scholarly journals Long-Term Potato Virus X (PVX)-Based Transient Expression of Recombinant GFP Protein in Nicotiana benthamiana Culture In Vitro

Plants ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2187
Author(s):  
Yana Sindarovska ◽  
Mykola Kuchuk
Keyword(s):  
Transient Expression ◽  
Nicotiana Benthamiana ◽  
Molecular Farming ◽  
Transient Gene Expression ◽  
Leaf Explants ◽  
Potato Virus X ◽  
Culture In Vitro ◽  
High Efficient

Plant molecular farming has a great potential to produce valuable proteins. Transient expression technology provides high yields of recombinant proteins in greenhouse-grown plants, but every plant must be artificially agroinfiltrated, and open greenhouse systems are less controlled. Here, we propose to propagate agrobacteria-free plants with high-efficient long-term self-replicated transient gene expression in a well-controlled closed in vitro system. Nicotiana benthamiana plant tissue culture in vitro, with transient expression of recombinant GFP, was obtained through shoot induction from leaf explants infected by a PVX-based vector. The transient expression occurs in new tissues and regenerants due to the natural systemic distribution of viral RNA carrying the target gene. Gene silencing was delayed in plants grown in vitro, and GFP was detected in plants for five to six months. Agrobacteria-free, GFP-expressing plants can be micropropagated in vitro (avoiding an agroinfiltration step), “rejuvenated” through regeneration (maintaining culture for years), or transferred in soil. The mean GFP in the regenerants was 18% of the total soluble proteins (TSP) (0.52 mg/g of fresh leaf weight (FW). The highest value reached 47% TSP (2 mg/g FW). This study proposes a new method for recombinant protein production combining the advantages of transient expression technology and closed cultural systems.

scholarly journals Transient expression and purification of β-caryophyllene synthase in Nicotiana benthamiana to produce β-caryophyllene in vitro

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e8904
Author(s):  
Saraladevi Muthusamy ◽  
Ramesh R. Vetukuri ◽  
Anneli Lundgren ◽  
Suresh Ganji ◽  
Li-Hua Zhu ◽  
...  
Keyword(s):  
Transient Expression ◽  
Nicotiana Benthamiana ◽  
Artemisia Annua ◽  
Molecular Farming ◽  
Expression System ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gas Chromatography Mass Spectrometry ◽  
Fresh Weight

The sesquiterpene β-caryophyllene is an ubiquitous component in many plants that has commercially been used as an aroma in cosmetics and perfumes. Recent studies have shown its potential use as a therapeutic agent and biofuel. Currently, β-caryophyllene is isolated from large amounts of plant material. Molecular farming based on the Nicotiana benthamiana transient expression system may be used for a more sustainable production of β-caryophyllene. In this study, a full-length cDNA of a new duplicated β-caryophyllene synthase from Artemisia annua (AaCPS1) was isolated and functionally characterized. In order to produce β-caryophyllene in vitro, the AaCPS1 was cloned into a plant viral-based vector pEAQ-HT. Subsequently, the plasmid was transferred into the Agrobacterium and agroinfiltrated into N. benthamiana leaves. The AaCPS1 expression was analyzed by quantitative PCR at different time points after agroinfiltration. The highest level of transcripts was observed at 9 days post infiltration (dpi). The AaCPS1 protein was extracted from the leaves at 9 dpi and purified by cobalt–nitrilotriacetate (Co-NTA) affinity chromatography using histidine tag with a yield of 89 mg kg−1 fresh weight of leaves. The protein expression of AaCPS1 was also confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analyses. AaCPS1 protein uses farnesyl diphosphate (FPP) as a substrate to produce β-caryophyllene. Product identification and determination of the activity of purified AaCPS1 were done by gas chromatography–mass spectrometry (GC–MS). GC–MS results revealed that the AaCPS1 produced maximum 26.5 ± 1 mg of β-caryophyllene per kilogram fresh weight of leaves after assaying with FPP for 6 h. Using AaCPS1 as a proof of concept, we demonstrate that N. benthamiana can be considered as an expression system for production of plant proteins that catalyze the formation of valuable chemicals for industrial applications.

