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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-015-0315-3) contains supplementary material, which is available to authorized users.
Highlights
Approaches 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
The mcherry sequence is fused at the N-terminus of the viral protein genome-linked (VPg) and the mcherry-VPg fusion gene is cloned between the P1 and HC-Pro coding sequences, at the cloning site currently used for Lettuce mosaic virus (LMV) engineering [25, 26]
We describe a significant methodological advance to simplify and accelerate the construction of infectious cDNA clones derived from the potyvirus Lettuce mosaic virus as a model example
Summary
Approaches 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. Approaches to simplify and accelerate the construction of FL-cDNAs for plant viruses were recently described, based on cloning strategies involving homologous recombination in yeast [3, 5] rather than more classical cloning approaches such as in vitro ligation. We report the development of a rapid and efficient in vitro recombination system based on assembling overlapping PCR-amplified DNA molecules in a single isothermal step, derived from the strategy based on in vitro Gibson assembly (GA) [9] This strategy was used to obtain modified infectious cDNA clones of a potyvirus, Lettuce mosaic virus (LMV). Such interactions should play crucial roles in many processes during the virus infection cycle, and probably during the eIF4E recruitment process
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
