Comment submitted by Dr J. Latham

EPA-HQ-OPP-2006-0642Re: EPA proposed rule: EPA-HQ-OPP-2006-0642 Date: Wednesday April 18, 2007 EPA is proposing the exemption of certain Plant viral coat protein genes under the Federal Insecticide, Fungicide, and Rodenticide Act. These include wild-type viral coat proteins found in the USA. I appreciate the opportunity to comment. I have detected a potential flaw in this proposal which I believe therefore requires amendment. This flaw is that, as proposed, the rule would allow the exemption of a Carmovirus coat proteins (family Tombusviridae) such as one of several that are known to infect plants in the USA, for example NLVCV (e.g. Robertson; 2004; Liu et al 2003). The coat protein of one carmovirus (Turnip Crinkle Virus, TCV) is known to inhibit host plant defence mechanisms by neutralising gene silencing (Thomas et al 2003; Qu and Morris 2003). Insertion of this coat protein, assuming it functions as a transgene as it does in the wild-type virus, would most likely result in the crop species into which it was inserted becoming susceptible to a wider range of plant viruses. This may have consequences for growers of the crop itself and it may also have consequences for nearby crops that are also susceptible to the same viruses since their prevalence would almost certainly increase. This hazard should be self-evident and in any case it is appropriately accepted by EPA that gene silencing inhibitors are not appropriate plant protectants (EPA-HQ-OPP-2006-0642). Further to the above concern, the presence of a protein that inhibits gene silencing in plants destined for consumption may also present a risk to the health of humans and other animals. This is because, as is well known, the pathways of gene silencing are significantly conserved between plants and animals. For example the NS1 protein of Human influenza virus inhibits gene silencing in plants (Delgadillo et al 2004). Humans and other animals rely on gene silencing as a primary defence against viral infections in general and the gut is a primary entry point for these pathogens. If they are consuming inhibitors of this defence along with their food these defences may plausibly be compromised. The fact that EPAs proposed rule is incapable of preventing this eventuality indicates that the proposed rule is insufficiently restrictive. It could perhaps be argued that an appropriate remedy to the rule would be to extend the rule to so as to exclude carmovirus coat proteins incorporated as plant protectants from the exemption. It is perhaps equally reasonable to argue that the coat proteins of some or all Tombusviridae should be restricted since it is unclear as yet whether the coat proteins of these viruses also inhibitors of gene silencing. A superior remedy however would be to recognise that the proposed rule has paid insufficient attention to the potential multifunctionality of plant viral coat proteins. Given that the additional function of the TCV coat protein mentioned above was discovered only in 2003, it is more than possible that coat proteins of other viruses have additional and as yet undiscovered functions, ones which also make them inappropriate as plant protectants. For example, as is the case with some plant replicases (Abbink et al 2002), coat proteins may inhibit other aspects of plant defences. Therefore, a superior remedy, and one which also should simultaneously prevent potential problems with heteroencapsidation and also recombination with superinfecting viruses, would be to amend the rule such as to restrict the exemption only to transgenes incapable of making coat protein molecules, either because they contain frameshift mutations or because they contain stop codons. It is of course possible to argue that coat proteins will not have the effects described here. Perhaps either because they are silenced or because inhibitors of gene silencing may not themselves be capable of being silenced. Such arguments are however far from convincing, they rely on various assumptions and are contradicted by significant pieces of evidence and (to be rigorous and watertight) any proposed rule needs to take these weaknesses into account. Thus inhibitors of gene silencing can themselves be silenced and provide resistance against homologous viruses (Savenkov and Valkonen 2002; Mlotshwa et al. 2002); and further, silenced genes can be expressed, function and transcomplement in the presence of infecting viruses (Farinelli et al. 1992; Hammond and Dienelt 1997; Mlotshwa et al. 2002). I therefore recommend that the rule be amended to prevent any possibility of coat protein expression. This course of action would in no way prevent the development of virus-resistant transgenic crops and is consistent with the recommendations of many authors who have considered this issue (Tepfer 1993; Hammond et al. 1999; Tepfer 2002; Power 2002). N. L. Robertson (2004) Biology of a new virus isolated from Lupinus nootkatensis plants in Alaska. Plant Pathology 53 (5), 569?576. First report of Calibrachoa mottle virus infecting petunia. Liu, H. Y., Sears, J. L., Bandla, M., Harness, A. M., Kulemeka, B. Plant Disease, 2003 (Vol. 87) (No. 12) 1538 Thomas, C. L., Leh, V., Lederer, C. and Maule, A. J. (2003). Turnip crinkle virus coat protein mediates suppression of RNA silencing in Nicotiana benthamiana. Virology 306(1): 33-41. Qu, F. and Morris, T. J. (2002). Efficient infection of Nicotiana benthamiana by Tomato bushy stunt virus is facilitated by the coat protein and maintained by p19 through suppression of gene silencing. Mol Plant Microbe Interact 15(3): 193-202. Delgadillo et al (2004) J Gen. Virol. 85 993-999 Abbink et al (2002) Silencing of a gene encoding a protein component of the oxygen-evolving complex of photosystem II enhances virus replication in plants. Virology 295: 307-319 Savenkov and Valkonen (2002) J Gen. Virol. 83: 2325-2335 Mlotshwa, S., Verver, J., Sithole-Niang, I., Prins, M., van Kammen, A. and Wellink, J. (2002). Transgenic plants expressing HC-pro show enhanced virus sensitivity while silencing of the transgene results in resistance. Virus Genes 25: 45-57. Farinelli, L., Malnoe, P. and Collet, G. F. (1992). Heterologous encapsidation of potato virus Y strain O (PVYo) with the transgenic coa protein of PVY strain N (PVYN) in Solanum tuberosum CV. Bintje. Biotechnology 10: 1020-1025. Hammond, J., Lecoq, H. and Raccah, B. (1999). Epidemiological risks from mixed infections and transgenic plants expressing viral genes. Adv Virus Res 54: 189-314. Tepfer, M. (1993). Viral genes and transgenic plants. Biotechnology 11:1125-1132. Tepfer, M. (2002). Risk assessment of virus-resistant transgenic plants. Ann. Rev. Phytopathol. 40: 467-91. Power, A. G. (2002). Ecological risks of transgenic virus-resistant crops. in Genetically engineered organisms: assessing environmental and health effects. D. K. a. B. Letourneau, B.E., CRC press.