Simultaneous equimolar expression of multiple proteins in plants from a disarmed potyvirus vector

Bedoya, Leonor C.; Martínez, Fernando; Rubio, Luis A.; Daròs, José‐Antonio

doi:10.1016/j.jbiotec.2010.08.006

Cited by 40 publications

(60 citation statements)

References 42 publications

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“…Virus clone TRV-Ros1 was produced from plasmids pTRV1 (Liu et al, 2002) and pTRV2-Ros1. Plasmid pGTEVa derives from pGTEV (Bedoya and Daròs, 2010) and contains the TEV cDNA corresponding to GenBank accession number DQ986288 under the control of Cauliflower mosaic virus 35S promoter and terminator, but includes two silent mutations (G273A and A1119G) to remove internal BsaI restriction sites. In pGTEV-Ros1(P1/HC-Pro), pGTEV-Ros1(NIb/CP), pGTEV-GFP, and pGTEV-GFP-Ros1, the Ros1 (DQ275529) and the enhanced GFP (AAB08060) cDNAs were inserted in different positions of pGTEVa by standard techniques based on the use of the type IIs restriction enzyme BsaI (New England Biolabs) and T4 DNA ligase (Fermentas; Engler et al, 2009).…”

Section: Plasmidsmentioning

confidence: 99%

“…Bacteria were grown in liquid culture, adjusted at 0.5 OD 600 in induction buffer (10 mM MES-NaOH, pH 5.6, 10 mM MgCl 2 , and 150 mM acetosyringone), cultured for 2 h at 28°C, and infiltrated in a plant leaf (Bedoya and Daròs, 2010). For agroinoculation of TRV-Ros1, two cultures of A. tumefaciens C58C1 each transformed with pTRV1 or pTRV2-Ros1 were treated as above, mixed, and infiltrated.…”

Section: Plant Inoculationmentioning

confidence: 99%

“…TuMV-WT and TuMV-Ros1 were mechanically inoculated from plasmids pTuMV (Martínez et al, 2011) and pTuMV-Ros1. Finally, TMV-Ros1 was mechanically inoculated from 5#-capped RNA transcripts obtained by in vitro transcription (Bedoya and Daròs, 2010) with T7 RNA polymerase (Epicentre) of plasmid pTMV-Ros1 digested by KpnI (Fermentas).…”

Section: Plant Inoculationmentioning

confidence: 99%

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Visual Tracking of Plant Virus Infection and Movement Using a Reporter MYB Transcription Factor That Activates Anthocyanin Biosynthesis

et al. 2012

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Insertion of reporter genes into plant virus genomes is a common experimental strategy to research many aspects of the viral infection dynamics. Their numerous advantages make fluorescent proteins the markers of choice in most studies. However, the use of fluorescent proteins still has some limitations, such as the need of specialized material and facilities to detect the fluorescence. Here, we demonstrate a visual reporter marker system to track virus infection and movement through the plant. The reporter system is based on expression of Antirrhinum majus MYB-related Rosea1 (Ros1) transcription factor (220 amino acids; 25.7 kD) that activates a series of biosynthetic genes leading to accumulation of colored anthocyanins. Using two different tobacco etch potyvirus recombinant clones tagged with Ros1, we show that infected tobacco (Nicotiana tabacum) tissues turn bright red, demonstrating that in this context, the sole expression of Ros1 is sufficient to induce pigment accumulation to a level readily detectable to the naked eye. This marker system also reports viral load qualitatively and quantitatively by means of a very simple extraction process. The Ros1 marker remained stable within the potyvirus genome through successive infectious passages from plant to plant. The main limitation of this marker system is that color output will depend on each particular plant host-virus combination and must be previously tested. However, our experiments demonstrate accurate tracking of turnip mosaic potyvirus infecting Arabidopsis (Arabidopsis thaliana) and either tobacco mosaic virus or potato X virus infecting Nicotiana benthamiana, stressing the general applicability of the method.

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Section: Plasmidsmentioning

confidence: 99%

Section: Plant Inoculationmentioning

confidence: 99%

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Visual Tracking of Plant Virus Infection and Movement Using a Reporter MYB Transcription Factor That Activates Anthocyanin Biosynthesis

et al. 2012

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“…Even though NIb can be provided in trans (8,9), it can only be relocated to the amino terminus of the polyprotein or in between P1 and HC-Pro without affecting virus viability. The relocation of NIb to the other seven positions renders nonviable viruses, even in a transgenic plant constitutively expressing TEV NIb that can be infected by a TEV mutant with a complete NIb deletion (8).…”

mentioning

confidence: 99%

Relocation of the NIb Gene in the Tobacco Etch Potyvirus Genome

Majer

Salvador

Zwart

et al. 2014

J Virol

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bPotyviruses express most of their proteins from a long open reading frame that is translated into a large polyprotein processed by three viral proteases. To understand the constraints on potyvirus genome organization, we relocated the viral RNA-dependent RNA polymerase (NIb) cistron to all possible intercistronic positions of the Tobacco etch virus (TEV) polyprotein. Only viruses with NIb at the amino terminus of the polyprotein or in between P1 and HC-Pro were viable in tobacco plants.

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“…The pGTEV plasmid contains a TEV infectious cDNA (Genbank accession number DQ986288) flanked by CaMV 35S promoter and terminator (Bedoya and Darós, 2010).…”

Section: Plasmid Constructionmentioning

confidence: 99%

Desarrollo De Un Vector Para La Expresión Simultánea De Múltiples Proteínas en Plantas Basado en El Potyvirus Del Grabado Del Tabaco

Rojas¹,

Leonor²

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Simultaneous equimolar expression of multiple proteins in plants from a disarmed potyvirus vector

Cited by 40 publications

References 42 publications

Visual Tracking of Plant Virus Infection and Movement Using a Reporter MYB Transcription Factor That Activates Anthocyanin Biosynthesis

Visual Tracking of Plant Virus Infection and Movement Using a Reporter MYB Transcription Factor That Activates Anthocyanin Biosynthesis

Relocation of the NIb Gene in the Tobacco Etch Potyvirus Genome

Desarrollo De Un Vector Para La Expresión Simultánea De Múltiples Proteínas en Plantas Basado en El Potyvirus Del Grabado Del Tabaco

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