Replication of Tomato bushy stunt virus RNA in a plant in vitro system

Gursinsky, Torsten; Schulz, Beate; Behrens, Sven-Erik

doi:10.1016/j.virol.2009.05.009

Cited by 26 publications

(39 citation statements)

References 44 publications

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“…The aim of this study was to establish a system, allowing the rapid preparation of lysates for cell-free synthesis of satisfactory recombinant protein yields. For this purpose, we further developed a system based on BY-2 cell suspension cultures designed for the replication of plant RNA viruses [15,16]. In contrast to the original protocol using batch-cultured cells, we used BY-2 cells growing continuously in a stirred-tank fermenter at a constant packed cell volume of 20% and a doubling time of around 32 h, to ensure a reproducible supply of homogeneous cell material.…”

Section: Resultsmentioning

confidence: 99%

Tobacco BY-2 cell-free lysate: an alternative and highly-productive plant-based in vitro translation system

et al. 2014

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BackgroundCell-free protein synthesis is a rapid and efficient method for the production of recombinant proteins. Usage of prokaryotic cell-free extracts often leads to non-functional proteins. Eukaryotic counterparts such as wheat germ extract (WGE) and rabbit reticulocyte lysate (RLL) may improve solubility and promote the correct folding of eukaryotic multi-domain proteins that are difficult to express in bacteria. However, the preparation of WGEs is complex and time-consuming, whereas RLLs suffer from low yields. Here we report the development of a novel cell-free system based on tobacco Bright Yellow 2 (BY-2) cells harvested in the exponential growth phase.ResultsThe highly-productive BY-2 lysate (BYL) can be prepared quickly within 4–5 h, compared to 4–5 d for WGE. The efficiency of the BYL was tested using three model proteins: enhanced yellow fluorescent protein (eYFP) and two versions of luciferase. The added mRNA was optimized by testing different 5’ and 3’ untranslated regions (UTRs). The protein yield in batch and dialysis reactions using BYL was much higher than that of a commercial Promega WGE preparation, achieving a maximum yield of 80 μg/mL of eYFP and 100 μg/mL of luciferase, compared to only 45 μg/mL of eYFP and 35 μg/mL of luciferase in WGEs. In dialysis reactions, the BYL yielded about 400 μg/mL eYFP, representing up to 50% more of the target protein than the Promega WGE, and equivalent to the amount using 5Prime WGE system.ConclusionsDue to the high yield and the short preparation time the BYL represents a remarkable improvement over current eukaryotic cell-free systems.

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Section: Resultsmentioning

confidence: 99%

Tobacco BY-2 cell-free lysate: an alternative and highly-productive plant-based in vitro translation system

et al. 2014

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“…Nicotiana tabacum BY-2 cells were cultured as described (Gursinsky et al, 2009) at 23°C in Murashige and Skoog liquid medium. Evacuolated BY-2 protoplasts to prepare cytoplasmic extract (BYL) were obtained by Percoll gradient centrifugation (Komoda et al, 2007;Gursinsky et al, 2009).…”

Section: Cell Culture and Preparation Of Cytoplasmic By-2 Cell Extractmentioning

confidence: 99%

“…Evacuolated BY-2 protoplasts to prepare cytoplasmic extract (BYL) were obtained by Percoll gradient centrifugation (Komoda et al, 2007;Gursinsky et al, 2009). …”

Section: Cell Culture and Preparation Of Cytoplasmic By-2 Cell Extractmentioning

confidence: 99%

“…To generate AGO1 variants and siRNA-programmed AGO/RISC in vitro, the AGO mRNAs were in vitro translated in 50% (v/v) BYL in the previously described conditions (Gursinsky et al, 2009). Briefly, 1.5 mg of AGO mRNA was translated in a 20-mL reaction in the presence of 50 nM synthetic siRNA for 60 min.…”

