Abstract
BackgroundWith the explosive numbers of sequences generated by next generation sequencing, the demand for high throughput screening to understand gene function has grown. Plant viral vectors have been widely used as tools in down-regulating plant gene expression. However, plant viral vectors can also express proteins in a very efficient manner and, therefore, can also serve as a valuable tool for characterizing proteins and their functions in metabolic pathways in planta.ResultsIn this study, we have developed a Gateway®-based high throughput viral vector cloning system from Narcissus Mosaic Virus (NMV). Using the reporter genes of GFP and GUS, and the plant genes PAP1 (an R2R3 MYB which activates the anthocyanin pathway) and selenium-binding protein 1 (SeBP), we show that NMV vectors and the model plant Nicotiana benthamiana can be used for efficient protein expression, protein subcellular localization and secondary metabolite production.ConclusionsOur results suggest that not only can the plant viral vector system be employed for protein work but also can potentially be amenable to producing valuable secondary metabolites on a large scale, as the system does not require plant regeneration from seed or calli, which are stages where certain secondary metabolites can interfere with development.
Highlights
Characterizing the functions of proteins encoded by newly identified DNA sequences is a fundamental aspect of molecular research
The AvrII-attR1-CmRccdB-attR2-ApaI Gateway® cassette was subsequently inserted in front of the Green Fluorescent Protein (GFP) sequence using the AvrII/ ApaI sites such that any sequence inserted into the vector encoded a protein fusion in frame with GFP attached at the C-terminus of the expressed protein of interest
For localization studies pNMV-GW was modified to result in formation of the expressed proteins with Green Fluorescent Protein (GFP) as a C-terminal fusion of a expressed protein
Summary
Construction of NMV expression vectors To enable NMV infection in plants without having to use in vitro reverse transcription, a construct was prepared that placed a DNA sequence for the viral RNA genome in a plant expression cassette, driven by the 35SCaMV promoter, with a duplicate subgenomic promoter for the coat protein of NMV placed in front of a cloning site immediately upstream of the native sequence for the coat protein (pNMV, Figure 1). GUS protein accumulation was confirmed in leaves of intact plants in the greenhouse that had been inoculated with the extracts of the pNMV-hGUS-bombarded leaves (Additional file 2: Figure S1), using a activity assay, coomassie Blue protein staining and the Western blot analysis (Figure 3). Commassie Blue staining (Figure 3C) and Western analysis (Figure 3D) showed that the Ni-affinity protocol was highly effective for purification of the GUS protein expressed from pNMV-hGUS-inoculated greenhouse plants. LC results showed that three flavonoid peaks were detected in the mock control and the leaves expressing GFP, and the patterns of the peaks appeared almost identical (Figures 7A and B), indicating that viral activity alone did not alter the flavonol profile. Based on the size detected, fulllength viral genomic RNAs were accumulated (Figure 8)
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