Functional expression and characterization of sesquiterpene synthases from Artemisia annua L. using transient expression system in Nicotiana benthamiana

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.

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Functional characterization of a glycine 185-to-valine substitution in human P-glycoprotein by using a vaccinia-based transient expression system.

Molecular Biology of the Cell ◽

10.1091/mbc.7.10.1485 ◽

1996 ◽

Vol 7 (10) ◽

pp. 1485-1498 ◽

Cited By ~ 43

Author(s):

M Ramachandra ◽

S V Ambudkar ◽

M M Gottesman ◽

I Pastan ◽

C A Hrycyna

Keyword(s):

Drug Resistance ◽

Atpase Activity ◽

Cyclosporin A ◽

Transient Expression ◽

Expression System ◽

Rhodamine 123 ◽

Wild Type ◽

P Glycoprotein ◽

Transient Expression System

Human P-glycoprotein (Pgp) is a 170-kDa plasma membrane protein that confers multidrug resistance to otherwise sensitive cells. A mutation in Pgp, G185-->V, originally identified as a spontaneous mutation, was shown previously to alter the drug resistance profiles in cell lines that are stably transfected with the mutant MDR1 cDNA and selected with cytotoxic agents. To understand the mechanism by which the V185 mutation leads to an altered drug resistance profile, we used a transient expression system that eliminates the need for drug selection to attain high expression levels and allows for the rapid characterization of many aspects of Pgp function and biosynthesis. The mutant and wild-type proteins were expressed at similar levels after 24-48 h in human osteosarcoma (HOS) cells by infection with a recombinant vaccinia virus encoding T7 RNA polymerase and simultaneous transfection with a plasmid containing MDR1 cDNA controlled by the T7 promoter. For both mutant and wild-type proteins, photolabeling with [3H]azidopine and [125I]iodoarylazidoprazosin, drug-stimulated ATPase activity, efflux of rhodamine 123, and accumulation of radiolabeled vinblastine and colchicine were evaluated. In crude membrane preparations from HOS cells, a higher level of basal Pgp-ATPase activity was observed for the V185 variant than for the wild-type, suggesting partial uncoupling of drug-dependent ATP hydrolysis by the mutant. Several compounds, including verapamil, nicardipine, tetraphenylphosphonium, and prazosin, stimulated ATPase activities of both the wild-type and mutant similarly, whereas cyclosporin A inhibited the ATPase activity of the mutant more efficiently than that of the wild-type. This latter observation explains the enhanced potency of cyclosporin A as an inhibitor of the mutant Pgp. No differences were seen in verapamil-inhibited rhodamine 123 efflux, but the rate of accumulation was slower for colchicine and faster for vinblastine in cells expressing the mutant protein, as compared with those expressing wild-type Pgp. We conclude that the G185-->V mutation confers pleiotropic alterations on Pgp, including an altered basal ATPase activity and altered interaction with substrates and the inhibitor cyclosporin A.

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Development of Agrobacterium-mediated transient expression system in Caragana intermedia and characterization of CiDREB1C in stress response

BMC Plant Biology ◽

10.1186/s12870-019-1800-4 ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 2

Author(s):

Kun Liu ◽

Qi Yang ◽

Tianrui Yang ◽

Yang Wu ◽

Guangxia Wang ◽

...

Keyword(s):

Stress Response ◽

Transient Expression ◽

Expression System ◽

Transient Expression System ◽

Caragana Intermedia

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Expression of active recombinant human gastric lipase in Nicotiana benthamiana using the CPMV- HT transient expression system

Protein Expression and Purification ◽

10.1016/j.pep.2011.09.005 ◽

2012 ◽

Vol 81 (1) ◽

pp. 69-74 ◽

Cited By ~ 28

Author(s):

Maria Vardakou ◽

Frank Sainsbury ◽

Neil Rigby ◽

Francis Mulholland ◽

George P. Lomonossoff

Keyword(s):

Transient Expression ◽

Nicotiana Benthamiana ◽

Expression System ◽

Transient Expression System ◽

Gastric Lipase

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Characterization of transient expression system for retroviral vector production

Journal of Bioscience and Bioengineering ◽

10.1263/jbb.101.361 ◽

2006 ◽

Vol 101 (4) ◽

pp. 361-368 ◽

Cited By ~ 17

Author(s):

Akitsu Hotta ◽

Yoshikazu Saito ◽

Kenji Kyogoku ◽

Yoshinori Kawabe ◽

Ken-ichi Nishijima ◽

...

