Journal articles: 'Nicotiana benthamiana – Genetic engineering'

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Hayashi, Shunya, Mutsumi Watanabe, Makoto Kobayashi, Takayuki Tohge, Takashi Hashimoto, and Tsubasa Shoji. "Genetic Manipulation of Transcriptional Regulators Alters Nicotine Biosynthesis in Tobacco." Plant and Cell Physiology 61, no. 6 (2020): 1041–53. http://dx.doi.org/10.1093/pcp/pcaa036.

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Abstract The toxic alkaloid nicotine is produced in the roots of Nicotiana species and primarily accumulates in leaves as a specialized metabolite. A series of metabolic and transport genes involved in the nicotine pathway are coordinately upregulated by a pair of jasmonate-responsive AP2/ERF-family transcription factors, NtERF189 and NtERF199, in the roots of Nicotiana tabacum (tobacco). In this study, we explored the potential of manipulating the expression of these transcriptional regulators to alter nicotine biosynthesis in tobacco. The transient overexpression of NtERF189 led to alkaloid production in the leaves of Nicotiana benthamiana and Nicotiana alata. This ectopic production was further enhanced by co-overexpressing a gene encoding a basic helix-loop-helix-family MYC2 transcription factor. Constitutive and leaf-specific overexpression of NtERF189 increased the accumulation of foliar alkaloids in transgenic tobacco plants but negatively affected plant growth. By contrast, in a knockout mutant of NtERF189 and NtERF199 obtained through CRISPR/Cas9-based genome editing, alkaloid levels were drastically reduced without causing major growth defects. Metabolite profiling revealed the impact of manipulating the nicotine pathway on a wide range of nitrogen- and carbon-containing metabolites. Our findings provide insights into the biotechnological applications of engineering metabolic pathways by targeting transcription factors.

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Hong Hanh, Ha, Le Thi Thu Hien, and Huynh Thi Thu Hue. "Transient expression of gene encoding ZmLEA14A protein in Nicotiana benthamiana plant." Vietnam Journal of Biotechnology 17, no. 3 (2020): 491–97. http://dx.doi.org/10.15625/1811-4989/17/3/13743.

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LEA protein family includes proteins accumulated in the late stage of embryogenesis and in vegetative tissues of stress-confronted plant. These proteins have been demontrated to play a major role in plant response to abiotic stresses, such as drought and salinity stress. The genes coding for LEA proteins in maize are divided into 9 groups including LEA 1, LEA 2, LEA 3, LEA 4, LEA 5, LEA 6, SMP, dehydrin, and AtM. The application of LEA genes to improve drought tolerance for plants by genetic engineering has also been studied extensively all over the world. In this study, pCAM/35S-ZmLEA14A-35S vector and pCAM/Ubi-ZmLEA14A-35S vector contained the ZmLEA14A gene isolated from Te vang 1, these vectors were used to transient express into Nicotiana benthamiana tobacco leaves by agro-infiltration method. The results of immunoassay between cmyc specific antibodies with proteins from infected leaves revealed the expression of recombinant ZmLEA14A protein in N. benthamiana leaves. Thereby, two constructs habouring the ZmLEA14A gene work at transcription and translation levels in the model plant that could harnessed for stable transformation in plants.

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Hao, Guixia, Marco Pitino, Yongping Duan, and Ed Stover. "Reduced Susceptibility to Xanthomonas citri in Transgenic Citrus Expressing the FLS2 Receptor From Nicotiana benthamiana." Molecular Plant-Microbe Interactions® 29, no. 2 (2016): 132–42. http://dx.doi.org/10.1094/mpmi-09-15-0211-r.

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Overexpression of plant pattern-recognition receptors by genetic engineering provides a novel approach to enhance plant immunity and broad-spectrum disease resistance. Citrus canker disease associated with Xanthomonas citri is one of the most important diseases damaging citrus production worldwide. In this study, we cloned the FLS2 gene from Nicotiana benthamiana cDNA and inserted it into the binary vector pBinPlus/ARS to transform Hamlin sweet orange and Carrizo citrange. Transgene presence was confirmed by polymerase chain reaction (PCR) and gene expression of NbFLS2 was compared by reverse transcription quantitative PCR. Reactive oxygen species (ROS) production in response to flg22Xcc was detected in transgenic Hamlin but not in nontransformed controls. Low or no ROS production was detected from nontransformed Hamlin seedlings challenged with flg22Xcc. Transgenic plants highly expressing NbFLS2 were selected and were evaluated for resistance to canker incited by X. citri 3213. Our results showed that the integration and expression of the NbFLS2 gene in citrus can increase canker resistance and defense-associated gene expression when challenged with X. citri. These results suggest that canker-susceptible Citrus genotypes lack strong basal defense induced by X. citri flagellin and the resistance of these genotypes can be enhanced by transgenic expression of the flagellin receptor from a resistant species.

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Khakhar, Arjun, Cecily Wang, Ryan Swanson, et al. "VipariNama: RNA viral vectors to rapidly elucidate the relationship between gene expression and phenotype." Plant Physiology 186, no. 4 (2021): 2222–38. http://dx.doi.org/10.1093/plphys/kiab197.

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Abstract Synthetic transcription factors have great promise as tools to help elucidate relationships between gene expression and phenotype by allowing tunable alterations of gene expression without genomic alterations of the loci being studied. However, the years-long timescales, high cost, and technical skill associated with plant transformation have limited their use. In this work, we developed a technology called VipariNama (ViN) in which vectors based on the tobacco rattle virus are used to rapidly deploy Cas9-based synthetic transcription factors and reprogram gene expression in planta. We demonstrate that ViN vectors can implement activation or repression of multiple genes systemically and persistently over several weeks in Nicotiana benthamiana, Arabidopsis (Arabidopsis thaliana), and tomato (Solanum lycopersicum). By exploring strategies including RNA scaffolding, viral vector ensembles, and viral engineering, we describe how the flexibility and efficacy of regulation can be improved. We also show how this transcriptional reprogramming can create predictable changes to metabolic phenotypes, such as gibberellin biosynthesis in N. benthamiana and anthocyanin accumulation in Arabidopsis, as well as developmental phenotypes, such as plant size in N. benthamiana, Arabidopsis, and tomato. These results demonstrate how ViN vector-based reprogramming of different aspects of gibberellin signaling can be used to engineer plant size in a range of plant species in a matter of weeks. In summary, ViN accelerates the timeline for generating phenotypes from over a year to just a few weeks, providing an attractive alternative to transgenesis for synthetic transcription factor-enabled hypothesis testing and crop engineering.

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Liu, Zongrang, Ralph Scorza, Jean-Michel Hily, Simon W. Scott, and Delano James. "Engineering Resistance to Multiple Prunus Fruit Viruses Through Expression of Chimeric Hairpins." Journal of the American Society for Horticultural Science 132, no. 3 (2007): 407–14. http://dx.doi.org/10.21273/jashs.132.3.407.

