Biomedical Engineering Reference
In-Depth Information
maize streak virus (MSV) and wheat dwarf virus
(WDV).
An important additional distinction between these
genera is that mastreviruses are not mechanically
transmissible. MSV, for example, has never been
introduced successfully into plants as native or
cloned DNA. Grimsley
et al.
(1987) were able to
overcome this problem using
Agrobacterium
, and
were the first to demonstrate the principle of agro-
infection. They constructed a plasmid containing a
tandem dimer of the MSV genome. This dimer was
inserted into a binary vector, and maize plants were
infected with
A. tumefaciens
containing the recom-
binant T-DNA. Viral symptoms appeared within
2 weeks of inoculation. Agroinfection has been used
to introduce the genomes of a number of different
viruses into plants. It can be demonstrated that, if
the T-DNA contains partially or completely duplic-
ated genomes, single copies of the genome can
escape and initiate infections. This may be mediated
by homologous recombination or a replicative
mechanism (Stenger
et al.
1991). The study of
Grimsley
et al.
(1987) incidentally provided the first
evidence that
Agrobacterium
could transfer T-DNA
to maize. Agroinfection is a very sensitive assay for
transfer to the plant cell because of the amplification
inherent in the virus infection and the resulting
visible symptoms.
A number of geminiviruses have been developed
as expression vectors, because of the possibility of
achieving high-level recombinant-protein expres-
sion as a function of viral replication (reviewed by
Stanley 1983, Timmermans
et al.
1994, Palmer &
Rybicki 1997). A generally useful strategy is the
replacement of the coat-protein gene, since this is
not required for replication and the strong promoter
can be used to drive transgene expression. In the
case of begomoviruses, which have bipartite genomes,
the coat-protein gene is located on DNA A, along
with all the functions required for DNA replication.
Replicons based on DNA A are therefore capable of
autonomous replication in protoplasts (e.g. Townsend
et al.
1986). Geminivirus replicon vectors can facilit-
ate the high-level transient expression of foreign
genes in protoplasts. There appears to be no intrinsic
limitation to the size of the insert, although larger
transgenes tend to reduce the replicon copy number
(e.g. Laufs
et al.
1990, Matzeit
et al.
1991). Generally,
it appears that mastrevirus replicons can achieve a
much higher copy number in protoplasts than re-
plicons based on begomiviruses. A WDV shuttle
vector capable of replicating in both
E. coli
and plants
was shown to achieve a copy number of greater than
3
10
4
in protoplasts derived from cultured maize
endosperm cells (Timmermans
et al.
1992), whereas
the typical copy number achieved by TGMV re-
plicons in tobacco protoplasts is less than 1000
(Kanevski
et al.
1992). This may, however, reflect
differences in the respective host cells, rather than
the intrinsic efficiencies of the vectors themselves.
Geminiviruses are also valuable as expression vec-
tors in whole plants. In the case of the mastreviruses,
all viral genes appear to be essential for systemic
infection, so coat-protein replacement vectors cannot
be used in this manner. In contrast, the coat-protein
genes of ACMV and TGMV are non-essential for sys-
temic infection, but they are required for insect trans-
mission (Briddon
et al.
1990). Therefore, replicon
vectors based on these viruses provide an
in planta
contained transient expression system. Note that
viral movement functions are supplied by DNA B, so
systemic infections occur only if DNA B is also pre-
sent in the plant.
In an early study, Ward
et al.
(1988) replaced
most of the ACMV AV1 gene with the
cat
reporter
gene. In infected tobacco plants, high level CAT
activity was detected for up to 4 weeks. Interest-
ingly, they found that deletion of the coat-protein
gene caused a loss of infectivity in plants, but this
was restored upon replacement with
cat
, which is
approximately the same size as the deleted gene.
This and many subsequent reports indicated that,
while there may be no intrinsic limit to the size of
replicon vectors in protoplasts, systemic infection
is dependent on preserving the size of the wild-
type DNA-A component. A further limitation to
this system is that the transmissibility of the re-
combinant genomes is poor, probably because they
are not packaged. One way in which this can be
addressed is to generate transgenic plants in which
recombinant viral genomes are produced in every
cell. This is achieved by transforming plants with
DNA constructs containing a partially duplicated
viral genome (Meyer
et al.
1992). Intact replicons can
excise from the delivered transgene in the same way
as the MSV genome escapes during agroinfection,
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