Comment submitted by Pamela Weathers, Society for In Vitro Biology Public Policy Committee

Society for In Vitro Biology Public Policy Committee Comments on EPA?s two proposed rule changes for the exemption of tolerance for virus coat proteins expressed in plants as part of a Plant-Incorporated Protectant (PIP) EPA-HQ-OPP-2006-0642 Public Comment on EPA?s proposed rule changes for the exemption of tolerance for virus coat proteins expressed in plants as part of a Plant-Incorporated Protectant (PIP). The Society for In Vitro Biology (SIVB) Public Policy Committee has read the proposed changes to 40 CFR Part 174, to exempt under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) certain plant incorporated protectants derived from plant viral coat protein genes (PVCP-PIPs) and to exempt from the requirement of a tolerance under the Federal Food, Drug and Cosmetic Act (FFDCA) certain residues of viral coat proteins expressed in plants as part of a Plant-Incorporated Protectant (PVC-Proteins). While the SIVB Public Policy Committee applauds and agrees with the concept of the exemption in principle, there are specific elements of the proposed changes that we think need reconsideration and revision. First, we believe the EPA should not limit the scope to only viral coat protein genes, but to all plant viral genes. It is important to note that not all plant viruses have coat proteins? some lack coats altogether; while others have envelopes, so geneticists need to have several tools at their disposal to achieve virus resistance. Furthermore, even when a plant virus has a coat protein, it is now possible to use DNA that codes for proteins other than coat proteins in order to create transgenic virus-resistant crops. Expressing a viral coat protein, therefore, represents only one approach of several that may be useful. It is thus incumbent on EPA to be able to exempt all plant viral sequences from FIFRA. The three issues of concern for EPA and its analysis of these issues in the present proposed rule apply equally well to other viral sequences, not just sequences encoding coat proteins. EPA cites 3 areas of potential concern: a) potential for increased weediness; b) potential for novel virus creation and c) potential for human toxicity. In no case was the EPA able to cite a real-world example (i.e., outside of very controlled laboratory or greenhouse conditions) where a problem has been manifest, so all these problems remain in the theoretical, rather than in the probable realm of possibilities. Furthermore, after years of experience with transgenic squash and papaya, none of EPA?s concerns have materialized. Therefore, EPA should take into consideration the experience of crops already in the field, which thus far, under real-world conditions, are manifestly low-risk. This experience has validated the risk assessments already completed by USDA/APHIS as part of that agency?s deregulation process, and therefore supports EPA?s proposal to exempt papaya under ? 174.27(a)(1) and also supports the use of option 4 as a valid approach for ? 174.27(a)(2) and the inclusion of squash as an exempt species under that option, if EPA retains its current proposal for exemption under ? 174.27. However, as EPA recognizes, likelihood is one of the components of a risk assessment, and in both proposed rules fails to identify any situations that have high likelihood and high hazard resulting from the incorporation of PVCP-PIP?s, or any other plant viral sequences for that matter. The SIVB Public Policy Committee argues that the current state of knowledge about PBCP-PIP?s and other plant viral sequences, does not warrant their treatment other than as low risk, and therefore can be categorically exempted by EPA from regulatory requirements under FIFRA and FFDCA. Potential for Increased Weediness (pertinent mainly to FIRFA) On the issue of weediness, the SIVB Public Policy Committee disagrees with some of the EPAs assumptions. While there is no doubt at all that many crops can hybridize with their wild relatives, there are many additional steps between hybridization and introgression. Hybridization does not, inexorably, lead to introgression and finally, to any negative impact resulting from it. Introgression requires survival of the F1 plants, the survival of hybrids between the F1 and the wild relatives, and survival and crossing of the progeny for a few more generations. There are precious few examples of bona-fide introgressions, and, in fact, the EPA complains about the lack of data on introgression. The reason for this lack of data is straightforward: introgression, to the extent it happens, has not created any noticeable problems, or at least problems worth studying. Indeed, over the years, extensive studies of the potential impacts of other transgenic traits into wild relatives?in this case sunflowers (see e.g. Arias and Rieseberg. 