Nicotiana benthamiana: its history and future as a model for plant-pathogen interactions.

Department of Plant Pathology, University of Kentucky, Lexington 40546, USA.
Molecular Plant-Microbe Interactions (Impact Factor: 4.46). 09/2008; 21(8):1015-26. DOI: 10.1094/MPMI-21-8-1015
Source: PubMed

ABSTRACT Nicotiana benthamiana is the most widely used experimental host in plant virology, due mainly to the large number of diverse plant viruses that can successfully infect it. Additionally, N. benthamiana is susceptible to a wide variety of other plant-pathogenic agents (such as bacteria, oomycetes, fungi, and so on), making this species a cornerstone of host-pathogen research, particularly in the context of innate immunity and defense signaling. Moreover, because it can be genetically transformed and regenerated with good efficiency and is amenable to facile methods for virus-induced gene silencing or transient protein expression, N. benthamiana is rapidly gaining popularity in plant biology, particularly in studies requiring protein localization, interaction, or plant-based systems for protein expression and purification. Paradoxically, despite being an indispensable research model, little is known about the origins, genetic variation, or ecology of the N. benthamiana accessions currently used by the research community. In addition to addressing these latter topics, the purpose of this review is to provide information regarding sources for tools and reagents that can be used to support research in N. benthamiana. Finally, we propose that N. benthamiana is well situated to become a premier plant cell biology model, particularly for the virology community, who as a group were the first to recognize the potential of this unique Australian native.

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    ABSTRACT: Hepatitis C virus (HCV) is major cause of liver cirrhosis in humans. HCV capsid (core) protein (HCVcp) is a highly demanded antigen for various diagnostic, immunization and pathogenesis studies. Plants are considered as an expression system for producing safe and inexpensive biopharmaceutical proteins. Although invention of transgenic (stable) tobacco plants expressing HCVcp with proper antigenic properties was recently reported, no data for "transient-expression" that is currently the method of choice for rapid, simple and lower-priced protein expression in plants is available for HCVcp. The purpose of this study was to design a highly codon-optimized HCVcp gene for construction of an efficient transient-plant expression system for production of HCVcp with proper antigenic properties in a regional tobacco plant (Iranian Jafarabadi-cultivar) by evaluation of different classes of vectors and suppression of gene-silencing in tobacco. A codon-optimized gene encoding the Kozak sequence, 6xHis-tag, HCVcp (1-122) and KDEL peptide in tandem (from N- to C-terminal) was designed and inserted into potato virus-X (PVX) and classic pBI121 binary vectors in separate cloning reactions. The resulted recombinant plasmids were transferred into Agrobacterium tumefaciens and vacuum infiltrated into tobacco leaves. The effect of gene silencing suppressor P19 protein derived from tomato bushy stunt virus on the expression yield of HCVcp by each construct was also evaluated by co-infiltration in separate groups. The expressed HCVcp was evaluated by dot and western blotting and ELISA assays. The codon-optimized gene had an increased adaptation index value (from 0.65 to 0.85) and reduced GC content (from 62.62 to 51.05) in tobacco and removed the possible deleterious effect of "GGTAAG" splice site in native HCVcp. Blotting assays via specific antibodies confirmed the expression of the 15 kDa HCVcp. The expression level of HCVcp was enhanced by 4-5 times in P19 co-agroinfiltrated plants with better outcomes for PVX, compared to pBI121 vector (0.022% versus 0.019% of the total soluble protein). The plant-derived HCVcp (pHCVcp) could properly identify the HCVcp antibody in HCV-infected human sera compared to Escherichia coli-derived HCVcp (eHCVcp), indicating its potential for diagnostic/immunization applications. By employment of gene optimization strategies, use of viral-based vectors and suppression of plant-derived gene silencing effect, efficient transient expression of HCVcp in tobacco with proper antigenic properties could be possible.
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    ABSTRACT: Plant-based transient overexpression systems enable rapid and scalable production of subunit vaccines. Previously, we have shown that cholera toxin B subunit (CTB), an oral cholera vaccine antigen, is N-glycosylated upon expression in transgenic Nicotiana benthamiana. Here, we found that overexpression of aglycosylated CTB by agroinfiltration of a tobamoviral vector causes massive tissue necrosis and poor accumulation unless retained in the endoplasmic reticulum (ER). However, the re-introduction of N-glycosylation to its original or an alternative site significantly relieved the necrosis and provided a high CTB yield without ER retention. Quantitative gene expression analysis of PDI, BiP, bZIP60, SKP1, 26Sα proteasome and PR1a, and the detection of ubiquitinated CTB polypeptides revealed that N-glycosylation significantly relieved ER stress and hypersensitive response, and facilitated the folding/assembly of CTB. The glycosylated CTB (gCTB) was characterized for potential vaccine use. Glycan profiling revealed that gCTB contained approximately 38% plant-specific glycans. gCTB retained nanomolar affinity to GM1-ganglioside with only marginal reduction of physicochemical stability and induced an anti-cholera holotoxin antibody response comparable to native CTB in a mouse oral immunization study. These findings demonstrated gCTB's potential as an oral immunogen and point to a potential role of N-glycosylation in increasing recombinant protein yields in plants.
    Scientific reports. 01/2015; 5:8003.
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    ABSTRACT: Nicotiana, a member of the Solanaceae family, is one of the most important research model plants, and of high agricultural and economic value worldwide. To better understand the substantial and rapid research progress with Nicotiana in recent years, its genomics, genetics, and nicotine gene studies are summarized, with useful web links. Several important genetic maps, including a high-density map of N. tabacum consisting of ~2,000 markers published in 2012, provide tools for genetics research. Four whole genome sequences are from allotetraploid species, including N. benthamiana in 2012, and three N. tabacum cultivars (TN90, K326, and BX) in 2014. Three whole genome sequences are from diploids, including progenitors N. sylvestris and N. tomentosiformis in 2013 and N. otophora in 2014. These and additional studies provide numerous insights into genome evolution after polyploidization, including changes in gene composition and transcriptome expression in N. tabacum. The major genes involved in the nicotine biosynthetic pathway have been identified and the genetic basis of the differences in nicotine levels among Nicotiana species has been revealed. In addition, other progress on chloroplast, mitochondrial, and NCBI-registered projects on Nicotiana are discussed. The challenges and prospects for genomic, genetic and application research are addressed. Hence, this review provides important resources and guidance for current and future research and application in Nicotiana.
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