Nanjing Agricultural University, Nanjing, Jiangsu Province, China) for providing the pTYs vectors and Liu Yule (Tsinghua University, Beijing, China) for providing the PCVA/PCVB vectors.
Funding
This study was supported by grants from the Key Projects of the National Key Research and Development Program of China (2016YFD0100307), the National Natural Science Foundation of China (31572139), Central Public-interest Scientific Institution Basal Research Fund (Y2020PT01), the Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences (CAAS-ASTIP-IVFCAAS) and the earmarked fund for the Modern Agro-Industry Technology Research System, China (CARS-23).
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Xiao, Z., Xing, M., Liu, X. et al. An efficient virus-induced gene silencing (VIGS) system for functional genomics in Brassicas using a cabbage leaf curl virus (CaLCuV)-based vector. Planta 252, 42 (2020). https://doi.org/10.1007/s00425-020-03454-7
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DOI: https://doi.org/10.1007/s00425-020-03454-7