scholarly journals Harnessing a Transient Gene Expression System in Nicotiana benthamiana to Explore Plant Agrochemical Transporters

Plants ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 524
Author(s):  
Bingqi Wu ◽  
Zhiting Chen ◽  
Xiaohui Xu ◽  
Ronghua Chen ◽  
Siwei Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transient Expression ◽  
Nicotiana Benthamiana ◽  
Heterologous Gene Expression ◽  
Expression System ◽  
Functional Characterization ◽  
Transient Gene Expression ◽  
High Mobility ◽  
Gene Expression System

Functional characterization of plant agrichemical transporters provided an opportunity to discover molecules that have a high mobility in plants and have the potential to increase the amount of pesticides reaching damage sites. Agrobacterium-mediated transient expression in tobacco is simple and fast, and its protein expression efficiency is high; this system is generally used to mediate heterologous gene expression. In this article, transient expression of tobacco nicotine uptake permease (NtNUP1) and rice polyamine uptake transporter 1 (OsPUT1) in Nicotiana benthamiana was performed to investigate whether this system is useful as a platform for studying the interactions between plant transporters and pesticides. The results showed that NtNUP1 increases nicotine uptake in N. benthamiana foliar discs and protoplasts, indicating that this transient gene expression system is feasible for studying gene function. Moreover, yeast expression of OsPUT1 apparently increases methomyl uptake. Overall, this method of constructing a transient gene expression system is useful for improving the efficiency of analyzing the functions of plant heterologous transporter-encoding genes and revealed that this system can be further used to study the functions of transporters and pesticides, especially their interactions.

scholarly journals Identification of a Movement Protein of the Tenuivirus Rice Stripe Virus

2008 ◽  
Vol 82 (24) ◽  
pp. 12304-12311 ◽  
Author(s):  
Ruyi Xiong ◽  
Jianxiang Wu ◽  
Yijun Zhou ◽  
Xueping Zhou
Keyword(s):  
Nicotiana Benthamiana ◽  
Fluorescent Protein ◽  
Severe Disease ◽  
Rice Stripe Virus ◽  
Potato Virus X ◽  
Infected Leaf ◽  
Biolistic Bombardment ◽  
Movement Proteins ◽  
Green Fluorescent

ABSTRACT Rice stripe virus (RSV) is the type member of the genus Tenuivirus. RSV has four single-stranded RNAs and causes severe disease in rice fields in different parts of China. To date, no reports have described how RSV spreads within host plants or the viral and/or host factor(s) required for tenuivirus movement. We investigated functions of six RSV-encoded proteins using trans-complementation experiments and biolistic bombardment. We demonstrate that NSvc4, encoded by RSV RNA4, supports the intercellular trafficking of a movement-deficient Potato virus X in Nicotiana benthamiana leaves. We also determined that upon biolistic bombardment or agroinfiltration, NSvc4:enhanced green fluorescent protein (eGFP) fusion proteins localize predominantly near or within the walls of onion and tobacco epidermal cells. In addition, the NSvc4:eGFP fusion protein can move from initially bombarded cells to neighboring cells in Nicotiana benthamiana leaves. Immunocytochemistry using tissue sections from RSV-infected rice leaves and an RSV NSvc4-specific antibody showed that the NSvc4 protein accumulated in walls of RSV-infected leaf cells. Gel retardation assays revealed that the NSvc4 protein interacts with single-stranded RNA in vitro, a common feature of many reported plant viral movement proteins (MPs). RSV NSvc4 failed to interact with the RSV nucleocapsid protein using yeast two-hybrid assays. Taken together, our data indicate that RSV NSvc4 is likely an MP of the virus. This is the first report describing a tenuivirus MP.

