This paper was presented to Workshop on the Ecological Risks of Trangenic
Crops, University of California, Berkeley, March 2-4, 2000
It gives evidence of recombination between
viral transgenes and viruses to generate new viruses. It also draws
attention to the danger of the cauliflower mosaic viral promoter which is
in practically all GM crops currently undergoing field trials or already
commercially released.
Key words: Viral resistant transgenic
plants, ecological risks, viral recombination, CaMV promoter, viral coat
protein, hazards of GM crops
Mae-Wan Ho and Angela Ryan -Institute of
Science in Society and Biology Department, Open University, Walton Hall,
Milton Keynes, MK7 6AA, UK
Joe Cummins -Department of Plant Sciences, University of Western Ontario,
Ontario, Canada
Recombination of viral transgenes with
viral genomes to generate new viruses
The first report that transgenic plants
expressing the coat protein of the tobacco mosaic virus (TMV) delayed the
development of disease appeared in 1986 (1). The same strategy was
subsequently used to create resistance to a range of other viruses (2),
but geneticists have questioned the safety of these transgenic crops since
the early days. The most obvious risk is the potential for generating new
infectious viruses by recombination, ie, the viral transgene joining up or
exchanging parts with the nucleic acid of other viruses. Because the
coat-protein does not block the virus entering into the plant cell, the
transgene will be exposed to the nucleic acids of many viruses that are
brought to the plant by insect vectors.
A number of studies have demonstrated that
plant viruses can acquire a variety of viral genes from transgenic plants.
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Defective red clover necrotic mosaic
virus lacking the gene enabling it to move from cell to cell, and
hence not infectious, recombined with a copy of that gene in
transgenic Nicotiana benthamiana plants, and regenerated
infectious viruses (3).
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Transgenic Brassica napus
containing gene VI, a translational activator, from the cauliflower
mosaic virus (CaMV), recombined with the complementary part of the
virus missing that gene (4), and gave infectious virus in 100% of the
transgenic plants.
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The same experiment carried out in Nicotiana
bigelovii (5) gave infectious recombinants that expanded the host
range of the virus.
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N. benthamiana plants expressing a
segment of the cowpea chlorotic mottle virus (CCMV) coat-protein gene
recombined with defective virus missing that gene (6). A later report
stated that recombination between transgenes and infecting virus in
CCMV was nevertheless much more frequent than recombination between
co-infecting viruses (7).
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N. benthamiana was transformed with
three different constructs containing the coat protein coding sequence
of African cassava mosaic virus (ACMV). Transformed plants were
inoculated with a coat protein deletion mutant of ACMV that induces
mild systemic symptoms in control plants. Several inoculated plants of
transgenic lines developed severe systemic symptoms typical of ACMV
(8). Recombination had occurred between the mutant viral DNA and the
integrated construct DNA, resulting in the production of recombinant
virus progeny with 'wild-type' characteristics As all these
experiments involved recombination between defective virus and
transgene, it was thought that under natural conditions, when viruses
are not defective, no recombinant viruses would be generated (9).
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However, recombination between
wild-type CaMV and transgene VI was demonstrated in N. bigelovii
(10). At least one of the recombinant virus was more virulent than the
wild type.
Green and Allison (11) found that trimming
the 3’ end of the transgene containing untranslated region (UTR) reduced
recombination to zero, as compared with 3% in the controls. As
ribonucleotide sequences within the 3' UTR are involved in initiating
viral replication, the presence of this sequence may encourage the
participation of the transgene in RNA recombination. This suggests that
most, if not all of the recombinations may involve template-switching
between homologous sequences during viral replication. Recent findings
also indicate that the viral RNA-dependent RNA polymerase of several
potyviruses and tomato aspermy virus have the ability to recognize
heterologous 3´ UTRs (from the lettuce mosaic virus and the cucumber
mosaic virus) included in transgene mRNAs, and to use them as
transcription promoters (12). These findings have important implications
for the safety of viral resistant transgenic plants in general.
It has been noted in experiments involving
CaMV (10) that the frequency of recombination is much higher than that for
other viruses. While recombinant CCMV was recovered from 3% of transgenic N.
benthamiana containing CCMV sequences (11), recombinant CaMV was
recovered from 36% of transgenic N. bigelovii. It was suspected
that double-stranded DNA breaks may be involved in the case of CaMV
recombination. This may be due to the fact that the transgenic DNA
included the CaMV 35S promoter.
The CaMV promoter – ubiquitous in
transgenic plants
One viral sequence is in practically all
first generation transgenic plants which are now either already
commercialized or undergoing field trials. This is the CaMV 35S promoter,
used to make transgenes over-express constitutively. Cummins first
questioned the safety of the CaMV 35S promoter back in 1994 (12), when the
first transgenic crop, the Flavr Savr tomato was being approved for
commercial release. He warned that the promoter could also recombine with
other viruses to generate new viruses. But that warning was almost
completely ignored.
Last year, two events provoked us to look
into the matter more carefully. First, scientists from John Innes Research
Institute published a paper showing that the CaMV 35S promoter has a
recombination hotspot, which means it is prone to break and join up with
other pieces of genetic material (13). Second, senior scientist Dr. Arpad
Pusztai of the UK Government-funded Rowett Institute in Scotland, who was
sacked from his job and vilified by the scientific establishment for
revealing the results of feeding experiments suggesting that certain
transgenic potatoes may be unsafe, finally published part of their results
in The Lancet (14). It aroused a fresh storm of attack and even
reported threats to the Editor of the Journal for publishing the paper
(15). The explosive claim in the paper is that "other parts of the
construct or the genetic transformation process" may be responsible
for the adverse effects observed in the young rats: changes in the small
and large intestine, with increase in lymphocytes (white blood cells) in
the gut lining, which indicates damage to the intestine and is also a
non-specific sign of viral infection.