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Prunus L. fruit production is seriously affected by several predominant viruses. The development of new cultivars resistant to these viruses is challenging but highly desired by breeders and growers. We report a posttranscriptional gene silencing-based approach for engineering multivirus resistance in plants. A single chimeric transgene, PTRAP6, was created by the fusion of 400 to 500-base pair (bp) gene fragments from six major Prunus fruit viruses, including american plum line pattern virus, peach mosaic virus, plum pox virus (PPV), prune dwarf virus (PDV), prunus necrotic ringspot virus, and tomato ringspot virus (ToRSV). Both strands of PTRAP6 were found being transcribed as an ≈2.5-kilobp transcript in planta without splicing interruption. To induce gene silencing/virus resistance, we placed two copies of PTRAP6 in an inverted repeat under the control of the cauliflower mosaic virus 35S promoter and separated by an intron spacer fragment to create PTRAP6i. Inoculation of the resulting transgenic Nicotiana benthamiana Domin. plants revealed that 12 of 28 R0 PTRAP6i transgenic lines (43%) were resistant to ToRSV ranging from mild symptoms to symptom-free phenotypes. Detailed analysis of two of three highly resistant homozygous R3 generation lines demonstrated that they were resistant to all three viruses tested, including PDV, PPV, and ToRSV. The remaining three viruses targeted by PTRAP6i were either unavailable for this study or were unable to systemically infect N. benthamiana. Transgene-wide and -specific small interfering RNA species were detected along with disappearance of transgene transcript in the resistant lines, indicating that posttranscriptional gene silencing underlies the mechanism of resistance. This work presents evidence that PTRAP6i is able to confer gene silencing-based resistance to multiple Prunus fruit viruses.

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Naim, Fatima, Kenlee Nakasugi, Ross N. Crowhurst, et al. "Advanced Engineering of Lipid Metabolism in Nicotiana benthamiana Using a Draft Genome and the V2 Viral Silencing-Suppressor Protein." PLoS ONE 7, no. 12 (2012): e52717. http://dx.doi.org/10.1371/journal.pone.0052717.

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Efremova, Larisa N., Svetlana R. Strelnikova, Guzel R. Gazizova, Elena A. Minkina, and Roman A. Komakhin. "A Synthetic Strong and Constitutive Promoter Derived from the Stellaria media pro-SmAMP1 and pro-SmAMP2 Promoters for Effective Transgene Expression in Plants." Genes 11, no. 12 (2020): 1407. http://dx.doi.org/10.3390/genes11121407.

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Synthetic promoters are vital for genetic engineering-based strategies for crop improvement, but effective methodologies for their creation and systematic testing are lacking. We report here on the comparative analysis of the promoters pro-SmAMP1 and pro-SmAMP2 from Stellaria media ANTIMICROBIAL PEPTIDE1 (AMP1) and ANTIMICROBIAL PEPTIDE2 (AMP2). These promoters are more effective than the well-known Cauliflower mosaic virus 35S promoter. Although these promoters share about 94% identity, the pro-SmAMP1 promoter demonstrated stronger transient expression of a reporter gene in Agrobacterium infiltration of Nicotiana benthamiana leaves, while the pro-SmAMP2 promoter was more effective for the selection of transgenic tobacco (Nicotiana tabacum) cells when driving a selectable marker. Using the cap analysis of gene expression method, we detected no differences in the structure of the transcription start sites for either promoter in transgenic plants. For both promoters, we used fine-scale deletion analysis to identify 160 bp-long sequences that retain the unique properties of each promoter. With the use of chimeric promoters and directed mutagenesis, we demonstrated that the superiority of the pro-SmAMP1 promoter for Agrobacterium-mediated infiltration is caused by the proline-inducible ACTCAT cis-element strictly positioned relative to the TATA box in the core promoter. Surprisingly, the ACTCAT cis-element not only activated but also suppressed the efficiency of the pro-SmAMP1 promoter under proline stress. The absence of the ACTCAT cis-element and CAANNNNATC motif (negative regulator) in the pro-SmAMP2 promoter provided a more constitutive gene expression profile and better selection of transgenic cells on selective medium. We created a new synthetic promoter that enjoys high effectiveness both in transient expression and in selection of transgenic cells. Intact promoters with differing properties and high degrees of sequence identity may thus be used as a basis for the creation of new synthetic promoters for precise and coordinated gene expression.

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Wang, Cuiwei, Christoph Crocoll, Niels Agerbirk, and Barbara Ann Halkier. "Engineering and optimization of the 2‐phenylethylglucosinolate production in Nicotiana benthamiana by combining biosynthetic genes from Barbarea vulgaris and Arabidopsis thaliana." Plant Journal 106, no. 4 (2021): 978–92. http://dx.doi.org/10.1111/tpj.15212.

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Röder, Juliane, Christina Dickmeis, Rainer Fischer, and Ulrich Commandeur. "Systemic Infection of Nicotiana benthamiana with Potato virus X Nanoparticles Presenting a Fluorescent iLOV Polypeptide Fused Directly to the Coat Protein." BioMed Research International 2018 (2018): 1–12. http://dx.doi.org/10.1155/2018/9328671.

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Plant virus-based nanoparticles can be produced in plants on a large scale and are easily modified to introduce new functions, making them suitable for applications such as vaccination and drug delivery, tissue engineering, and in vivo imaging. The latter is often achieved using green fluorescent protein and its derivatives, but the monovalent fluorescent protein iLOV is smaller and more robust. Here, we fused the iLOV polypeptide to the N-terminus of the Potato virus X (PVX) coat protein, directly or via the Foot-and-mouth disease virus 2A sequence, for expression in Nicotiana benthamiana. Direct fusion of the iLOV polypeptide did not prevent the assembly or systemic spread of the virus and we verified the presence of fusion proteins and iLOV hybrid virus particles in leaf extracts. Compared to wild-type PVX virions, the PVX particles displaying the iLOV peptide showed an atypical, intertwined morphology. Our results confirm that a direct fusion of the iLOV fluorescent protein to filamentous PVX nanoparticles offers a promising tool for imaging applications.

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Gao, L., R. Zhai, Y. K. Zhong, et al. "Screening Isolates of Soybean mosaic virus for Infectivity in a Model Plant, Nicotiana benthamiana." Plant Disease 99, no. 4 (2015): 442–46. http://dx.doi.org/10.1094/pdis-04-14-0405-re.

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Soybean mosaic virus (SMV), belonging to the genus Potyvirus of the family Potyviridae, has a relatively narrow host range almost exclusively confined to leguminous hosts. While disease management through genetic transformation can be an effective approach, soybean remains recalcitrant to routine genetic transformation. In this context, it is important to identify new hosts for SMV that can be used to develop effective transgenic resistance strategies. Transformation in Nicotiana benthamiana is simple and highly efficient; hence, here we demonstrate the infectivity of SMV strain SC7 in N. benthamiana plants. To identify an SMV strain infectious in N. benthamiana, we mechanically inoculated N. benthamiana plants with 37 isolates from 21 (SC1 to SC21) SMV strains. Plants inoculated with isolates of strain SC7 produced mosaic symptoms on leaves. However, N. benthamiana plants inoculated with the 20 other SMV strains showed no visible symptoms. Furthermore, soybean cv. Nannong 1138-2 inoculated with sap prepared from symptomatic N. benthamiana leaves showed typical SMV mosaic symptoms 2 weeks after inoculation. In addition, SMV was detected in symptomatic N. benthamiana and soybean leaves by RT-PCR, DAS-ELISA, and further identified by sequencing. Together, the results indicate that N. benthamiana plants could support multiplication of SMV strain SC7. The findings of this study would be useful for the investigation of SMV resistance using the model plant N. benthamiana.