1994 ; Whitton et al. 1997 ; Linder et al. 1998; Snow et al. 1998 ; Rieseberg et al. 1999; Pilson, 2000 ; Pilson and Decker. 2002: Burke and Rieseberg. 2003; Snow et al. 2003), including disease resistance transgenes, have not demonstrated that introgression of traits, while potentially enhancing fitness, would enhance weediness of wild or feral populations receiving the transgene. Transgenes, viral coat protein or otherwise, do not exist independently in the plant?s genome. They are integrated into a chromosome, and thus physically linked to many other genes. In a crop plant, these genes have been selected to enhance the plant?s performance under cultivation, i.e., they condition a domestication syndrome (e.g., lack of dormancy, lack of thorns, lack of bitterness, lack of shattering, etc). Domestication typically leaves a plant unadapted to survival in the wild; any of these traits can counteract any beneficial impact a transgene may have. Undoubtedly, there is a chance a resistance transgene might get introgressed into a wild population; however, the probability is likely similar to a resistance gene arising spontaneously in the wild population. As we have gotten to know the nature of plant resistance alleles, we now realize they can and do rearrange themselves rather frequently to produce new alleles. Plant populations, after all, are genetically dynamic. Novel Virus Creation With respect to novel virus creation, the EPA recaps at great length its initial assessment of why it originally decided viral interactions were of negligible consequence. Years of experience with transgenic crops in the field have proven EPA?s initial assessment to be accurate. However, in a stance that is contrary to its original assessment and the accumulated data, the EPA contrives a precautionary, non-scientific reason as to why it should not exempt several construct designs that confer viral resistance. The proposed rules are based on recommendations from the 2004 and 2005 SAPs. An enormous amount of information on resistance mechanisms has come to light since then, and the SAP comments are simply outdated based on the biological discoveries of the past several years. We now know that transgenes confer viral resistance via RNA interference (RNAi), a process whereby mRNA gets degraded in the cytoplasm. Thus, the absence of mRNA in the cytoplasm will, in fact, decrease the chances of viral recombinations or transencapsidations. Likewise, the docket expresses concerns that constitutive promoters can increase the amount of mRNA available in the cytoplasm. In fact, quite the opposite happens. High levels of a given mRNA in the cytoplasm stimulate an RNA-dependent RNA polymerase that creates double stranded RNA, which is the trigger for the RNAi mechanism. Alternatively, a construct designed to produce double stranded RNA via antisense RNA will trigger the RNAi mechanism, again, destroying mRNA in the cytoplasm. It is precisely because of the existence of RNAi that the EPA proposal to only exempt events whereby ?the genetic material that encodes the pesticidal substance is inserted only in an inverted repeat....? is misguided and counterproductive. Antisense constructs and sense constructs expressed at high levels can be very effective at triggering RNA silencing and should not be precluded a priori. The exemption, therefore, needs to be targeted to any construct capable of producing silencing RNAs, without dictating the need for inverted repeats, lack of ATG codon, etc. Based on the extensive history of safe use, there is no reason to exclude plants producing viral coat proteins from exemption. In addition, the proposed exclusions to the exemption, outlined below, would also be counterproductive and as they would preclude the ability to use RNAi, the latest, most effective, and safest technology available. 1. Reducing the extent of shared sequence similarity between the infecting virus and the transgene to reduce the opportunities for homologous recombination. This is ill-advised because homology is needed to trigger RNA silencing; furthermore, once silencing is triggered, there are no RNA molecules left to engage in recombination. In fact, this recommendation will have the opposite of the desired effect? it will make silencing less likely, and therefore increase the possibility of heterologous recombination. 