Abstract
Potyviruses are plus-strand RNA viruses that can be easily transformed into expression vectors to quickly express one carotenogenic enzyme or transcription factor, or more, in plant tissues. Unlike the technically challenging and time-consuming process of plant transformation, manipulation of a roughly 10,000 nt-long viral genome is rather straightforward via common molecular biology techniques. Here I describe how to insert the cDNAs of the proteins of interest into two particular positions of the cDNA of a Tobacco etch virus (TEV) mutant that lacks the viral NIb cistron and only infects the plants in which this protein is expressed. This deletion increases the space to harbor foreign sequences. The selection of the expression site must be made according to subcellular localization requirements. The recombinant virus is then inoculated into Nicotiana benthamiana plants by means of Agrobacterium tumefaciens. The expression of the viral genome entails the production of carotenogenic proteins in the plant tissues with a consequent effect on the plant carotenoid pathway.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Pogue GP, Lindbo JA, Garger SJ, Fitzmaurice WP (2002) Making an ally from an enemy: plant virology and the new agriculture. Annu Rev Phytopathol 40:45–74. https://doi.org/10.1146/annurev.phyto.40.021102.150133
Hefferon K (2017) Plant virus expression vectors: a powerhouse for Global Health. Biomedicine 5(3). https://doi.org/10.3390/biomedicines5030044
Pasin F, Menzel W, Daròs JA (2019) Harnessed viruses in the age of metagenomics and synthetic biology: an update on infectious clone assembly and biotechnologies of plant viruses. Plant Biotechnol J. https://doi.org/10.1111/pbi.13084
Krenek P, Samajova O, Luptovciak I, Doskocilova A, Komis G, Samaj J (2015) Transient plant transformation mediated by Agrobacterium tumefaciens: principles, methods and applications. Biotechnol Adv 33(6 Pt 2):1024–1042. https://doi.org/10.1016/j.biotechadv.2015.03.012
Majer E, Llorente B, Rodríguez-Concepción M, Daròs JA (2017) Rewiring carotenoid biosynthesis in plants using a viral vector. Sci Rep 7:41645. https://doi.org/10.1038/srep41645
Aragonés V, Pérez-de-Castro A, Cordero T, Cebolla-Cornejo J, López C, Picó B, Daròs JA (2019) A Watermelon mosaic virus clone tagged with the yellow visual maker phytoene synthase facilitates scoring infectivity in melon breeding programs. Eur J Plant Pathol 153:317. https://doi.org/10.1007/s10658-018-01621-x
Wylie SJ, Adams M, Chalam C, Kreuze J, López-Moya JJ, Ohshima K, Praveen S, Rabenstein F, Stenger D, Wang A, Zerbini FM, Ictv Report C (2017) ICTV Virus Taxonomy Profile: Potyviridae. J Gen Virol 98(3):352–354. https://doi.org/10.1099/jgv.0.000740
Revers F, García JA (2015) Molecular biology of potyviruses. Adv Virus Res 92:101–199. https://doi.org/10.1016/bs.aivir.2014.11.006
Bedoya L, Martínez F, Rubio L, Daròs JA (2010) Simultaneous equimolar expression of multiple proteins in plants from a disarmed potyvirus vector. J Biotechnol 150(2):268–275. https://doi.org/10.1016/j.jbiotec.2010.08.006
Majer E, Salvador Z, Zwart MP, Willemsen A, Elena SF, Daròs JA (2014) Relocation of the NIb gene in the tobacco etch potyvirus genome. J Virol 88(8):4586–4590. https://doi.org/10.1128/JVI.03336-13
Carrington JC, Haldeman R, Dolja VV, Restrepo-Hartwig MA (1993) Internal cleavage and trans-proteolytic activities of the VPg-proteinase (NIa) of tobacco etch potyvirus in vivo. J Virol 67(12):6995–7000
Majer E, Navarro JA, Daròs JA (2015) A potyvirus vector efficiently targets recombinant proteins to chloroplasts, mitochondria and nuclei in plant cells when expressed at the amino terminus of the polyprotein. Biotechnol J. https://doi.org/10.1002/biot.201500042
Li XH, Carrington JC (1995) Complementation of tobacco etch potyvirus mutants by active RNA polymerase expressed in transgenic cells. Proc Natl Acad Sci U S A 92(2):457–461
Martí M, Diretto G, Aragonés V, Ahrazem O, Gómez-Gómez L, Daròs JA (2019) Efficient production of saffron crocins and picrocrocin in Nicotiana benthamiana using a virus-driven system. Submitted
Thole V, Worland B, Snape JW, Vain P (2007) The pCLEAN dual binary vector system for Agrobacterium-mediated plant transformation. Plant Physiol 145(4):1211–1219
Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, Smith HO (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6(5):343–345. https://doi.org/10.1038/nmeth.1318
Acknowledgments
This work was supported by the Ministerio de Ciencia, Innovación y Universidades (Spain) grant BIO2017-83184-R (AEI/FEDER UE).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2020 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Daròs, JA. (2020). Use of Potyvirus Vectors to Produce Carotenoids in Plants. In: Rodríguez-Concepción, M., Welsch, R. (eds) Plant and Food Carotenoids. Methods in Molecular Biology, vol 2083. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9952-1_23
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9952-1_23
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9951-4
Online ISBN: 978-1-4939-9952-1
eBook Packages: Springer Protocols