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Ambrós, Silvia, Choaa El-Mohtar, Susana Ruiz-Ruiz, et al. "Agroinoculation of Citrus tristeza virus Causes Systemic Infection and Symptoms in the Presumed Nonhost Nicotiana benthamiana." Molecular Plant-Microbe Interactions® 24, no. 10 (2011): 1119–31. http://dx.doi.org/10.1094/mpmi-05-11-0110.

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Citrus tristeza virus (CTV) naturally infects only some citrus species and relatives and within these it only invades phloem tissues. Failure to agroinfect citrus plants and the lack of an experimental herbaceous host hindered development of a workable genetic system. A full-genome cDNA of CTV isolate T36 was cloned in binary plasmids and was used to agroinfiltrate Nicotiana benthamiana leaves, with or without coinfiltration with plasmids expressing different silencing-suppressor proteins. A time course analysis in agroinfiltrated leaves indicated that CTV accumulates and moves cell-to-cell for at least three weeks postinoculation (wpi), and then, it moves systemically and infects the upper leaves with symptom expression. Silencing suppressors expedited systemic infection and often increased infectivity. In systemically infected Nicotiana benthamiana plants, CTV invaded first the phloem, but after 7 wpi, it was also found in other tissues and reached a high viral titer in upper leaves, thus allowing efficient transmission to citrus by stem-slash inoculation. Infected citrus plants showed the symptoms, virion morphology, and phloem restriction characteristic of the wild T36 isolate. Therefore, agroinfiltration of Nicotiana benthamiana provided the first experimental herbaceous host for CTV and an easy and efficient genetic system for this closterovirus.

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Goodin, Michael M., David Zaitlin, Rayapati A. Naidu, and Steven A. Lommel. "Nicotiana benthamiana: Its History and Future as a Model for Plant–Pathogen Interactions." Molecular Plant-Microbe Interactions® 21, no. 8 (2008): 1015–26. http://dx.doi.org/10.1094/mpmi-21-8-1015.

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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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Reed, James, and Anne Osbourn. "Engineering terpenoid production through transient expression in Nicotiana benthamiana." Plant Cell Reports 37, no. 10 (2018): 1431–41. http://dx.doi.org/10.1007/s00299-018-2296-3.

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Nguyen, Hanh P., Suma Chakravarthy, André C. Velásquez, et al. "Methods to Study PAMP-Triggered Immunity Using Tomato and Nicotiana benthamiana." Molecular Plant-Microbe Interactions® 23, no. 8 (2010): 991–99. http://dx.doi.org/10.1094/mpmi-23-8-0991.

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Understanding the molecular basis of plant responses to pathogen-associated molecular patterns (PAMPs) is an active area of research in the field of plant–microbe interactions. A growing number of plant genes involved in various steps of PAMP-triggered immunity (PTI) pathways and microbial factors involved in the elicitation or suppression of PTI have been identified. These studies have largely relied on Arabidopsis thaliana and, therefore, most of the PTI assays have been developed and optimized for that model plant system. Although PTI is a conserved feature among plants, the response spectra vary across different species. Thus, there is a need for robust PTI assays in other pathosystems, such as those involving Solanaceae plant–pathogen interactions, which include many economically important plants and their diseases. We have optimized molecular, cellular, and whole-plant methods to measure PTI responses in two widely studied solanaceous species, tomato (Solanum lycopersicum) and Nicotiana benthamiana. Here, we provide detailed protocols for measuring various PTI-associated phenotypes, including bacterial populations after pretreatment of leaves with PAMPs, induction of reporter genes, callose deposition, activation of mitogen-activated protein kinases, and a luciferase-based reporter system. These methods will facilitate limited genetic screens and detailed characterization of potential PTI-related genes in model and economically important Solanaceae spp.–pathogen interactions.

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Zhao, Meiwei, Tao Zhang, Lei Yang, Hongtao Feng, and Zhengxiong Zhao. "Initial Characterization and Expression Pattern Analysis of Tobacco (Nicotiana Tabacum) HMGS Gene." E3S Web of Conferences 292 (2021): 03094. http://dx.doi.org/10.1051/e3sconf/202129203094.

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3-hydroxy-3-methylglutaryl coenzyme A synthase (HMGS) is a member of condensing enzymes that catalyze a Claisen-like condensation reaction.The tobacco (nicotiana tabacum) HMGS gene was firstly characterized using the rapid amplification of cDNA ends methods based on one tobacco EST. The full-length tobacco HMGS gene mRNA was 1,773bp containing a 1389 bp open reading frame, which encodes a protein of 462 amino acids. Sequence analysis revealed that the HMGS of tobacco shares high homology with the HMGS of nicotiana tomentosiformis (96%), nicotiana attenuata (95%), Nicotiana sylvestris (95%), nicotiana benthamiana(94%), solanum lycopersicum(94%), solanum tuberosum(93%) and withania somnifera(93%). Results also showed that tobacco HMGS gene has a closer genetic relationship with the HMGS gene of withania somnifera. Tissue expression profile analysis revealed that the tobacco HMGS gene was highly expressed in flower, but moderately expressed in leaf and stem, and weakly expressed in root. Our experiment established the foundation for further research on this tobacco gene.

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Ge, Linmei, Jiangtao Zhang, Xueping Zhou, and Hongye Li. "Genetic Structure and Population Variability of Tomato Yellow Leaf Curl China Virus." Journal of Virology 81, no. 11 (2007): 5902–7. http://dx.doi.org/10.1128/jvi.02431-06.

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ABSTRACT Geminiviruses have circular single-stranded DNA genomes and are important pathogens in tropical and subtropical regions, but their population diversity and variability are poorly understood. Here, we have investigated variations accumulating in Tomato yellow leaf curl China virus (TYLCCNV), a geminivirus in the genus Begomovirus of the family Geminiviridae. The population variation was analyzed in a naturally infected tomato (Solanum lycopersicom) plant and in Nicotiana benthamiana and tomato plants experimentally infected with a swarm of TYLCCNV DNA clones to provide an identical sequence for initiation of infection. Our results demonstrate that the population of TYLCCNV in a naturally infected tomato plant was genetically heterogeneous and that rapid mutation occurred in the populations amplified from N. benthamiana and tomato plants that had been infected with cloned DNA. This feature of the population of TYLCCNV in these plants consisted of the consensus sequence and a pool of mutants that are not identical but are closely related to the consensus sequence, and it coincides with the quasispecies concept described for many RNA viruses. The mutation frequency was circa 10−4 in N. benthamiana and tomato at 60 days postinoculation, a value comparable to that reported for plant RNA viruses. The quasispecies-like nature of the TYLCCNV populations suggested that TYLCCNV is capable of rapid evolution and adaptation in response to changing agricultural practices.

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Hasan, Md Mohidul, Hyun-Soon Kim, Jae-Heung Jeon, et al. "Metabolic engineering of Nicotiana benthamiana for the increased production of taxadiene." Plant Cell Reports 33, no. 6 (2014): 895–904. http://dx.doi.org/10.1007/s00299-014-1568-9.

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Jutras, Philippe V., Isobel Dodds, and Renier AL van der Hoorn. "Proteases of Nicotiana benthamiana: an emerging battle for molecular farming." Current Opinion in Biotechnology 61 (February 2020): 60–65. http://dx.doi.org/10.1016/j.copbio.2019.10.006.