2. Excluding any sequences containing replicase recognition sites that are potential sites and any sequences known or thought to be recombinatorial hotspots In the absence of mRNA in the cytoplasm, these recommendations become moot. 3. Avoiding potential hairpin structures in the transgene. Again, this is an absolutely counterproductive recommendation, as the most effective silencing constructs are designed to create a hairpin between inverted repeats. As mentioned previously, the most effective silencing strategies are those which eliminate all viral mRNA from the cytoplasm. Not only is this the most effective in terms of resistance, it is also the most effective strategy if the goal is to eliminate any chance of recombination between viruses. Potential for Human Toxicity - Production of Proteins As is the case with the section on viral interactions, the EPA?s initial assessment on the safety of viral proteins was correct, as evidenced by the history of safe use which the technology has accrued in transgenic fruits and vegetables, and by the safe consumption of an almost infinite array of naturally occurring plant viral proteins in the human diet. There is no scientific reason why the EPA cannot exempt all plant viral proteins. Furthermore, the recommendations EPA is making here are, again, counterproductive, and are guaranteed to make the technology less effective and less safe, rather than safer. The recommendations are also contradictory and, as such, require revision. First, the EPA recommends that constructs rely only on ?inverted repeats.? Yet, this strategy is only effective with a hairpin between the repeats. As we have already pointed out, however, the previous section advises against hairpins. Second, the EPA also demands that the viral sequence must be ?virtually unmodified.? As defined, virtual modification precludes truncations. However, for the construction of effective silencing constructs, is neither necessary nor desirable to use the whole coding sequence? 200 to 400 bp are sufficient and effective. Last, the docket goes on to call for allergenicity testing of viral coat proteins, particularly if there has been an insertion or deletion. The EPA acknowledges that there is no evidence whatsoever that viral proteins or their derivatives are allergenic, and in fact, the recommendation for allergenicity testing is contrary to the weight of all available scientific evidence. Current allergenicity evaluation paradigms use criteria that have been well validated, and are, furthermore, independent of whether a gene has a deletion or not. The SIVB Public Policy Committee recommends that EPA extends the exemption to all plant viral proteins. Inert Ingredients that may be used in PIPs The SIVB Public Policy Committee endorses the recommendations as outlined by EPA. We applaud the addition of tratracycline resistance to the list. We would also recommend the addition of the hph gene (hygromycin phosphotransferase) for hygromycin resistance, and mutant, resistant versions of the AHAS gene (acetohydroxy acid synthase) for resistance to imidazalinone herbicides. Respectfully submitted, Public Policy Committee of the Society for In Vitro Biology Pamela J. Weathers, Ph.D., Chair Todd Jones, Ph.D., Committee Member June Bradlaw, Ph.D., Committee Member Wayne Parrot, Ph.D., ad hoc Committee member References Arias, D.M. and L.H. Rieseberg. 1994. Gene flow between cultivated and wild sunflower. Theoretical and Applied Genetics 89:655-660. Whitton, J.D. et al. 1997. The persistence of cultivar alleles in wild populations of sunflowers five generations after hybridization. Theoretical and Applied Genetics 95:33-40. Linder, C.R., et al. 1998. Long-term introgression of crop genes into wild sunflower populations. Theoretical and Applied Genetics 96(March):339-347. Snow A.A. et al. 1998. Fecundity, phenology, and seed of F1 wild-crop hybrids in sunflower (Helianthus annuus, Asteraceae). American Journal of Botany 85:794- 801. Rieseberg, L.H. et al. 1999. Introgression between cultivated sunflowers and a sympatric wild relative, Helianthus petiolaris (Asteraceae). International Journal of Plant Science 160:102-108. Pilson, D. 2000. Herbivory and natural selection on flowering phenology in wild sunflower, Helianthus annuus. Oecologia 122(1):72-82. Pilson, D. and K.L. Decker. 2002. Compensation for herbivory in wild sunflower: response to simulated damage by the head-clipping weevil. Ecology 83:3097-3107. Burke, J.M., and L.H. Rieseberg. 2003. Fitness effects of transgenic disease resistance in sunflowers. Science 300(May 23):1250. Snow, A.A., et al. 2003. A Bt transgene reduces herbivory and enhances fecundity in wild sunflowers. Ecological Applications 13(April):279-286.