Section: In Vitro Slicer Assaymentioning

confidence: 99%

“…While Arabidopsis is not permissive to a tombusvirus infection, CymRSV and TBSV replicate in heterologous systems such as Saccharomyces cerevisiae (Panavas and Nagy, 2003;Pantaleo et al, 2003;Navarro et al, 2006). TBSV also was recently applied in a plant cytoplasm-based system to reproduce viral replication as well as antiviral RNA silencing in vitro (Gursinsky et al, 2009;Schuck et al, 2013).…”

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confidence: 99%

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Homeologs of the Nicotiana benthamiana Antiviral ARGONAUTE1 Show Different Susceptibilities to microRNA168-Mediated Control

et al. 2015

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The plant ARGONAUTE1 protein (AGO1) is a central functional component of the posttranscriptional regulation of gene expression and the RNA silencing-based antiviral defense. By genomic and molecular approaches, we here reveal the presence of two homeologs of the AGO1-like gene in Nicotiana benthamiana, NbAGO1-1H and NbAGO1-1L. Both homeologs retain the capacity to transcribe messenger RNAs (mRNAs), which mainly differ in one 18-nucleotide insertion/deletion (indel). The indel does not modify the frame of the open reading frame, and it is located eight nucleotides upstream of the target site of a microRNA, miR168, which is an important modulator of AGO1 expression. We demonstrate that there is a differential accumulation of the two NbAGO1-1 homeolog mRNAs at conditions where miR168 is up-regulated, such as during a tombusvirus infection. The data reported suggest that the indel affects the miR168-guided regulation of NbAGO1 mRNA. The two AGO1 homeologs show full functionality in reconstituted, catalytically active RNA-induced silencing complexes following the incorporation of small interfering RNAs. Virus-induced gene silencing experiments suggest a specific involvement of the NbAGO1 homeologs in symptom development. The results provide an example of the diversity of microRNA target regions in NbAGO1 homeolog genes, which has important implications for improving resilience measures of the plant during viral infections.

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A versatile coupled cell‐free transcription–translation system based on tobacco BY‐2 cell lysates

Buntru

Vogel

Stoff

et al. 2015

Biotech & Bioengineering

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Cell-free protein synthesis is a powerful method for the high-throughput production of recombinant proteins, especially proteins that are difficult to express in living cells. Here we describe a coupled cell-free transcription-translation system based on tobacco BY-2 cell lysates (BYLs). Using a combination of fractional factorial designs and response surface models, we developed a cap-independent system that produces more than 250 μg/mL of functional enhanced yellow fluorescent protein (eYFP) and about 270 μg/mL of firefly luciferase using plasmid templates, and up to 180 μg/mL eYFP using linear templates (PCR products) in 18 h batch reactions. The BYL contains actively-translocating microsomal vesicles derived from the endoplasmic reticulum, promoting the formation of disulfide bonds, glycosylation and the cotranslational integration of membrane proteins. This was demonstrated by expressing a functional full-size antibody (∼ 150 μg/mL), the model enzyme glucose oxidase (GOx) (∼ 7.3 U/mL), and a transmembrane growth factor (∼ 25 μg/mL). Subsequent in vitro treatment of GOx with peptide-N-glycosidase F confirmed the presence of N-glycans. Our results show that the BYL can be used as a high-throughput expression and screening platform that is particularly suitable for complex and cytotoxic proteins.

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Replication of Tomato bushy stunt virus RNA in a plant in vitro system

Cited by 26 publications

References 44 publications

Tobacco BY-2 cell-free lysate: an alternative and highly-productive plant-based in vitro translation system

Tobacco BY-2 cell-free lysate: an alternative and highly-productive plant-based in vitro translation system

Homeologs of the Nicotiana benthamiana Antiviral ARGONAUTE1 Show Different Susceptibilities to microRNA168-Mediated Control

A versatile coupled cell‐free transcription–translation system based on tobacco BY‐2 cell lysates

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