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Liu, Junying, Huiyan Fan, Ying Wang, et al. "Genome-Wide microRNA Profiling Using Oligonucleotide Microarray Reveals Regulatory Networks of microRNAs in Nicotiana benthamiana During Beet Necrotic Yellow Vein Virus Infection." Viruses 12, no. 3 (2020): 310. http://dx.doi.org/10.3390/v12030310.

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Beet necrotic yellow vein virus (BNYVV) infections induce stunting and leaf curling, as well as root and floral developmental defects and leaf senescence in Nicotiana benthamiana. A microarray analysis with probes capable of detecting 1596 candidate microRNAs (miRNAs) was conducted to investigate differentially expressed miRNAs and their targets upon BNYVV infection of N. benthamiana plants. Eight species-specific miRNAs of N. benthamiana were identified. Comprehensive characterization of the N. benthamiana microRNA profile in response to the BNYVV infection revealed that 129 miRNAs were altered, including four species-specific miRNAs. The targets of the differentially expressed miRNAs were predicted accordingly. The expressions of miR164, 160, and 393 were up-regulated by BNYVV infection, and those of their target genes, NAC21/22, ARF17/18, and TIR, were down-regulated. GRF1, which is a target of miR396, was also down-regulated. Further genetic analysis of GRF1, by Tobacco rattle virus-induced gene silencing, assay confirmed the involvement of GRF1 in the symptom development during BNYVV infection. BNYVV infection also induced the up-regulation of miR168 and miR398. The miR398 was predicted to target umecyanin, and silencing of umecyanin could enhance plant resistance against viruses, suggesting the activation of primary defense response to BNYVV infection in N. benthamiana. These results provide a global profile of miRNA changes induced by BNYVV infection and enhance our understanding of the mechanisms underlying BNYVV pathogenesis.

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Souza, Tiago Alves Jorge de, Greice Lubini, Andrea Carla Quiapim, and Tiago Campos Pereira. "Nicotiana benthamiana seeds tolerate hyperaccelerations up to 400,000 x g." Research, Society and Development 10, no. 8 (2021): e27510817323. http://dx.doi.org/10.33448/rsd-v10i8.17323.

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Exposure to hypergravity can alter the viability, morphology, development and behavior of living beings. Thus, the analysis of these factors is essential when considering life on supermassive planets, as well as in 'ballistic panspermia' scenarios related to the ejection of rocks from the surface of a planet, which could serve as transfer vehicles to spread the life between planets within a solar system. Studies analyzing the effects of hypergravity regimes are abundant in the literature, however, only a few researches carried out experiments using conditions of the order of 105 x g. In addition, the only plant species tested so far, as an entire structure instead of detached parts, exposed to gravity stress of this order of magnitude in its entirety was Oryza sativa, whose seeds were able to germinate after being exposed to 450,000 x g. Recently, our research group demonstrated that some free-living nematode species can support 400,000 x g. In the present study, we report that seeds of the plant model Nicotiana benthamiana exposed to 400,000 x g for 1h are able to germinate into fully normal young seedlings, with no apparent morphological alterations. Since N. benthamiana is used in laboratories worldwide and an easy to cultivate plant model, theoretical and experimental models of lithopanspermia and life in supermassive planets may benefit from it.

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Lorang, J. M., C. H. Hagerty, R. Lee, P. E. McClean, and T. J. Wolpert. "Genetic Analysis of Victorin Sensitivity and Identification of a Causal Nucleotide-Binding Site Leucine-Rich Repeat Gene in Phaseolus vulgaris." Molecular Plant-Microbe Interactions® 31, no. 10 (2018): 1069–74. http://dx.doi.org/10.1094/mpmi-12-17-0328-r.

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Cochliobolus victoria, the causal agent of Victoria blight, is pathogenic due to its production of a toxin called victorin. Victorin sensitivity in oats, barley, Brachypodium spp., and Arabidopsis has been associated with nucleotide-binding site leucine-rich repeat (NLR) genes, a class of genes known for conferring disease resistance. In this work, we investigated the sensitivity of Phaseolus vulgaris to victorin. We found that victorin sensivity in Phaseolus vulgaris is a developmentally regulated, quantitative trait. A single quantitative trait locus (QTL) accounted for 34% of the phenotypic variability in victorin sensitivity among Stampede × Red Hawk (S×R) recombinant inbred lines. We cloned two NLR-encoding genes within this QTL and showed one, Phvul05G031200 (PvLOV), confers victorin-dependent cell death when overexpressed in Nicotiana benthamiana. Protein sequences of PvLOV from victorin-sensitive and the victorin-resistant bean parents differ by two amino acids in the leucine-rich repeat region, but both proteins confer victorin-dependent cell death when overexpressed in N. benthamiana.

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Ilgekbaeva,, G. D., E. Sh Makhashov, G. Tulepova, D. Yessimkhankyzy, and S. T. Sadiev. "EXPRESSION OF THE SURFACE ANTIGEN BRUCELLA ABORTUS OMR16 IN NICOTIANA BENTHAMIANA PLANT." REPORTS 5, no. 333 (2020): 81–85. http://dx.doi.org/10.32014/2020.2518-1483.122.

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Brucellosis is one of the most contagious and infectious diseases with high incidence rates of cattle and humans in Kazakhstan. Using modern biotechnology techniques to develop vaccines that are reliable and affordable for farmers is an alternative solution to the problem. Plant viruses are often used as a vector for obtaining the expression of antigens of the pathogen. The grape virus A (BAB) is widely used among plant viruses. Brucella membrane proteins are the main objects of this research for futher development of vaccines or diagnostic texts against brucellosis, Membrane proteins (OMPs) are cell specific surface antigens that are immunogenic. OMPs are ideal candidates for the production of recombinant brucellosis vaccines. The object of the study was the outer membrane protein (Omp16), which plays an important role in the suppression of TNF-α production in macrophages. In this study, molecular cloning and analysis of the expression of the Omp16 gene, which was used to express the recombinant protein in plants, was carried out. We selected brucella from the vaccine strain of Brucella abortus 19, and the plant Nicotiana benthamiana, as the subjects for our research, since they widely used for the production of recombinant proteins, and they both appropriate for molecular genetic research. A viral vector was constructed to express the brucellosis antigen Omp16 in Nicotiana benthamiana plants. Nineteen explants were used for the regeneration of transgenic plants. As a result of this studies, the introduced gene of Omp16 was under the subgenomic promoter control of the ORF4 and was successfully expressed while maintaining the efficiency of expression in transgenic plants. The efficiency of viral vectors was evaluated at the level of transcription during expression of the protein Omp16 with viral proteins. The entire leaf blade was infiltrated; the density of Agrobacteria was 0.7. We were able to obtained transgenic plants Nicotiana benthamiana carrying the gene of capsid protein BAB, and the expression of the membrane antigen Omp16 in the viral vector was achieved by replacing the ORF4 with the Omp16 gene. The development of transgenic plants was carried out using agrobacterial transformation.

