A novel virus induced gene silencing method and its application in functional analysis of cotton BAX inhibitor 1 in cotton

Working group session: 
Breeding and Applied Genomics
Presentation type: 
5 minute Oral and Poster
Authors: 
Zhang, Jingxia
Wang, Furong
Zhang, Chuanyun
Zhang, Junhao
Liu, Guodong
Chen, Yu
Hao, Fushun
Zhang, Jun
Author Affliation: 
Key Laboratory of Cotton Breeding and Cultivation in Huang-Huai-Hai Plain, Ministry of Agriculture, Cotton Research Center of Shandong Academy of Agricultural Sciences, Jinan 250100, China
Key Laboratory of Cotton Breeding and Cultivation in Huang-Huai-Hai Plain, Ministry of Agriculture, Cotton Research Center of Shandong Academy of Agricultural Sciences, Jinan 250100, China
Key Laboratory of Cotton Breeding and Cultivation in Huang-Huai-Hai Plain, Ministry of Agriculture, Cotton Research Center of Shandong Academy of Agricultural Sciences, Jinan 250100, China
Nanjing Agricultural University, Nanjing 210095, China
Key Laboratory of Cotton Breeding and Cultivation in Huang-Huai-Hai Plain, Ministry of Agriculture, Cotton Research Center of Shandong Academy of Agricultural Sciences, Jinan 250100, China
Key Laboratory of Cotton Breeding and Cultivation in Huang-Huai-Hai Plain, Ministry of Agriculture, Cotton Research Center of Shandong Academy of Agricultural Sciences, Jinan 250100, China
State Key Laboratory of Cotton Biology, Henan Key Laboratory of Plant Stress Biology, College of Life Science, Henan University, Kaifeng 475004, China
Key Laboratory of Cotton Breeding and Cultivation in Huang-Huai-Hai Plain, Ministry of Agriculture, Cotton Research Center of Shandong Academy of Agricultural Sciences, Jinan 250100, China
Abstract: 
Virus-induced gene silencing (VIGS) has been widely used to investigate the functions of genes expressed in mature leaves, but not in very young seedlings and roots in cotton. Here, we developed a simple seed soak inoculation (SSI) agroinoculation VIGS (SSI-VIGS) method by soaking naked cotton seeds into Agrobacterium cultures carrying tobacco rattle virus (TRV)-VIGS vectors. Obvious photo-bleaching symptom in newly emerging leaves inoculated with pTRV:GhCLA1 vectors by SSI-VIGS method was observed after 12-14 day post inoculation, while the virus control plants remained green at the same period. Molecular analysis indicated that the photo-bleaching symptom resulted from the reduction of endogenous GhCLA1 transcript abundances, suggesting that SSI-VIGS was able to elicit gene silencing in young seedlings. Naked seeds, Agrobacterium cultures with an OD 600 of 1.5 and 90 min for inoculation time were optimal for SSI-VIGS efficiency of GhCLA1. Besides, SSI-VIGS was able to knock down the expression of gene expressed in roots at the early stage of development, by employing cotton GhPGF (pigment gland formation) as a target. In addition, we readily reduced the expression of cotton Bax inhibitor 1 (GhBI-1) using SSI-VIGS system, The seedlings inoculated with pTRV:GhBI-1 vectors were more sensitive to salt stress compared with viral controls, indicating that GhBI-1 played a positive role in salt tolerance in cotton. Collectively, SSI is a novel and simple agroinoculation method for VIGS, and very useful in elucidating functions of genes, especially genes expressed in roots and young seedlings, in cotton and other seed plants.