Construction of inducible expression systems for controlled transfer of Agrobacterium tumefaciens T-DNA in plant transient expression systems

2021 ◽  
Author(s):  
◽  
Erna Denkovskienė
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Transient Expression ◽  
Inducible Expression ◽  
Expression Systems

Inducible expression of Agrobacterium virulence gene VirE2 for stringent regulation of T-DNA transfer in biosafe plant transient expression systems

2014 ◽  
Vol 31 ◽  
pp. S183
Author(s):  
Erna Denkovskiene ◽  
Sarunas Paskevicius ◽  
Stefan Werner ◽  
Anatoli Giritch ◽  
Ausra Razanskiene
Keyword(s):  
Transient Expression ◽  
Virulence Gene ◽  
Inducible Expression ◽  
Dna Transfer ◽  
Expression Systems

scholarly journals Control of T-DNA Transfer from Agrobacterium tumefaciens to Plants Based on an Inducible Bacterial Toxin-Antitoxin System

2020 ◽  
Vol 33 (9) ◽  
pp. 1142-1149
Author(s):  
Erna Denkovskienė ◽  
Šarūnas Paškevičius ◽  
Justina Stankevičiūtė ◽  
Yuri Gleba ◽  
Aušra Ražanskienė
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Open Field ◽  
Transient Expression ◽  
Genetically Modified ◽  
Pharmaceutical Products ◽  
Inducible Expression ◽  
Dna Transfer ◽  
Potential Usefulness ◽  
Bacterial Toxin ◽  
Transient Transformation

High-value pharmaceutical products are already successfully produced in contained facilities using Agrobacterium-mediated transient transformation of plants. However, transfection methods suitable for open field applications are still desirable as a cheaper alternative. Biosafety concerns related to the use of recombinant agrobacteria in an industrial transfection process include possible transformation or transfection of unintended hosts or spread of the genetically modified agrobacteria in the environment. In this paper, we explored a novel biocontrol approach resulting in greater biosafety of the transient expression process in plants. Our proposed solution involves inducible expression of Agrobacterium tumefaciens toxin PemK and antitoxin PemI that provides for strictly regulated T-DNA transfer from agrobacteria to plants. We also identified several other toxins from putative Agrobacterium toxin-antitoxin modules and demonstrate their potential usefulness in the control of Agrobacterium tumefaciens as a DNA vector.

Production of pharmaceutical-grade recombinant aprotinin and a monoclonal antibody product using plant-based transient expression systems

2010 ◽  
Vol 8 (5) ◽  
pp. 638-654 ◽  
Author(s):  
Gregory P. Pogue ◽  
Fakhrieh Vojdani ◽  
Kenneth E. Palmer ◽  
Ernie Hiatt ◽  
Steve Hume ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Transient Expression ◽  
Expression Systems ◽  
Monoclonal Antibody Product

scholarly journals Ready‐to‐Use Stocks of Agrobacterium tumefaciens Can Simplify Process Development for the Production of Recombinant Proteins by Transient Expression in Plants

2019 ◽  
Vol 14 (10) ◽  
pp. 1900113 ◽  
Author(s):  
Holger Spiegel ◽  
Alexander Boes ◽  
Camil Perales Morales ◽  
Thomas Rademacher ◽  
Johannes F. Buyel
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Transient Expression ◽  
Recombinant Proteins ◽  
Process Development

scholarly journals Application of sonication-assisted Agrobacterium-mediated transformation in Chenopodium rubrum L

2011 ◽  
Vol 49 (No. 6) ◽  
pp. 255-260 ◽  
Author(s):  
J.I. Flores Solís ◽  
P. Mlejnek ◽  
K. Studená ◽  
S. Procházka
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Transient Expression ◽  
Chenopodium Rubrum ◽  
Limiting Factors ◽  
Gus Gene ◽  
Efficient Tool ◽  
Factors Affecting ◽  
Agrobacterium Mediated Transformation ◽  
Glucuronidase Activity ◽  
Gene Coding

Chenopodium rubrum belongs to the plant species in which standard Agrobacterium-mediated transformation procedures remain inefficient. We demonstrate that the employment of sonication-assisted Agrobacterium-mediated transformation (SAAT) effectively enhanced transient expression of GUS gene coding for b-glucuronidase in Chenopodium rubrum. Further the results indicated that the age of seedlings is one of the limiting factors affecting the potency of Agrobacterium tumefaciens infection. Histochemical detection of b-glucuronidase activity revealed that two-days-old seedlings were much more susceptible to infection than ten-days-old ones. According to our results SAAT technology could provide an efficient tool for obtaining stable transformants when applied to two-days-old seedlings of Chenopodium rubrum.