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Wang, A., L. Tian, T. S. Huang, et al. "THE DEVELOPMENT OF GENETIC RESISTANCE TO PLUM POX VIRUS IN TRANSGENIC NICOTIANA BENTHAMIANA AND PRUNUS DOMESTICA." Acta Horticulturae, no. 839 (July 2009): 665–72. http://dx.doi.org/10.17660/actahortic.2009.839.91.

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Mittelberger, Cecilia, Hagen Stellmach, Bettina Hause, et al. "A Novel Effector Protein of Apple Proliferation Phytoplasma Disrupts Cell Integrity of Nicotiana spp. Protoplasts." International Journal of Molecular Sciences 20, no. 18 (2019): 4613. http://dx.doi.org/10.3390/ijms20184613.

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Effector proteins play an important role in the virulence of plant pathogens such as phytoplasma, which are the causative agents of hundreds of different plant diseases. The plant hosts comprise economically relevant crops such as apples (Malus × domestica), which can be infected by ‘Candidatus Phytoplasma mali’ (P. mali), a highly genetically dynamic plant pathogen. As the result of the genetic and functional analyses in this study, a new putative P. mali effector protein was revealed. The so-called “Protein in Malus Expressed 2” (PME2), which is expressed in apples during P. mali infection but not in the insect vector, shows regional genetic differences. In a heterologous expression assay using Nicotiana benthamiana and Nicotiana occidentalis mesophyll protoplasts, translocation of both PME2 variants in the cell nucleus was observed. Overexpression of the effector protein affected cell integrity in Nicotiana spp. protoplasts, indicating a potential role of this protein in pathogenic virulence. Interestingly, the two genetic variants of PME2 differ regarding their potential to manipulate cell integrity. However, the exact function of PME2 during disease manifestation and symptom development remains to be further elucidated. Aside from the first description of the function of a novel effector of P. mali, the results of this study underline the necessity for a more comprehensive description and understanding of the genetic diversity of P. mali as an indispensable basis for a functional understanding of apple proliferation disease.

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Ali, Md Sarafat, and Kwang-Hyun Baek. "Co-Suppression of NbClpC1 and NbClpC2, Encoding Clp Protease Chaperons, Elicits Significant Changes in the Metabolic Profile of Nicotiana benthamiana." Plants 9, no. 2 (2020): 259. http://dx.doi.org/10.3390/plants9020259.

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Metabolites in plants are the products of cellular metabolic processes, and their differential amount can be regarded as the final responses of plants to genetic, epigenetic, or environmental stresses. The Clp protease complex, composed of the chaperonic parts and degradation proteases, is the major degradation system for proteins in plastids. ClpC1 and ClpC2 are the two chaperonic proteins for the Clp protease complex and share more than 90% nucleotide and amino acid sequence similarities. In this study, we employed virus-induced gene silencing to simultaneously suppress the expression of ClpC1 and ClpC2 in Nicotiana benthamiana (NbClpC1/C2). The co-suppression of NbClpC1/C2 in N. benthamiana resulted in aberrant development, with severely chlorotic leaves and stunted growth. A comparison of the control and NbClpC1/C2 co-suppressed N. benthamiana metabolomes revealed a total of 152 metabolites identified by capillary electrophoresis time-of-flight mass spectrometry. The co-suppression of NbClpC1/C2 significantly altered the levels of metabolites in glycolysis, the tricarboxylic acid cycle, the pentose phosphate pathway, and the purine biosynthetic pathway, as well as polyamine and antioxidant metabolites. Our results show that the simultaneous suppression of ClpC1 and ClpC2 leads to aberrant morphological changes in chloroplasts and that these changes are related to changes in the contents of major metabolites acting in cellular metabolism and biosynthetic pathways.

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Verchot, Jeanmarie, Venura Herath, Cesar D. Urrutia, et al. "Development of a Reverse Genetic System for Studying Rose Rosette Virus in Whole Plants." Molecular Plant-Microbe Interactions® 33, no. 10 (2020): 1209–21. http://dx.doi.org/10.1094/mpmi-04-20-0094-r.

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Rose rosette virus (RRV) is a negative-sense RNA virus with a seven-segmented genome that is enclosed by a double membrane. We constructed an unconventional minireplicon system encoding the antigenomic (ag)RNA1 (encoding the viral RNA-dependent RNA polymerase [RdRp]), agRNA3 (encoding the nucleocapsid protein [N]), and a modified agRNA5 containing the coding sequence for the iLOV protein in place of the P5 open reading frame (R5-iLOV). iLOV expression from the R5-iLOV template was amplified by activities of the RdRp and N proteins in Nicotiana benthamiana leaves. A mutation was introduced into the RdRp catalytic domain and iLOV expression was eliminated, indicating RNA1-encoded polymerase activity drives iLOV expression from the R5-iLOV template. Fluorescence from the replicon was highest at 3 days postinoculation (dpi) and declined at 7 and 13 dpi. Addition of the tomato bushy stunt virus (TBSV) P19 silencing-suppressor protein prolonged expression until 7 dpi. A full-length infectious clone system was constructed of seven binary plasmids encoding each of the seven genome segments. Agro-delivery of constructs encoding RRV RNAs 1 through 4 or RNAs 1 through 7 to N. benthamiana plants produced systemic infection. Finally, agro-delivery of the full-length RRV infectious clone including all segments produced systemic infection within 60 dpi. This advance opens new opportunities for studying RRV infection biology.

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Ahmad, Aktsar Roskiana, Pornjira Kaewpungsup, Narach Khorattanakulchai, Kaewta Rattanapisit, Prasit Pavasant, and Waranyoo Phoolcharoen. "Recombinant Human Dentin Matrix Protein 1 (hDMP1) Expressed in Nicotiana benthamiana Potentially Induces Osteogenic Differentiation." Plants 8, no. 12 (2019): 566. http://dx.doi.org/10.3390/plants8120566.

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Inductive molecules are critical components for successful bone tissue engineering. Dentin matrix protein-1 (DMP1), a non-collagenous protein in the bone matrix, has been shown to play roles in osteogenic differentiation and phosphate homeostasis. This study aimed to produce recombinant human dentin matrix protein-1 (hDMP1) in Nicotiana benthamiana and investigated the ability of this plant-produced DMP1 to induce osteogenesis in human periodontal ligament stem cells (hPDLSCs). The hDMP1 gene was cloned into the geminiviral vector for transient expression in N. benthamiana. We found that hDMP1 was transiently expressed in N. benthamiana leaves and could be purified by ammonium sulphate precipitation followed by nickel affinity chromatography. The effects of hDMP1 on the induction of cell proliferation and osteogenic differentiation were investigated. The results indicated that plant-produced hDMP1 could induce the cell proliferation of hPDLSCs and increase the expression levels of osteogenic genes, including osterix (OSX), type I collagen (COL1), bone morphogenetic protein-2 (BMP2), and Wnt3a. Moreover, the plant-produced hDMP1 promoted calcium deposition in hPDLSCs as determined by alizarin red S staining. In conclusion, our results indicated that plant-produced hDMP1 could induce osteogenic differentiation in hPDLSCs and could potentially be used as a bone inducer in bone tissue engineering.

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Pfalz, Marina, Michael Dalgaard Mikkelsen, Paweł Bednarek, Carl Erik Olsen, Barbara Ann Halkier, and Juergen Kroymann. "Metabolic Engineering in Nicotiana benthamiana Reveals Key Enzyme Functions in Arabidopsis Indole Glucosinolate Modification." Plant Cell 23, no. 2 (2011): 716–29. http://dx.doi.org/10.1105/tpc.110.081711.

