recombinant viruses may add some advantages to a particular
expression project as all the regulatory elements that the virus
provides have been shaped by evolution to work coordinately to
optimize the expression of the viral proteins and, expectantly, foreign proteins. In our recent research, we found that potyviruses are
particularly helpful for expressing carotenogenic enzymes in plant
tissues, since a substantial accumulation of products of this metabolic pathway can take place in infected tissues a few days after
inoculation [5, 6]. Potyviruses (genus Potyvirus) compose the largest group of RNA viruses that infect plants. They consist of an
approximately 10,000 nt-long RNA genome of plus polarity that
is attached to a viral protein genome-linked (VPg) at the 5
0 end,
containing a poly(A) tail at the 3
0 end, and encapsidated into an
elongated and flexuous virion [7]. Potyviruses have a unique strategy to express their genomes. Basically, genomic RNA encodes a
large polyprotein that is processed after translation into about
10 mature proteins by three viral-encoded proteases [8]. Conversion of a potyvirus into an expression vector is quite a straight
forward process that only requires the insertion of the cDNAs
corresponding to one protein of interest, or more, in some particular intercistronic positions of the viral genome, provided that these
cDNAs are flanked by the right sequences to mediate the release of
the heterologous proteins from the viral polyprotein [9, 10]. In
practice, nuclear inclusion a (NIa) protease (NIaPro), which recognizes a À6/+1 seven amino-acid motif with high specificity, is used
for this purpose [11]. Attention to signal peptides and the right
position of the heterologous proteins in the viral polyprotein must
be also paid if these proteins need to be targeted to any particular
subcellular compartments [12].
Here I describe a method to express one or more carotenogenic
enzymes or transcription factors in Nicotiana benthamiana plants
using Tobacco etch virus (TEV; genus Potyvirus) (Fig. 1). More
specifically, in this method we use a TEV version that lacks the
NIb cistron, which encodes for the viral RNA-dependent RNA
polymerase and is only able to infect the N. benthamiana plants
that stably express NIb [9, 13, 14]. Deletion of viral NIb cistron
increases the space to harbor foreign RNA, the same strategy herein
described can also be used with a full-length TEV or any other
potyvirus vector. After construction of the recombinant virus
(Figs. 2 and 3), plants are agroinoculated. To replicate its genome
in infected tissues, the recombinant virus expresses a polyprotein
from which foreign proteins are released (Fig. 2). This method can
be useful for producing a particular carotenoid in N. benthamiana
tissues (Fig. 4), but to also quickly analyze the effect of the expressions of native, heterologous or engineered proteins in the carotenoid biosynthesis pathway.
304
Jose ´ -Antonio Daro ` s
expression project as all the regulatory elements that the virus
provides have been shaped by evolution to work coordinately to
optimize the expression of the viral proteins and, expectantly, foreign proteins. In our recent research, we found that potyviruses are
particularly helpful for expressing carotenogenic enzymes in plant
tissues, since a substantial accumulation of products of this metabolic pathway can take place in infected tissues a few days after
inoculation [5, 6]. Potyviruses (genus Potyvirus) compose the largest group of RNA viruses that infect plants. They consist of an
approximately 10,000 nt-long RNA genome of plus polarity that
is attached to a viral protein genome-linked (VPg) at the 5
0 end,
containing a poly(A) tail at the 3
0 end, and encapsidated into an
elongated and flexuous virion [7]. Potyviruses have a unique strategy to express their genomes. Basically, genomic RNA encodes a
large polyprotein that is processed after translation into about
10 mature proteins by three viral-encoded proteases [8]. Conversion of a potyvirus into an expression vector is quite a straight
forward process that only requires the insertion of the cDNAs
corresponding to one protein of interest, or more, in some particular intercistronic positions of the viral genome, provided that these
cDNAs are flanked by the right sequences to mediate the release of
the heterologous proteins from the viral polyprotein [9, 10]. In
practice, nuclear inclusion a (NIa) protease (NIaPro), which recognizes a À6/+1 seven amino-acid motif with high specificity, is used
for this purpose [11]. Attention to signal peptides and the right
position of the heterologous proteins in the viral polyprotein must
be also paid if these proteins need to be targeted to any particular
subcellular compartments [12].
Here I describe a method to express one or more carotenogenic
enzymes or transcription factors in Nicotiana benthamiana plants
using Tobacco etch virus (TEV; genus Potyvirus) (Fig. 1). More
specifically, in this method we use a TEV version that lacks the
NIb cistron, which encodes for the viral RNA-dependent RNA
polymerase and is only able to infect the N. benthamiana plants
that stably express NIb [9, 13, 14]. Deletion of viral NIb cistron
increases the space to harbor foreign RNA, the same strategy herein
described can also be used with a full-length TEV or any other
potyvirus vector. After construction of the recombinant virus
(Figs. 2 and 3), plants are agroinoculated. To replicate its genome
in infected tissues, the recombinant virus expresses a polyprotein
from which foreign proteins are released (Fig. 2). This method can
be useful for producing a particular carotenoid in N. benthamiana
tissues (Fig. 4), but to also quickly analyze the effect of the expressions of native, heterologous or engineered proteins in the carotenoid biosynthesis pathway.
304
Jose ´ -Antonio Daro ` s