A simplified approach to construct infectious cDNA clones of a tobamovirus in a binary vector

Junqueira, Bruna Rayane Teodoro; Nicolini, Cícero; Lucinda, Natália; Orílio, Anelise F.; Nagata, Tatsuya

doi:10.1016/j.jviromet.2013.12.024

Cited by 12 publications

(7 citation statements)

References 38 publications

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“…On the other hand, in vivo infectious transcripts are driven by Cauliflower mosaic virus 35S promoter in a binary vector. Cells transformed with the plasmids containing virus cDNA clones are then introduced into plants through agroinfiltration, particle bombardment, or rubbing onto the leaf’s surface [ 24 , 25 ]. In this review, we describe and summarise the application of reverse genetics in potyviral studies based on the potyvirus infectious clones developed to date.…”

Section: Introductionmentioning

confidence: 99%

Application of Reverse Genetics in Functional Genomics of Potyvirus

Kannan

Zainal

Ismail

et al. 2020

Viruses

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Numerous potyvirus studies, including virus biology, transmission, viral protein function, as well as virus–host interaction, have greatly benefited from the utilization of reverse genetic techniques. Reverse genetics of RNA viruses refers to the manipulation of viral genomes, transfection of the modified cDNAs into cells, and the production of live infectious progenies, either wild-type or mutated. Reverse genetic technology provides an opportunity of developing potyviruses into vectors for improving agronomic traits in plants, as a reporter system for tracking virus infection in hosts or a production system for target proteins. Therefore, this review provides an overview on the breakthroughs achieved in potyvirus research through the implementation of reverse genetic systems.

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Section: Introductionmentioning

confidence: 99%

Application of Reverse Genetics in Functional Genomics of Potyvirus

Kannan

Zainal

Ismail

et al. 2020

Viruses

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“…The availability of full-length infectious cDNA clones (FL-cDNAs) is crucial for reverse genetics studies on plant viruses. Since the first description of the cloning of an infectious clone of Brome mosaic virus [ 1 ], many other infectious clones of plant RNA viruses were obtained by cloning the full-length genomic cDNA under T7 RNA polymerase or 35S promoter sequences [ 2 – 4 ]. 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.…”

Section: Introductionmentioning

confidence: 99%

Gibson assembly: an easy way to clone potyviral full-length infectious cDNA clones expressing an ectopic VPg

Bordat

Houvenaghel

German-Retana

2015

Virol J

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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.

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“…To date, 10 infectious cDNA clones for Tobamovirus have been obtained: tobacco mosaic virus (TMV) (Meshi et al, 1986), tomato mosaic virus (ToMV) (Weber et al, 1992), odontoglossum ringspot virus (ORSV) (Yu and Wong, 1998), Kyuri green mottle mosaic virus (KGMMV) (Yoon et al, 2001), zucchini green mottle mosaic virus (ZGMMV) (Yoon et al, 2002), obuda pepper virus (ObPV) (Junqueira et al, 2014 ), turnip vein-clearing virus (TVCV) (Zhang et al, 1999), pepper mild mottle virus (PMMoV) (Ichiki et al, 2009), ribgrass mosaic virus (RMV) (Ryu et al, 2012), maracuja mosaic virus (MarMV) (Song et al, 2006), and Chinese rape mosaic virus (CRMV) (Aguilar et al, 1996).…”

Section: Introductionmentioning

confidence: 99%

Completion sequence and cloning of the infectious cDNA of a chb isolate of cucumber green mottle mosaic virus

Zhong¹,

Zhao²,

Liu³

et al. 2015

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Cucumber green mottle mosaic virus (CGMMV) is an important and widespread seed-borne virus that infects Cucurbitaceous plants. It is a member of the genus Tobamovirus in the family Virgaviridae with a monopartite (+) ssRNA genome. Here we report the complete genome sequence, construction and testing of the infectious clones of a chb isolate of CGMMV. Full-length CGMMV cDNA was cloned into the vector pUC19. The linearized vector containing full-length cDNA was used as template for in vitro transcription, and the synthesized capped transcript was highly infectious in Chenopodium amaranticolor and cucumber (Cucumis sativus). Inoculated plants showed symptoms typical of CGMMV infection. The infectivity was confirmed by mechanical transmission to new plants, RT-PCR and western blot. Progeny virus derived from infectious transcripts had the same biological and biochemical properties as wild-type virus. To our knowledge, this is the first detailed report of a biologically active transcript from CGMMV.

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A simplified approach to construct infectious cDNA clones of a tobamovirus in a binary vector

Cited by 12 publications

References 38 publications

Application of Reverse Genetics in Functional Genomics of Potyvirus

Application of Reverse Genetics in Functional Genomics of Potyvirus

Gibson assembly: an easy way to clone potyviral full-length infectious cDNA clones expressing an ectopic VPg

Completion sequence and cloning of the infectious cDNA of a chb isolate of cucumber green mottle mosaic virus

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