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Zhou, Yang, Meiqi Ding, Xiaodong Duan, Kai R. Konrad, Georg Nagel, and Shiqiang Gao. "Extending the Anion Channelrhodopsin-Based Toolbox for Plant Optogenetics." Membranes 11, no. 4 (2021): 287. http://dx.doi.org/10.3390/membranes11040287.

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Optogenetics was developed in the field of neuroscience and is most commonly using light-sensitive rhodopsins to control the neural activities. Lately, we have expanded this technique into plant science by co-expression of a chloroplast-targeted β-carotene dioxygenase and an improved anion channelrhodopsin GtACR1 from the green alga Guillardia theta. The growth of Nicotiana tabacum pollen tube can then be manipulated by localized green light illumination. To extend the application of analogous optogenetic tools in the pollen tube system, we engineered another two ACRs, GtACR2, and ZipACR, which have different action spectra, light sensitivity and kinetic features, and characterized them in Xenopus laevis oocytes, Nicotiana benthamiana leaves and N. tabacum pollen tubes. We found that the similar molecular engineering method used to improve GtACR1 also enhanced GtACR2 and ZipACR performance in Xenopus laevis oocytes. The ZipACR1 performed in N. benthamiana mesophyll cells and N. tabacum pollen tubes with faster kinetics and reduced light sensitivity, allowing for optogenetic control of anion fluxes with better temporal resolution. The reduced light sensitivity would potentially facilitate future application in plants, grown under low ambient white light, combined with an optogenetic manipulation triggered by stronger green light.

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Balmuth, Alexi, and John P. Rathjen. "Genetic and molecular requirements for function of the Pto/Prf effector recognition complex in tomato and Nicotiana benthamiana." Plant Journal 51, no. 6 (2007): 978–90. http://dx.doi.org/10.1111/j.1365-313x.2007.03199.x.

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Alkanaimsh, Salem, Jasmine M. Corbin, Muchena J. Kailemia, et al. "Purification and site-specific N-glycosylation analysis of human recombinant butyrylcholinesterase from Nicotiana benthamiana." Biochemical Engineering Journal 142 (February 2019): 58–67. http://dx.doi.org/10.1016/j.bej.2018.11.004.

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Nekrasov, Vladimir, Brian Staskawicz, Detlef Weigel, Jonathan D. G. Jones, and Sophien Kamoun. "Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease." Nature Biotechnology 31, no. 8 (2013): 691–93. http://dx.doi.org/10.1038/nbt.2655.

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Aslan, Selcuk, Chuanxin Sun, Svetlana Leonova, et al. "Wax esters of different compositions produced via engineering of leaf chloroplast metabolism in Nicotiana benthamiana." Metabolic Engineering 25 (September 2014): 103–12. http://dx.doi.org/10.1016/j.ymben.2014.07.001.

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Slocombe, Stephen P., Ines Schauvinhold, Ryan P. McQuinn, et al. "Transcriptomic and Reverse Genetic Analysesof Branched-Chain Fatty Acid and Acyl Sugar Production in Solanum pennellii and Nicotiana benthamiana." Plant Physiology 148, no. 4 (2008): 1830–46. http://dx.doi.org/10.1104/pp.108.129510.

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Leister, R. Todd, Douglas Dahlbeck, Brad Day, Yi Li, Olga Chesnokova, and Brian J. Staskawicz. "Molecular Genetic Evidence for the Role of SGT1 in the Intramolecular Complementation of Bs2 Protein Activity in Nicotiana benthamiana." Plant Cell 17, no. 4 (2005): 1268–78. http://dx.doi.org/10.1105/tpc.104.029637.

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Lee, Wing-Sham, Shih-Feng Fu, Jeanmarie Verchot-Lubicz, and John P. Carr. "Genetic modification of alternative respiration in Nicotiana benthamiana affects basal and salicylic acid-induced resistance to potato virus X." BMC Plant Biology 11, no. 1 (2011): 41. http://dx.doi.org/10.1186/1471-2229-11-41.

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Saunders, Keith, Christina Wege, Karuppannan Veluthambi, Holger Jeske, and John Stanley. "The distinct disease phenotypes of the common and yellow vein strains of Tomato golden mosaic virus are determined by nucleotide differences in the 3′-terminal region of the gene encoding the movement protein." Journal of General Virology 82, no. 1 (2001): 45–51. http://dx.doi.org/10.1099/0022-1317-82-1-45.

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In Nicotiana benthamiana, the common strain of the bipartite geminivirus Tomato golden mosaic virus (csTGMV) induces extensive chlorosis whereas the yellow vein strain (yvTGMV) produces veinal chlorosis on systemically infected leaves. In Datura stramonium, csTGMV produces leaf distortion and a severe chlorotic mosaic whereas yvTGMV produces only small chlorotic lesions on systemically infected leaves. Genetic recombination and site-directed mutagenesis studies using infectious clones of csTGMV and yvTGMV have identified a role in symptom production for the gene encoding the movement protein (MP). The MP amino acid at position 272, either valine (csTGMV) or isoleucine (yvTGMV), influenced symptoms in both hosts by inducing an intermediate phenotype when exchanged between the two strains. Exchange of an additional strain-specific MP amino acid at position 288, either glutamine (csTGMV) or lysine (yvTGMV), resulted in the change of symptom phenotype to that of the other strain. In situ hybridization analysis in N. benthamiana demonstrated that there was no qualitative difference in the tissue distribution of the two strains although csTGMV accumulated in higher amounts, suggesting that the efficiency of virus movement rather than distinct differences in tissue specificity of the strains is responsible for the symptom phenotypes.

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Yelina, Natalia E., Eugene I. Savenkov, Andrey G. Solovyev, Sergey Y. Morozov, and Jari P. T. Valkonen. "Long-Distance Movement, Virulence, and RNA Silencing Suppression Controlled by a Single Protein in Hordei- and Potyviruses: Complementary Functions between Virus Families." Journal of Virology 76, no. 24 (2002): 12981–91. http://dx.doi.org/10.1128/jvi.76.24.12981-12991.2002.

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ABSTRACT RNA silencing is a natural defense mechanism against genetic stress factors, including viruses. A mutant hordeivirus (Barley stripe mosaic virus [BSMV]) lacking the γb gene was confined to inoculated leaves in Nicotiana benthamiana, but systemic infection was observed in transgenic N. benthamiana expressing the potyviral silencing suppressor protein HCpro, suggesting that the γb protein may be a long-distance movement factor and have antisilencing activity. This was shown for γb proteins of both BSMV and Poa semilatent virus (PSLV), a related hordeivirus. Besides the functions in RNA silencing suppression, γb and HCpro had analogous effects on symptoms induced by the hordeiviruses. Severe BSMV-induced symptoms were correlated with high HCpro concentrations in the HCpro-transgenic plants, and substitution of the γb cistron of BSMV with that of PSLV led to greatly increased symptom severity and an altered pattern of viral gene expression. The efficient systemic infection with the chimera was followed by the development of dark green islands (localized recovery from infection) in leaves and exemption of new developing leaves from infection. Recovery and the accumulation of short RNAs diagnostic of RNA silencing in the recovered tissues in wild-type N. benthamiana were suppressed in HCpro-transgenic plants. These results provide evidence that potyviral HCpro and hordeivirus γb proteins contribute to systemic viral infection, symptom severity, and RNA silencing suppression. HCpro's ability to suppress the recovery of plants from viral infection emphasizes recovery as a manifestation of RNA silencing.