Keyword(s):

Transient Expression ◽

Expression System ◽

Retroviral Vector ◽

Transient Expression System

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

PeerJ ◽

10.7717/peerj.8904 ◽

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.

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Functional characterization of a stilbene synthase gene using a transient expression system in planta

Plant Cell Reports ◽

10.1007/s00299-008-0664-0 ◽

2008 ◽

Vol 28 (4) ◽

pp. 589-599 ◽

Cited By ~ 17

Author(s):

Jose Condori ◽

Giuliana Medrano ◽

Ganapathy Sivakumar ◽

Vipin Nair ◽

Carole Cramer ◽

...

Keyword(s):

Transient Expression ◽

Expression System ◽

Functional Characterization ◽

Stilbene Synthase ◽

In Planta ◽

Transient Expression System ◽

Stilbene Synthase Gene

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A high-efficiency Agrobacterium-mediated transient expression system in the leaves of Artemisia annua L.

Plant Methods ◽

10.1186/s13007-021-00807-5 ◽

2021 ◽

Vol 17 (1) ◽

Author(s):

Yongpeng Li ◽

Tiantian Chen ◽

Wei Wang ◽

Hang Liu ◽

Xin Yan ◽

...

Keyword(s):

Transient Expression ◽

High Throughput ◽

High Efficiency ◽

Artemisia Annua ◽

Expression System ◽

Functional Characterization ◽

Cauliflower Mosaic Virus ◽

Transient Transformation ◽

Transformation System ◽

Transient Expression System

Abstract Background The Agrobacterium-mediated transient transformation, which proved effective in diverse plant species, has been widely applied for high-throughput gene function studies due to its simplicity, rapidity, and high efficiency. Despite the efforts have made on Artemisia annua transient expression, achieving high-throughput gene functional characterization basing on a fast and easy-manipulated transient transformation system in A. annua remains challenging. Results The first pair of true leaves of A. annua is an ideal candidate for Agrobacterium injection. EHA105 was the optimal strain that can be used for the development of the transient expression system. The supplementation of Triton X-100 at a concentration of 0.005% greatly improved the transient expression frequency. According to the histochemical β-Glucuronidase (GUS) staining assay, high transient expression level of the reporter gene (GUS) maintained at least a week. Dual-luciferase (Dual-LUC) transient assays showed that the activity of cauliflower mosaic virus 35S (CaMV35S) promoter and its derivates varied between A. annua and tobacco. In A. annua, the CaMV35S promoter had comparable activity with double CaMV35S promoter, while in tobacco, CaMV35S exhibited approximately 50% activity of double CaMV35S promoter. Otherwise, despite the CaMV35S promoter and double CaMV35S promoter from GoldenBraid Kit 2.0 displayed high activity strength in tobacco, they demonstrated a very low activity in transiently expressed A. annua. The activity of UBQ10 promoter and endogenous UBQb promoter was investigated as well. Additionally, using our transient expression system, the transactivation of AaGSW1 and AaORA on AaCYP71AV1 promoter was confirmed. Dual-LUC assays demonstrated that AaHD8 activated the expression of two glandular secreting trichomes-specific lipid transfer protein genes AaLTP1 and AaLTP2, indicating that AaLTP1 and AaLTP2 might serve as downstream components of AaHD8-involved glandular trichome initiation and cuticle formation, as well as artemisinin secretion in A. annua. Conclusions A simple, rapid, good-reproducibility, high-efficiency and low-cost transient transformation system in A. annua was developed. Our method offered a new way for gene functional characterization studies such as gene subcellular localization, promoter activity and transcription activation assays in A. annua, avoiding the aberrant phenotypes resulting from gene expression in a heterologous system.

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