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
Dr. Mae-Wan Ho and Angela Ryan -Institute of Science in Society and Biology Department, Open University, UK
Prof. Joe Cummins -Department of Plant Sciences, University of Western Ontario, Canada
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.
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).
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.
The same experiment carried out in Nicotiana bigelovii (5) gave infectious recombinants that expanded the host range of the virus.
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).
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).
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.
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.
Article first published 10/10/00
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