Reprogramming of Oncogene Expression in Gingival Mesenchymal Stem Cells Following Long-Term Culture In Vitro

2017 ◽  
Vol 19 (3) ◽  
pp. 159-170 ◽  
Author(s):  
Agnese Gugliandolo ◽  
Thangavelu Soundara Rajan ◽  
Domenico Scionti ◽  
Francesca Diomede ◽  
Placido Bramanti ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Culture In Vitro ◽  
Long Term Culture ◽  
Oncogene Expression

Efficient transient expression of human GM-CSF protein in Nicotiana benthamiana using potato virus X vector

2006 ◽  
Vol 72 (4) ◽  
pp. 756-762 ◽  
Author(s):  
Fengyong Zhou ◽  
Ming-Li Wang ◽  
Henrik H. Albert ◽  
Paul H. Moore ◽  
Yun J. Zhu
Keyword(s):  
Transient Expression ◽  
Potato Virus ◽  
Nicotiana Benthamiana ◽  
Potato Virus X ◽  
Gm Csf

scholarly journals Multiple Coat Protein Mutations Abolish Recognition of Pepino mosaic potexvirus (PepMV) by the Potato Rx Resistance Gene in Transgenic Tomatoes

2010 ◽  
Vol 23 (4) ◽  
pp. 376-383 ◽  
Author(s):  
Thierry Candresse ◽  
Armelle Marais ◽  
Chantal Faure ◽  
Marie Pierre Dubrana ◽  
Julie Gombert ◽  
...  
Keyword(s):  
Resistance Gene ◽  
Transient Expression ◽  
Nicotiana Benthamiana ◽  
Potato Virus X ◽  
Hypersensitive Reaction ◽  
Coat Proteins ◽  
Pepino Mosaic Virus ◽  
Sensing System ◽  
Resistance Breaking ◽  
Transgenic Tomatoes

Despite the fact that Pepino mosaic virus (PepMV) and Potato virus X (PVX) share less than 40% identity in their coat proteins (CP), the known PVX elicitor of Rx, transgenic tomato (cv. Microtom) plants expressing a functional potato Rx resistance gene showed resistance toward PepMV. However, in a low percentage of plants, PepMV accumulation was observed and back inoculation experiments demonstrated that these plants contained resistance-breaking PepMV variants. Sequencing of the CP gene of these variants showed the accumulation of mutations in the amino acid 41 to 125 region the CP, whereas no mutations were observed in the nonevolved isolates. Agroinfiltration-mediated transient expression of the mutant CP demonstrated that they had a greatly attenuated or abolished ability to induce a hypersensitive reaction in Rx-expressing Nicotiana benthamiana leaves. The transient expression of truncated forms of the PepMV CP allowed the identification of a minimal elicitor domain (amino acids 30 to 136). These results demonstrate that the Rx-based sensing system is able to recognize the PepMV CP but, contrary to the situation with PVX, for which only two closely spaced resistance-breaking mutations are known, many mutations over a significant stretch of the PepMV CP allow escape from recognition by Rx.

scholarly journals In Vivo Glycan Engineering via the Mannosidase I Inhibitor (Kifunensine) Improves Efficacy of Rituximab Manufactured in Nicotiana benthamiana Plants

2018 ◽  
Author(s):  
Vally Kommineni ◽  
Matthew Markert ◽  
Zhongjie Ren ◽  
Sreenath Palle ◽  
Berenice Carrillo ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Transient Expression ◽  
Nicotiana Benthamiana ◽  
Cost Effective ◽  
Fold Increase ◽  
Expression Platform ◽  
Dependent Cell ◽  
Anti Cd20

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.

scholarly journals Long-term culture in vitro impairs the immunosuppressive activity of mesenchymal stem cells on T cells

2012 ◽  
Vol 6 (5) ◽  
pp. 1183-1189 ◽  
Author(s):  
XUE-YI LI ◽  
JIN DING ◽  
ZHAO-HUI ZHENG ◽  
XIAO-YAN LI ◽  
ZHEN-BIAO WU ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Mesenchymal Stem Cells ◽  
Immunosuppressive Activity ◽  
Culture In Vitro ◽  
Long Term Culture

Human placental decidua basalis-derived mesenchymal stem cells differentiate into dopamine neuron-like cells with no response to long-term culture in vitro

Neuroreport ◽  
2012 ◽  
Vol 23 (8) ◽  
pp. 513-518 ◽  
Author(s):  
Guo-hui Lu ◽  
Wang-shi Yong ◽  
Zhi-min Xu ◽  
Yi-quan Ke ◽  
Xiao-dan Jiang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Dopamine Neuron ◽  
Culture In Vitro ◽  
Long Term Culture ◽  
Decidua Basalis
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