scholarly journals Inducible Expression of Agrobacterium Virulence Gene VirE2 for Stringent Regulation of T-DNA Transfer in Plant Transient Expression Systems

2015 ◽  
Vol 28 (11) ◽  
pp. 1247-1255 ◽  
Author(s):  
Erna Denkovskienė ◽  
Šarūnas Paškevičius ◽  
Stefan Werner ◽  
Yuri Gleba ◽  
Aušra Ražanskienė
Keyword(s):  
Transient Expression ◽  
Viral Vectors ◽  
Fluorescent Protein ◽  
Virulence Gene ◽  
High Volume ◽  
Inducible Expression ◽  
Dna Transfer ◽  
Expression Vectors ◽  
Inducible Promoters ◽  
Regulated Promoters

Agrotransfection with viral vectors is an effective solution for the transient production of valuable proteins in plants grown in contained facilities. Transfection methods suitable for field applications are desirable for the production of high-volume products and for the transient molecular reprogramming of plants. The use of genetically modified (GM) Agrobacterium strains for plant transfections faces substantial biosafety issues. The environmental biosafety of GM Agrobacterium strains could be improved by regulating their T-DNA transfer via chemically inducible expression of virE2, one of the essential Agrobacterium virulence genes. In order to identify strong and stringently regulated promoters in Agrobacterium strains, we evaluated isopropyl-β-d-thiogalactoside–inducible promoters Plac, Ptac, PT7/lacO, and PT5/lacOlacO and cumic acid–inducible promoters PlacUV5/CuO, Ptac/CuO, PT5/CuO, and PvirE/CuO. Nicotiana benthamiana plants were transfected with a virE2-deficient A. tumefaciens strain containing transient expression vectors harboring inducible virE2 expression cassettes and containing a marker green fluorescent protein (GFP) gene in their T-DNA region. Evaluation of T-DNA transfer was achieved by counting GFP expression foci on plant leaves. The virE2 expression from cumic acid–induced promoters resulted in 47 to 72% of wild-type T-DNA transfer. Here, we present efficient and tightly regulated promoters for gene expression in A. tumefaciens and a novel approach to address environmental biosafety concerns in agrobiotechnology.

scholarly journals Improving plant transient expression through the rational design of synthetic 5′ and 3′ untranslated regions

Plant Methods ◽  
2019 ◽  
Vol 15 (1) ◽  
Author(s):  
Hadrien Peyret ◽  
James K. M. Brown ◽  
George P. Lomonossoff
Keyword(s):  
Recombinant Protein ◽  
Transient Expression ◽  
Recombinant Proteins ◽  
Rational Design ◽  
Critical Role ◽  
Regulatory Elements ◽  
Plant Viruses ◽  
Untranslated Regions ◽  
Expression Vectors ◽  
Expression Systems

Abstract Background The growing field of plant molecular farming relies on expression vectors that allow high yields of recombinant proteins to be produced through transient gene expression. While numerous expression vectors currently exist for this purpose, there are very few examples of systematic efforts to improve upon these. Moreover, the current generation of expression systems makes use of naturally-occurring regulatory elements, typically selected from plant viruses, to maximise yields. This study aims to use rational design to generate synthetic sequences that can rival existing ones. Results In this work, we present the rational design of novel synthetic 5′ and 3′ untranslated regions (UTRs) which can be used in various combinations to modulate accumulation levels of transiently-expressed recombinant proteins. Using the pEAQ-HT expression vector as a point of comparison, we show that pre-existing expression systems can be improved by the deployment of rationally designed synthetic UTRs. Notably, we show that a suite of short, synthetic 5′UTRs behave as expression enhancers that outperform the HT 5′UTR present in the CPMV-HT expression system. Furthermore, we confirm the critical role played by the 3′UTR of cowpea mosaic virus RNA-2 in the performance of the CPMV-HT system. Finally, we use the knowledge obtained from these results to develop novel expression vectors (named pHRE and pHREAC) that equal or outperform pEAQ-HT in terms of recombinant protein yield. These new vectors are also domesticated for the use of certain Type IIS restriction enzymes, which allows for quicker cloning and straightforward assessment of different combinations of UTRs. Conclusions We have shown that it is possible to rationally design a suite of expression modulators in the form of synthetic UTRs. We have created novel expression vectors that allow very high levels of recombinant protein expression in a transient expression context. This will have important consequences for future efforts to develop ever-better plant transient overexpression vectors for research or industrial applications.

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