ArticleVirology journal2015
Gibson assembly: an easy way to clone potyviral full-length infectious cDNA clones expressing an ectopic VPg.
Article in Virology journal, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 26 papers.
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Who cites it
26 citing papers in PubMed.
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- Enzyme-substrate co-display on yeast empowers engineering of tobacco etch virus protease activity.Protein engineering, design & selection : PEDS · 2025Article
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- Identification of a TuMV isolate (TuMV-ZR) from Pseudostellaria heterophylla and its development into a viral expression vector.Virus research · 2023Article
- Trends on Human Norovirus Virus-like Particles (HuNoV-VLPs) and Strategies for the Construction of Infectious Viral Clones toward In Vitro Replication.Life (Basel, Switzerland) · 2023Review
- Generation of a Triple-Shuttling Vector and the Application in Plant Plus-Strand RNA Virus Infectious cDNA Clone Construction.International journal of molecular sciences · 2023Article
- BigDNA: Primer Design Software for Overlap-Based Assembly of Phage Genomes and Larger DNAs.PHAGE (New Rochelle, N.Y.) · 2022Article
- PlasmidMaker is a versatile, automated, and high throughput end-to-end platform for plasmid construction.Nature communications · 2022Article
- RUFY3 and RUFY4 are ARL8 effectors that promote coupling of endolysosomes to dynein-dynactin.Nature communications · 2022Article
- Synthesis of Full-Length cDNA Infectious Clones ofViruses · 2020Article
- A suicide enzyme catalyzes multiple reactions for biotin biosynthesis in cyanobacteria.Nature chemical biology · 2020Article
- Home-made enzymatic premix and Illumina sequencing allow for one-step Gibson assembly and verification of virus infectious clones.Phytopathology research · 2020Article
- Harnessed viruses in the age of metagenomics and synthetic biology: an update on infectious clone assembly and biotechnologies of plant viruses.Plant biotechnology journal · 2019Review
- Agroinoculation of Grapevine Pinot Gris Virus in tobacco and grapevine provides insights on viral pathogenesis.PloS one · 2019Article
- Rapid Construction of a Replication-Competent Infectious Clone of Human Adenovirus Type 14 by Gibson Assembly.Viruses · 2018Article
- Modified Roller Tube Method for Precisely Localized and Repetitive Intermittent Imaging During Long-term Culture of Brain Slices in an Enclosed System.Journal of visualized experiments : JoVE · 2017Article
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3 authors.
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No grant is acknowledged in the PubMed record.
Abstract
backgroundApproaches to simplify and accelerate the construction of full-length infectious cDNA clones for plant potyviruses have been described, based on cloning strategies involving in vitro ligation or homologous recombination in yeast. In the present study, we developed a faster and more efficient in vitro recombination system using Gibson assembly (GA), to engineer a Lettuce mosaic virus (LMV) infectious clone expressing an ectopic mcherry-tagged VPg (Viral protein genome-linked) for in planta subcellular localization of the viral protein in an infection context.
methodsThree overlapping long distance PCR fragments were amplified and assembled in a single-step process based on in vitro recombination (Gibson assembly). The resulting 17.5 kbp recombinant plasmids (LMVmchVPg_Ec) were inoculated by biolistic on lettuce plants and then propagated mechanically on Nicotiana benthamiana. Confocal microscopy was used to analyze the subcellular localization of the ectopically expressed mcherry-VPg fusion protein.
resultsThe Gibson assembly allowed the cloning of the expected plasmids without any deletion. All the inoculated plants displayed symptoms characteristic of LMV infection. The majority of the mcherry fluorescent signal observed using confocal microscopy was located in the nucleus and nucleolus as expected for a potyviral VPg.
conclusionsThis is the first report of the use of the Gibson assembly method to construct full-length infectious cDNA clones of a potyvirus genome. This is also the first description of the ectopic expression of a tagged version of a potyviral VPg without affecting the viability of the recombinant potyvirus.
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