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Kim, Tae-Geum, Bang-Geul Kim, Dong-Keun Jeong, Yong-Suk Jang, Jin-Yong Lee, and Moon-Sik Yang. "Production of monoclonal antibodies against the FimA protein of Porphyromonas gingivalis in Nicotiana benthamiana." Biotechnology and Bioprocess Engineering 17, no. 2 (2012): 420–26. http://dx.doi.org/10.1007/s12257-011-0636-z.

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Kommineni, Vally, Matthew Markert, Zhongjie Ren, et al. "In Vivo Glycan Engineering via the Mannosidase I Inhibitor (Kifunensine) Improves Efficacy of Rituximab Manufactured in Nicotiana benthamiana Plants." International Journal of Molecular Sciences 20, no. 1 (2019): 194. http://dx.doi.org/10.3390/ijms20010194.

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N-glycosylation has been shown to affect the pharmacokinetic properties of several classes of biologics, including monoclonal antibodies, blood factors, and lysosomal enzymes. In the last two decades, N-glycan engineering has been employed to achieve a N-glycosylation profile that is either more consistent or aligned with a specific improved activity (i.e., effector function or serum half-life). In particular, attention has focused on engineering processes in vivo or in vitro to alter the structure of the N-glycosylation of the Fc region of anti-cancer monoclonal antibodies in order to increase antibody-dependent cell-mediated cytotoxicity (ADCC). Here, we applied the mannosidase I inhibitor kifunensine to the Nicotiana benthamiana transient expression platform to produce an afucosylated anti-CD20 antibody (rituximab). We determined the optimal concentration of kifunensine used in the infiltration solution, 0.375 µM, which was sufficient to produce exclusively oligomannose glycoforms, at a concentration 14 times lower than previously published levels. The resulting afucosylated rituximab revealed a 14-fold increase in ADCC activity targeting the lymphoma cell line Wil2-S when compared with rituximab produced in the absence of kifunensine. When applied to the cost-effective and scalable N. benthamiana transient expression platform, the use of kifunensine allows simple in-process glycan engineering without the need for transgenic hosts.

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Reyes, F., N. Fiore, M. A. Reyes, P. Sepúlveda, V. Paredes, and H. Prieto. "Biological Behavior and Partial Molecular Characterization of Six Chilean Isolates of Plum pox virus." Plant Disease 87, no. 1 (2003): 15–20. http://dx.doi.org/10.1094/pdis.2003.87.1.15.

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Plum pox virus (PPV) strain D was first detected in Chile in 1992 infecting Prunus trees including peaches, nectarines, apricots, and plums. Since then, quarantine efforts have included periodic surveys in the central zone of the country, the main region for stone fruit production. This work describes the characterization of six PPV isolates from this area of Chile, using biological and molecular approaches. PPV isolates were introduced into Prunus tomentosa and Nicotiana benthamiana hosts by grafting and mechanical inoculation, respectively. Symptoms were evaluated by following the appearance of circular necrotic spots and mosaic in leaves of P. tomentosa and mosaic and some leaf deformation in N. benthamiana. Molecular analysis was carried out using reverse transcription-polymerase chain reaction, allowing the cloning and sequencing of 1.34-kb fragments corresponding to the 3' region of the replicase gene, the complete coat protein (CP) gene, and the 3' nontranslated region of the PPV genome. Evolutionary distance analysis of these nucleotide sequences and their deduced coat protein amino acid sequences grouped the six Chilean isolates among strain D isolates, with closest genetic distances to those of Central Germany and Poland. Representative sources of these isolates suggest that strain D could be the only type of PPV currently present in Chile.

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Zhu, Feng, De-Hui Xi, Shu Yuan, Fei Xu, Da-Wei Zhang, and Hong-Hui Lin. "Salicylic Acid and Jasmonic Acid Are Essential for Systemic Resistance Against Tobacco mosaic virus in Nicotiana benthamiana." Molecular Plant-Microbe Interactions® 27, no. 6 (2014): 567–77. http://dx.doi.org/10.1094/mpmi-11-13-0349-r.

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Systemic resistance is induced by pathogens and confers protection against a broad range of pathogens. Recent studies have indicated that salicylic acid (SA) derivative methyl salicylate (MeSA) serves as a long-distance phloem-mobile systemic resistance signal in tobacco, Arabidopsis, and potato. However, other experiments indicate that jasmonic acid (JA) is a critical mobile signal. Here, we present evidence suggesting both MeSA and methyl jasmonate (MeJA) are essential for systemic resistance against Tobacco mosaic virus (TMV), possibly acting as the initiating signals for systemic resistance. Foliar application of JA followed by SA triggered the strongest systemic resistance against TMV. Furthermore, we use a virus-induced gene-silencing–based genetics approach to investigate the function of JA and SA biosynthesis or signaling genes in systemic response against TMV infection. Silencing of SA or JA biosynthetic and signaling genes in Nicotiana benthamiana plants increased susceptibility to TMV. Genetic experiments also proved the irreplaceable roles of MeSA and MeJA in systemic resistance response. Systemic resistance was compromised when SA methyl transferase or JA carboxyl methyltransferase, which are required for MeSA and MeJA formation, respectively, were silenced. Moreover, high-performance liquid chromatography–mass spectrometry analysis indicated that JA and MeJA accumulated in phloem exudates of leaves at early stages and SA and MeSA accumulated at later stages, after TMV infection. Our data also indicated that JA and MeJA could regulate MeSA and SA production. Taken together, our results demonstrate that (Me)JA and (Me)SA are required for systemic resistance response against TMV.

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van den Boogaart, Tom, Fuijang Wen, Jeffrey W. Davies, and George P. Lomonossoff. "Replicase-Derived Resistance Against Pea early browning virus in Nicotiana benthamiana Is an Unstable Resistance Based upon Posttranscriptional Gene Silencing." Molecular Plant-Microbe Interactions® 14, no. 2 (2001): 196–203. http://dx.doi.org/10.1094/mpmi.2001.14.2.196.

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Virus resistance in Nicotiana benthamiana plants containing a translatable Pea early browning virus (PEBV) 54K sequence from the 201K replicase gene has been reported previously. Resistant plants contain multiple transgene copies divided between two loci. Analysis of a genetic series containing the two loci in separate homozygous or heterozygous condition suggest that only one of the loci is necessary to induce the resistance. The resistance observed in R2 and R3 generations of lines containing both transgene loci in homozygous condition became less consistent in R4 and R5 generations. This inversely correlated with steady-state transgene transcript levels of the segregating populations. The use of recombinant Potato virus X vectors carrying PEBV 54K sequences showed that the resistance is based upon posttranscriptional gene silencing, is non-strand specific, and recognizes 3′ located sequences within the PEBV 54K sequence.

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Ferro, Camila G., F. Murilo Zerbini, Jesús Navas-Castillo, and Elvira Fiallo-Olivé. "Revealing the Complexity of Sweepovirus-Deltasatellite–Plant Host Interactions: Expanded Natural and Experimental Helper Virus Range and Effect Dependence on Virus-Host Combination." Microorganisms 9, no. 5 (2021): 1018. http://dx.doi.org/10.3390/microorganisms9051018.