We submitted a paper last October, to the
Journal, Microbial Ecology in Health and Disease, whose Editor,
Prof. Tore Midvedt is a medical microbial ecologist in the Karolinska
Institute of Sweden. He put it out on the Journal website before the paper
was printed, and within two days, someone managed to collect ten critiques
on our paper, including one from Monsanto, which ranged from polite to
very rude. We rebutted the criticisms in full, and posted that on the web,
and no reply from our critics had appeared since. In January, Nature
Biotechnology published an offensive report which concentrated on the
criticisms and ignored our rebuttal completely.
Our manuscript (16) reviews and synthesizes
the scientific literature on and around the CaMV 35S promoter. The
promoter is promiscuous and functions efficiently in all plants, as well
as green algae, yeast and E. coli. It has a modular structure, with
parts common to, and interchangeable with promoters of other plant and
animal viruses. It also has a recombination hotspot, flanked by multiple
motifs involved in recombination, and is similar to other recombination
hotspots including the borders of the Agrobacterium T DNA vector
most frequently used in making transgenic plants. The suspected mechanism
of recombination – double-stranded DNA break-repair - requires little or
no DNA sequence homologies. Finally, recombination between viral
transgenes and infecting viruses has been demonstrated in the laboratory.
Transgenic constructs are already
well-known to be unstable; the structural stability of artificial vectors,
for example, is a text-book topic (17), and the existence of a
recombination hotspot will only exacerbate the problem. Consequently,
transgenic constructs containing the CaMV promoter may be more prone to
horizontal gene transfer and recombination. The potential hazards are
mutagenesis and carcinogenesis due to random insertion of foreign invasive
DNA into genomes, the reactivation of dormant viruses and generation of
new viruses (reviewed in refs. 18 and 19). Consequently, we have called
for all transgenic crops and products containing the CaMV promoter to be
withdrawn and banned, which in accordance with the precautionary principle
as well as sound science.
Our critics claim the CaMV 35S promoter is
not harmful because people have been eating the virus in infected cabbages
and cauliflower for many years. However, what we have been consuming is
predominantly intact virus and not naked viral genomes. Naked viral
genomes have been found to give full-blown infections in non-host species
that are not susceptible to the intact virus. Moreover, the 35S promoter
in the CaMV is a stable, integral part of the virus, and cannot be
compared to the 35S promoter in artificial transgenic constructs, which
are well-known to be structurally unstable. The 35S promoter in the virus
does not transfer into genomes because pararetroviruses, such as CaMV, do
not integrate into host genomes to complete their lifecycle; and the virus
replicates in the cytoplasm (20). This is completely different from the
35S promoter in transgenic constructs that are already integrated into
host genomes, and were designed to do so.
Proviral sequences (21) and related
retrotransposons are now found to be present in all genomes, including
those of higher plants (22). And as all viral promoters are modular, and
have at least one module – the TATA box - in common, if not more, it is
not inconceivable that the 35S promoter in transgenic constructs can
reactivate dormant viruses or generate new viruses by recombination. The
CaMV 35S promoter has been joined artificially to the cDNAs of a wide
range of viral genomes, and infectious viruses produced in the laboratory
(23, 24). There is also evidence that proviral sequence in the banana
genome can be reactivated, especially in tissue culture (25). This
research was done by Roger Hull, one of our critics who was none too
polite. He himself had earlier warned that viral coat proteins in
transgenic plants not only can offer disguise to related viruses to move
around the plant and infect it, but also that the protein may wrap up
retrotransposons in plants and allow them to be transmitted horizontally
to other species (26).
The fact that plants are "loaded"
with potentially mobile elements, such as retrotransposons, can only make
things worse. Most, if not all, of the elements will have been ‘tamed’
in the course of evolution and hence no longer mobile. But integration of
transgenic constructs containing the 35S promoter may mobilize the
elements. The elements may in turn provide helper-functions to destabilize
the transgenic DNA, and may also serve as substrates for recombination to
generate more exotic invasive elements.
New findings are revealing how plants
naturally resist viral infections by making small antisense RNA of 25
nucleotides against viral genes. Exactly the same mechanism is directed
against transgenes to silence them (27). The authors remark that the
gene-silencing "may represent a natural antiviral defence mechanism
and transgenes might be targeted because they, or their RNA are perceived
as viruses."
In signing on to the International
Biosafety Protocol in Montreal in January, the US, UK and more than 150
other governments agreed to implement the precautionary principle. In last
week’s Independent on Sunday, UK Prime Minister Tony Blair, who a
year ago was so confident of the safety of GE foods that he was
photographed eating a GE tomato himself, dramatically changed his tune and
now admits GE may damage both human health and biodiversity (28). He vows
to put those concerns above jobs and profit.
The available evidence clearly indicates
that there are serious potential hazards associated with the use of the
CaMV promoter. All GM crops and products containing the CaMV promoter
should therefore be withdrawn both from commercial use and from field
trials unless and until they can be shown to be safe. |