Biomedical Engineering Reference
In-Depth Information
virus-resistant transgenic crop to reach commercial
production (Tricoli
et al.
1995).
While the heterologous coat-protein approach
can be successful, it has been demonstrated that, in
many cases, the effect of transgene expression is
mediated at the RNA rather than the protein level.
This can be proved by generating transgenic plants
carrying coat-protein genes that cannot be trans-
lated to yield functional protein, as first shown by
Lindbo and Dougherty (1992) using the tobacco
etch virus coat-protein gene. The transgene RNA
apparently interferes with viral replication (a
phenomenon called RNA-mediated viral resistance
(RMVR)). This requires homology between the
transgene and the target virus, and involves high-
level transgene transcription but low-level accumu-
lation of the transcript. This has much in common
with post-transcriptional gene silencing (discussed
in more detail in Chapter 13), which can lead to
transgene silencing and the cosuppression of homo-
logous endogenous genes, as well as viral resistance.
The interested reader can consult several excellent
reviews covering this and related phenomena
(Waterhouse
et al.
1998, Grant 1999, Plasterk &
Ketting 2000, Hammond
et al.
2001).
A different method of minimizing the effects of
plant virus infection was developed by Gehrlach
et al.
(1987). They generated plants that expressed
the satellite RNA of tobacco ringspot virus and such
plants were resistant to infection with tobacco
ringspot virus itself. Another potential method of
inducing resistance to viruses is the production
of antiviral proteins in transgenic plants. American
pokeweed produces an antiviral protein called
dianthrin that functions as a ribosome-inactivating
protein. The cDNA for this protein has been cloned
(Lin
et al.
1991) and expressed in
Nicotiana bentham-
iana
(a relative of tobacco), providing resistance
against African cassava mosaic virus (ACMV)
(Hong
et al.
1996). Interestingly in this experiment,
the dianthrin gene was expressed under the control
of an ACMV promoter, such that the antiviral pro-
tein was expressed only upon viral infection. In this
manner, the toxic effects of constitutive transgene
expression were avoided.
Antibodies specific for virion proteins have also
been used to protect plants from viruses. In the first
demonstration of this approach, Tavladoraki
et al.
(1993) expressed a single-chain Fv fragment (scFv)
specific for ACMV in transgenic
N. benthamiana
, and
demonstrated resistance to viral infection. Other
groups have generated transgenic tobacco plants
expressing antibodies specific for TMV, resulting in
reduced infectivity. Voss
et al.
(1995) expressed full-
size IgGs, while Zimmermann
et al.
(1998) expressed
scFv fragments. Targeting scFv fragments to the
plasma membrane also provides protection against
virus infection (Schillberg
et al.
2001).
Resistance to microbial pathogens
Progress has also been made in developing resistance
to plant-pathogenic fungi which are traditionally
controlled by appropriate farming practices (e.g.
crop rotation) and the application of expensive and
environmentally harmful fungicides. A straightfor-
ward approach is to engineer plants with antifungal
proteins from heterologous species. This was first
demonstrated by Broglie
et al.
(1991) who showed
that expression of bean chitinase can protect tobacco
and oil-seed rape from post-emergent damping off
caused by
Rhizoctonia solani.
Plants synthesize a
wide range of so-called 'pathogenesis-related pro-
teins' (PR proteins), such as chitinase, which are
induced by microbial infection. They also synthesize
antifungal peptides called defensins and other
antifungal proteins. As the genes for more of these
proteins have been cloned and characterized, the
number of transgenic plants constitutively express-
ing such proteins continues to rise. For example,
tobacco osmotin has been expressed in transgenic
potato, providing resistance to
Phytophthora infes-
tans
(Liu
et al.
1994), and in transgenic rice, provid-
ing resistance to
R. solani
(Lin
et al.
1995). Instead
of using a protein to provide direct protection,
a metabolic-engineering strategy can be utilized.
Phytoallexins are alkaloids with antifungal activity,
and transforming plants with genes encoding the
appropriate biosynthetic enzymes can increase
their synthesis. Hain
et al.
(1993) generated tomato
plants expressing the grapevine gene for stilbene
synthase, and these plants demonstrated increased
resistance to infection by
Botrytis cinerea.
Similarly,
Anzai
et al.
(1989) have used a bacterial gene
facilitating tabtoxin detoxification to protect tomato
plants against
Pseudomonas syringae
infection.
