Use of TRV-mediated VIGS for functional genomics research in citrus
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Virus induced gene silencing (VIGS) is a powerful tool for identifying gene functions in plants. Tobacco rattle virus (TRV)-mediated VIGS has been reported for silencing of endogenous genes in many plants. In citrus, analysis of gene function is mainly relied on the table genetic transformation, even though this technique is low-efficiency and time-consuming. In this study, a TRV-mediated VIGS was successfully applied for verification of gene function in citrus. A reporter gene, Magnesium chelatase subunit I (ChlI) from C. grandis was constructed into TRV2 vector to induce gene silencing with visible photobleaching symptom. TRV VIGS induced photobleaching symptom was observed on the leaves of Nicotiana benthamiana and Citrus after infiltration. The susceptibility to VIGS vector was found to be genotype dependent. Mexican lemon is the most susceptible one. The chlorosis area per leaf was 65.5% of the whole leaf area. GXSTY pummelo exhibited less susceptible to VIGS with only tiny patches of chlorosis appeared on the leaves of treated plants. The parameters were optimized to improve the efficiency. The tiny young leaves and half expanded leaves were more susceptible to VIGS infection. Young seedlings were highly susceptible to vacuum infiltration. The expression of fluorescence from GFP reporter gene was observed in the young seedlings of GXSTY after infiltration with TRV. Close to 100% of infiltration efficiency was achieved by vacuum infiltration. The results indicated that TRV mediated VIGS with vacuum infiltration could be an ideal tool for gene function studies in citrus.
A TRV mediated VIGS has been optimized to use in the citrus for verification of gene function. The sensitivity to VIGS vector was genotype dependent. Young seedling and young leaf were the suitable materials for VIGS infection. Vacuum infiltration was a high efficiency infiltration method.
KeywordsTRV-mediated VIGS Vacuum infiltration Gene silencing Citrus
This study was supported by the Earmarked Fund for China Agriculture Research System (CARS-26); the Earmarked Fund for Chongqing Special & Economic Agriculture Research System on Late Maturation Citrus; Cooperative Research Program for Agriculture Science & Technology Development (Grant No. PJ01211201); the “Double World-classes” Development Plan of Southwest University; China Postdoctoral Science Foundation (Grant No. 2017M622946); Chongqing Special Postdoctoral Science Foundation under Grant (No. XmT2018062); the National Natural Science Foundation of China (Grant No. 31901995). We thank Deqiang Tai (Shanxi Agricultural University) for technical assistant on vaccum infiltration method of VIGS.
XZ and FW designed this research. FW, YX, XL, WS, SZ carried out the experiments and data analyses. FW prepared manuscript. XZ revised manuscript.
Compliance with ethical standards
Conflict of interest
All the authors declare that they have no conflict of interest.
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