Chapter 23
Use of Potyvirus Vectors to Produce Carotenoids in Plants
Jose ´ -Antonio Daro ` s
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 ntlong 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.
Key words Viral vector, Potyvirus, Transient expression, Protein expression, Polyprotein processing,
Carotenoid biosynthesis
1 Introduction
Plant viruses, if properly manipulated, can be converted into vectors to produce compounds of interest in plant tissues [1, 2]. In
contrast to the complex genomes of host plants, which are technically difficult and time-consuming to manipulate, the small genomes of most plant viruses can be cloned in a single plasmid, or a
few, and can be easily manipulated by common molecular biology
techniques [3]. Once engineered and converted into expression
vectors, most recombinant viruses can be quickly and efficiently
delivered to plant tissues by means of Agrobacterium tumefaciens.
In essence, this is no different from the most general method of the
A. tumefaciens-mediated transient expression of RNAs and proteins
in plant tissues [4], a technique that also frequently uses elements of
a viral origin, such as promoters, suppressors of RNA silencing
pathways, and other regulatory elements. However, the use of
Manuel Rodrı ´guez-Concepcio ´ n and Ralf Welsch (eds.), Plant and Food Carotenoids: Methods and Protocols,
Methods in Molecular Biology, vol. 2083, https://doi.org/10.1007/978-1-4939-9952-1_23,
© Springer Science+Business Media, LLC, part of Springer Nature 2020
303
Use of Potyvirus Vectors to Produce Carotenoids in Plants
Jose ´ -Antonio Daro ` s
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 ntlong 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.
Key words Viral vector, Potyvirus, Transient expression, Protein expression, Polyprotein processing,
Carotenoid biosynthesis
1 Introduction
Plant viruses, if properly manipulated, can be converted into vectors to produce compounds of interest in plant tissues [1, 2]. In
contrast to the complex genomes of host plants, which are technically difficult and time-consuming to manipulate, the small genomes of most plant viruses can be cloned in a single plasmid, or a
few, and can be easily manipulated by common molecular biology
techniques [3]. Once engineered and converted into expression
vectors, most recombinant viruses can be quickly and efficiently
delivered to plant tissues by means of Agrobacterium tumefaciens.
In essence, this is no different from the most general method of the
A. tumefaciens-mediated transient expression of RNAs and proteins
in plant tissues [4], a technique that also frequently uses elements of
a viral origin, such as promoters, suppressors of RNA silencing
pathways, and other regulatory elements. However, the use of
Manuel Rodrı ´guez-Concepcio ´ n and Ralf Welsch (eds.), Plant and Food Carotenoids: Methods and Protocols,
Methods in Molecular Biology, vol. 2083, https://doi.org/10.1007/978-1-4939-9952-1_23,
© Springer Science+Business Media, LLC, part of Springer Nature 2020
303