A Narcissus mosaic viral vector system for protein expression and flavonoid production

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.

Harnessed viruses in the age of metagenomics and synthetic biology: an update on infectious clone assembly and biotechnologies of plant viruses

“…On these same lines and as suggested by Gleba et al (2014), what if we could use engineered plant viruses as gene therapy tools to reprogramme field-grown crops? As proof of principle, viral vectors have been used to rewire plant biosynthetic pathways by delivery of transcription factors, targeted knock-down of metabolic genes or overexpression of heterologous enzymes (Bedoya et al, 2012;Kumagai et al, 1995;Majer et al, 2017;Mozes-Koch et al, 2012;Zhang et al, 2013). Such approaches might be applied for crop biofortification as an alternative or in conjunction to standard breeding and transgenic strategies.…”

When plant virology met Agrobacterium: the rise of the deconstructed clones

“…Similarly, Zhang et al . () created a nondeconstructed expression vector based on the potexvirus narcissus mosaic virus (NMV), which allowed for systemic spread of transgenic viral RNA, but only succeeded in initiating low numbers of small infection foci in which transgene expression was relatively low. On the other hand, Liu and Kearney () developed the FECT vector series based on fully deconstructed foxtail mosaic virus (FoMV), yet another potexvirus (Figure ).…”

“…They found that the optimal use of the vector involved fusing GFP to the N-terminus of the CP, which resulted in GFP yields of 0.2-0.4 mg/g FWT, which is inferior to most other replicating viral overexpression systems. Similarly, Zhang et al (2013) created a nondeconstructed expression vector based on the potexvirus narcissus mosaic virus (NMV), which allowed for systemic spread of transgenic viral RNA, but only succeeded in initiating low numbers of small infection foci in which transgene expression was relatively low. On the other hand, Liu and Kearney (2010) developed the FECT vector series based on fully deconstructed foxtail mosaic virus (FoMV), yet another potexvirus (Figure 2).…”

Development of Beet necrotic yellow vein virus‐based vectors for multiple‐gene expression and guide RNA delivery in plant genome editing

“…Since then, plant virus-based vectors have been widely used as effective tools for recombinant protein expression and genomic research (Palmer and Gleba, 2014), especially for some plant species that are difficult to transform. So far, a number of plant viruses have been developed as delivery vectors for multiple purposes, such as tobamoviruses (Takamatsu et al, 1987;Yusibov et al, 1999), potexviruses (Baulcombe et al, 1995;Chapman et al, 1992;Zhang et al, 2013), Potyvirus (Jarugula et al, 2016;Lellis et al, 2002;Majer et al, 2015;Seo et al, 2016), Comoviruses (Sainsbury et al, 2008;Zhang et al, 2010), Geminiviruses (Stanley, 1993), Caulimovirus (Brisson et al, 1984) and Necrovirus (Zhou et al, 2010) in dicotyledonous plants, Barley stripe mosaic virus (Cheuk and Houde, 2018;Lee et al, 2012;Yuan et al, 2011), Soilborne wheat mosaic virus (Jarugula et al, 2016), Wheat steak mosaic virus (Choi et al, 2000), and Foxtail mosaic virus (Bouton et al, 2018) in monocotyledonous plants. However, some studies in basic and applied plant biology needing expression of functional complex heterologous proteins, production of antibodies and pharmaceutical peptides, require simultaneous expression of two or more genes within single cells.…”