Studies on Tomato bushy stunt virus and Cucumber necrosis virus as gene expression vectors
Date Issued
2005
Date
2005
Author(s)
Lin, I-Jeng
DOI
zh-TW
Abstract
Successful infection in plants by plant viruses requires that viruses can accomplish a series of infection steps including replication in plant cells, cell-to-cell movement through plasmodesmata, and long-distance movement via vascular tissue. Most RNA viruses need coat protein expression to facilitate efficiently systemic movement and some viruses have to form virions in order to move within plants. Tomato bushy stunt virus (TBSV) is in a special group of plant viruses that coat protein is dispensable for systemic movement in some specific hosts. In this study, TBSV and Cucumber necrosis virus (CNV) that have the same genome organization and high sequence homology were chosen to construct viral vectors. Enhanced green fluorescent protein (EGFP) was used as a reporter gene. Insertion and replacement of the coat protein gene were applied to construct four mutants: pTBSV-EGFPa, pCNV*-EGFPa, pTBSV-EGFPb, and pCNV*-EGFPb. The wild-type TBSV and CNV clones and their derived mutant clones were used as DNA templates to perform in vitro transcription. Protoplasts and plants of Nicotiana benthamiana and different varieties of tomato plants were inoculated by the transcripts, respectively. Northern hybridization and Real-Time PCR were used to compare genomic RNA accumulation of the wild-type and mutants of TBSV and CNV in vivo. The EGFP expression in protoplasts as well as the movement of four viral mutants in plant were observed by detection of fluorescence with an epifluorescence microscope. The results revealed that all mutants could replicate in N. benthamiana protoplasts. The level of genomic RNA accumulation of CNV*-EGFPb in protoplast was about 1/10 of TBSV and CNV. Whereas, TBSV-EGFPa, CNV*-EGFPa, and TBSV-EGFPb were only about 1/465 amount of RNA in TBSV or CNV-infected protoplasts. Analysis of genomic RNA accumulation of these mutants in N. benthamiana plants showed that TBSV-EGFPa, CNV*-EGFPb, and TBSV-EGFPb accumulated to similar level as CNV, but about 1/8 amount of TBSV in inoculated leaves at three days post inoculation (dpi). The amount of CNV*-EGFPa RNA accumulated in inoculated leaves was about 1/865 amount of TBSV at 3 dpi. The symptoms caused by mutants were delayed as compared to wild type viruses. In addition, TBSV-EGFPa was able to move systemically and green fluorescence was observed in systemic leaves. Four cultivars of tomato plants could be infected with TBSV and TBSV-EGFPa. Both TBSV and TBSV-EGFPa infected tomato plant cultivar ‘Shin-Guang’ systemically and green fluorescence was observed in TBSV-EGFPa-infected systemic leaves. According to the experimental results, TBSV seems more suitable than CNV to be engineered as a gene expression vector. Furthermore, the mutant construct derived from TBSV that had foreign gene inserted into the 5’ end of coat protein gene could systemically infect N. benthamiana and tomato plants, and the foreign gene was able to express during infection. Accordingly, a TBSV-based vector can be used to express the interested genes in plants in the future.
Subjects
蕃茄叢生矮化病毒
胡瓜壞疽病毒
基因表現載體
gene expression vectors
Cucumber necrosis virus
Tomato bushy stunt virus
Type
other
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