Development of a new tobamovirus-based viral vector for protein expression in plants

Vasques, Raquel Medeiros; Lacorte, Cristiano; Luz, L; Aranda, Miguel A.; Nagata, Tatsuya

doi:10.1007/s11033-018-4449-4

Cited by 7 publications

(5 citation statements)

References 33 publications

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“…In contrast to In‐Fusion, Gibson assembly yields closed circular DNA molecules. Applied for reverse genetic studies of potyvirus clones (Bordat et al ., ; Pasin et al ., ), Gibson assembly has been used to generate binary infectious clones of members of genera Tymovirus (Blawid and Nagata, ), Carlavirus (Carvalho et al ., ), Comovirus (Bijora et al ., ), Potyvirus (Rose and Maiss, ), Polerovirus (Wetzel et al ., ), Benyvirus (Laufer et al ., ), Tobamovirus (Vasques et al ., ) and Begomovirus (Ferro et al ., ). Gibson assembly versions with improved fidelity are commercially available (e.g.…”

Section: Advanced Methods For Binary Infectious Clone Assemblymentioning

confidence: 99%

Harnessed viruses in the age of metagenomics and synthetic biology: an update on infectious clone assembly and biotechnologies of plant viruses

Pasin

Menzel

Daròs

2019

Plant Biotechnology Journal

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Summary Recent metagenomic studies have provided an unprecedented wealth of data, which are revolutionizing our understanding of virus diversity. A redrawn landscape highlights viruses as active players in the phytobiome, and surveys have uncovered their positive roles in environmental stress tolerance of plants. Viral infectious clones are key tools for functional characterization of known and newly identified viruses. Knowledge of viruses and their components has been instrumental for the development of modern plant molecular biology and biotechnology. In this review, we provide extensive guidelines built on current synthetic biology advances that streamline infectious clone assembly, thus lessening a major technical constraint of plant virology. The focus is on generation of infectious clones in binary T‐ DNA vectors, which are delivered efficiently to plants by Agrobacterium . We then summarize recent applications of plant viruses and explore emerging trends in microbiology, bacterial and human virology that, once translated to plant virology, could lead to the development of virus‐based gene therapies for ad hoc engineering of plant traits. The systematic characterization of plant virus roles in the phytobiome and next‐generation virus‐based tools will be indispensable landmarks in the synthetic biology roadmap to better crops.

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Section: Advanced Methods For Binary Infectious Clone Assemblymentioning

confidence: 99%

Harnessed viruses in the age of metagenomics and synthetic biology: an update on infectious clone assembly and biotechnologies of plant viruses

Pasin

Menzel

Daròs

2019

Plant Biotechnology Journal

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“…By coinfiltrating the target construct with another one containing a silencing suppressor gene it is expected to increase the yield of the target proteins, as reported in other vector systems (Lindbo, 2007a; Rhee et al, 2016; Vasques et al, 2019). However, our study did not show a relevant difference in the production level of GFP and XynGH10, when coinfiltrated with the potyvirus HC‐Pro construct.…”

Section: Discussionmentioning

confidence: 97%

“…In many cases, the 5′ and 3′ ends of the CP gene of RNA viruses contain important secondary structures, such as a subgenomic RNA promoter (Grdzelishvili, Chapman, Dawson, & Lewandowski, 2000; Man & Epel, 2004; Sempere, Gómez, Truniger, & Aranda, 2011; Vasques, Lacorte, da Luz, Aranda, & Nagata, 2019). Thus, modifications of the CP gene in these regions may reduce the productivity of the target protein.…”

Section: Discussionmentioning

confidence: 99%

Development of a heterologous gene expression vector in plants based on an infectious clone of a tobravirus, pepper ringspot virus

Tavares‐Esashika

Campos

Maeda

et al. 2022

Annals of Applied Biology

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Heterologous gene expression systems are important tools for biotechnology, especially due to the increasing demands for mass production of pharmaceutical proteins.In this study, we assessed the use of the infectious clone of a tobravirus, pepper ringspot virus (PepRSV), as a heterologous gene expression tool in Nicotiana benthamiana plants. The insert region of gene of interest was evaluated in the infectious clone of PepRSV using the reporter gene encoding the green fluorescent protein (GFP). The insertion of the GFP gene in the coat protein gene region resulted in a higher protein yield than the previous system. Then, the reporter gene was replaced by the xylanase gene, and enzymatically active xylanase was produced. This study provides an efficient tool for the production of heterologous proteins using a plant system.

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“…Recently, a study using recombinant CHIKV E2 antigens, produced in both prokaryotic and eukaryotic expression systems, showed a strong reaction to anti-CHIKV IgG antibodies, with an accuracy higher than 90% [ 62 ]. An assay for chikungunya diagnosis, using recombinant multi-epitope proteins expressed in plants, reported a good quality expression and confirmation by Western-blotting [ 63 ]. The same multi-epitope strategy, using an eukaryotic system, was used to produce recombinant antigens, useful for the differentiation between dengue and Zika infections [ 33 ].…”

Section: Discussionmentioning

confidence: 99%

A Chikungunya Virus Multiepitope Recombinant Protein Expressed from the Binary System Insect Cell/Recombinant Baculovirus Is Useful for Laboratorial Diagnosis of Chikungunya

et al. 2022

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Chikungunya virus (CHIKV) is an arbovirus currently distributed worldwide, causing a disease that shares clinical signs and symptoms with other illnesses, such as dengue and Zika and leading to a challenging clinical differential diagnosis. In Brazil, CHIKV emerged in 2014 with the simultaneous introduction of both Asian and East/Central/South African (ECSA) genotypes. Laboratorial diagnosis of CHIKV is mainly performed by molecular and serological assays, with the latter more widely used. Although many commercial kits are available, their costs are still high for many underdeveloped and developing countries where the virus circulates. Here we described the development and evaluation of a multi-epitope recombinant protein-based IgG-ELISA (MULTREC IgG-ELISA) test for the specific detection of anti-CHIKV antibodies in clinical samples, as an alternative approach for laboratorial diagnosis. The MULTREC IgG-ELISA showed 86.36% of sensitivity and 100% of specificity, and no cross-reactivity with other exanthematic diseases was observed. The recombinant protein was expressed from the binary system insect cell/baculovirus using the crystal-forming baculoviral protein polyhedrin as a carrier of the target recombinant protein to facilitate recovery. The crystals were at least 10 times smaller in size and had an amorphous shape when compared to the polyhedrin wild-type crystal. The assay uses a multi-epitope antigen, representing two replicates of 18 amino acid sequences from the E2 region and a sequence of 17 amino acids from the nsP3 region of CHIKV. The recombinant protein was highly expressed, easy to purify and has demonstrated its usefulness in confirming chikungunya exposure, indeed showing a good potential tool for epidemiological surveillance.

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Development of a new tobamovirus-based viral vector for protein expression in plants

Cited by 7 publications

References 33 publications

Harnessed viruses in the age of metagenomics and synthetic biology: an update on infectious clone assembly and biotechnologies of plant viruses

Harnessed viruses in the age of metagenomics and synthetic biology: an update on infectious clone assembly and biotechnologies of plant viruses

Development of a heterologous gene expression vector in plants based on an infectious clone of a tobravirus, pepper ringspot virus

A Chikungunya Virus Multiepitope Recombinant Protein Expressed from the Binary System Insect Cell/Recombinant Baculovirus Is Useful for Laboratorial Diagnosis of Chikungunya

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