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Sweepoviruses are begomoviruses (genus Begomovirus, family Geminiviridae) with ssDNA genomes infecting sweet potato and other species of the family Convolvulaceae. Deltasatellites (genus Deltasatellite, family Tolecusatellitidae) are small-size non-coding DNA satellites associated with begomoviruses. In this study, the genetic diversity of deltasatellites associated with sweepoviruses infecting Ipomoea indica plants was analyzed by further sampling the populations where the deltasatellite sweet potato leaf curl deltasatellite 1 (SPLCD1) was initially found, expanding the search to other geographical areas in southern continental Spain and the Canary Islands. The sweepoviruses present in the samples coinfected with deltasatellites were also fully characterized by sequencing in order to define the range of viruses that could act as helper viruses in nature. Additionally, experiments were performed to assess the ability of a number of geminivirids (the monopartite tomato leaf deformation virus and the bipartite NW begomovirus Sida golden yellow vein virus, the bipartite OW begomovirus tomato leaf curl New Delhi virus, and the curtovirus beet curly top virus) to transreplicate SPLCD1 in their natural plant hosts or the experimental host Nicotiana benthamiana. The results show that SPLCD1 can be transreplicated by all the geminivirids assayed in N. benthamiana and by tomato leaf curl New Delhi virus in zucchini. The presence of SPLCD1 did not affect the symptomatology caused by the helper viruses, and its effect on viral DNA accumulation depended on the helper virus–host plant combination.

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Adeel, Muhammad, Tahir Farooq, Jason C. White, Yi Hao, Zifu He, and Yukui Rui. "Carbon-based nanomaterials suppress tobacco mosaic virus (TMV) infection and induce resistance in Nicotiana benthamiana." Journal of Hazardous Materials 404 (February 2021): 124167. http://dx.doi.org/10.1016/j.jhazmat.2020.124167.

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Burger, C. "Virus-induced silencing of sterol biosynthetic genes: identification of a Nicotiana tabacum L. obtusifoliol-14 -demethylase (CYP51) by genetic manipulation of the sterol biosynthetic pathway in Nicotiana benthamiana L." Journal of Experimental Botany 54, no. 388 (2003): 1675–83. http://dx.doi.org/10.1093/jxb/erg184.

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Kung, Yi-Jung, Pin-Chun Lin, Shyi-Dong Yeh, et al. "Genetic Analyses of the FRNK Motif Function of Turnip mosaic virus Uncover Multiple and Potentially Interactive Pathways of Cross-Protection." Molecular Plant-Microbe Interactions® 27, no. 9 (2014): 944–55. http://dx.doi.org/10.1094/mpmi-04-14-0116-r.

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Cross-protection triggered by a mild strain of virus acts as a prophylaxis to prevent subsequent infections by related viruses in plants; however, the underling mechanisms are not fully understood. Through mutagenesis, we isolated a mutant strain of Turnip mosaic virus (TuMV), named Tu-GK, that contains an Arg182Lys substitution in helper component-proteinase (HC-ProK) that confers complete cross-protection against infection by a severe strain of TuMV in Nicotiana benthamiana, Arabidopsis thaliana Col-0, and the Arabidopsis dcl2-4/dcl4-1 double mutant defective in DICER-like ribonuclease (DCL)2/DCL4-mediated silencing. Our analyses showed that HC-ProK loses the ability to interfere with microRNA pathways, although it retains a partial capability for RNA silencing suppression triggered by DCL. We further showed that Tu-GK infection triggers strong salicylic acid (SA)-dependent and SA-independent innate immunity responses. Our data suggest that DCL2/4-dependent and –independent RNA silencing pathways are involved, and may crosstalk with basal innate immunity pathways, in host defense and in cross-protection.

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Han, Zhao-Fen, David M. Hunter, Susan Sibbald, Ji-Shu Zhang, and Lining Tian. "Biological Activity of the tzs Gene of Nopaline Agrobacterium tumefaciens GV3101 in Plant Regeneration and Genetic Transformation." Molecular Plant-Microbe Interactions® 26, no. 11 (2013): 1359–65. http://dx.doi.org/10.1094/mpmi-04-13-0106-r.

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Agrobacterium tumefaciens has been widely used in plant genetic transformation. Hormone-encoding genes residing in the T-DNA region have been removed, resulting in disarmed Agrobacterium strains that are used in various transformation experiments. Nopaline Agrobacterium strains, however, carry another hormone gene, trans-zeatin synthesizing (tzs), that codes for trans-zeatin in the virulence region of the tumor-inducing plasmids. We investigated the activity and function of the tzs gene of a nopaline Agrobacterium sp. strain GV3101 in plant in vitro regeneration. Leaf explants of tobacco and Nicotiana benthamiana co-cultured with strain GV3101 exhibited active shoot regeneration in media without added plant growth regulators. On medium without plant growth regulators, transgenic shoots were also induced from explants co-cultured with GV3101 containing a binary vector. Enzyme-linked immunosorbent assay showed that cell-free extracts of Agrobacterium sp. strain GV3101 culture contained the trans-zeatin at 860 ng/liter. Polymerase chain reaction using tzs-specific primers showed that the tzs gene was present in strain GV3101 but not in other Agrobacterium strains. The study showed that the tzs gene in GV3101 was actively expressed, and that trans-zeatin produced in the Agrobacterium strain can promote plant shoot regeneration.

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Crivelli, G., M. Ciuffo, A. Genre, V. Masenga, and M. Turina. "Reverse Genetic Analysis of Ourmiaviruses Reveals the Nucleolar Localization of the Coat Protein in Nicotiana benthamiana and Unusual Requirements for Virion Formation." Journal of Virology 85, no. 10 (2011): 5091–104. http://dx.doi.org/10.1128/jvi.02565-10.

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Zhao, Conghui, Zhenming Yu, Jaime A. Teixeira da Silva, et al. "Functional Characterization of a Dendrobium officinale Geraniol Synthase DoGES1 Involved in Floral Scent Formation." International Journal of Molecular Sciences 21, no. 19 (2020): 7005. http://dx.doi.org/10.3390/ijms21197005.

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Floral scent is a key ornamental trait that determines the quality and commercial value of orchids. Geraniol, an important volatile monoterpene in orchids that attracts pollinators, is also involved in responses to stresses but the geraniol synthase (GES) responsible for its synthesis in the medicinal orchid Dendrobium officinale has not yet been identified. In this study, three potential geraniol synthases were mined from the D. officinale genome. DoGES1, which was localized in chloroplasts, was characterized as a geraniol synthase. DoGES1 was highly expressed in flowers, especially in petals. DoGES1 transcript levels were high in the budding stage of D. officinale flowers at 11:00 a.m. DoGES1 catalyzed geraniol in vitro, and transient expression of DoGES1 in Nicotiana benthamiana leaves resulted in the accumulation of geraniol in vivo. These findings on DoGES1 advance our understanding of geraniol biosynthesis in orchids, and lay the basis for genetic modification of floral scent in D. officinale or in other ornamental orchids.

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Journal articles on the topic 'Nicotiana benthamiana – Genetic engineering'

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