On August 5, 1997, APHIS held a public meeting to discuss issues related to the use of transgenes from plant viruses in the development of genetically engineered plants.
1:00 Stacking of resistance genes
Also, make recommendations on any data that should be submitted by applicants to address any of the issues raised during the discussion.
Additions and modifications to each issue will be allowed within the defined scope and time constraints.
This summary does not comprise a comprehensive record of all the ideas discussed during the meeting. Rather, it attempts to document key points made during the discussion. These points were written and agreed upon by the scientists invited by APHIS: M. Zaitlin, B. Falk, G. De Zoeten, R. Allison, D. Rochon, J. Culver, and L. Hanley-Bowdoin; J. White, R. Dobert, and D. Heron from USDA, APHIS; and S. Charlton from the Canadian Food Inspection Agency. list of participants).
The meeting opened with a discussion of strategies for designing transgenes derived from plant viruses that might reduce the likelihood of a recombination event. The proposed recommendations were discussed and modified as shown below. It was noted that the time frame for designing a transgene through commercialization of a plant expressing that gene was at least seven years and that an effective resistance strategy may work in one plant and not in another.
ISSUE I. Although the perceived risk of producing a recombinant virus is considered to be very low, when designing a transgene from a viral gene, scientists should consider the following recommendations (the original recommendations are listed in next document):
There was significant discussion on one of the proposed strategy (item two above), i.e., "The transgene should not be expressed in cells that the virus gene is not replicated in." Comments ranged from, "This statement is inane" to "Current research suggests that virus taxa (luteoviruses and geminiviruses) that previously were thought to replicate only in the phloem and associated cells (phloem limited), may replicate to some degree in mesophyll cells (phloem enhanced)."
ISSUE II. USE OF FULL LENGTH REPLICASE AS A TRANSGENE and TRANSGENE EXPRESSION IN CELLS WHERE THE VIRUS DOES NOT REPLICATE.
This discussion focused on using luteoviruses and their replicases as model system. Luteoviruses are thought to replicate only in the phloem.
Question. Satellite RNAs and ST9a RNA have been identified with certain luteoviruses. Could satellite RNAs be replicated by replicase transgene? What potential impacts could be envisioned?
ST9a RNA is an RNA associated with beet western yellows luteovirus (BWYV). This RNA enhances the replication of BWYV and causes more severe symptoms in infected plants. ST9a RNA encodes its own replicase and can replicate independently in protoplasts. It depends on BWYV for its coat and movement proteins. With respect to luteoviral replicase expressing plant and ST9a RNA several points were made:
Question: Could host cell RNAs be transcribed by the replicase transgene? Is there evidence that this could occur? What potential impacts could be envisioned?
One of the quintessential characteristics of viral replicases is their specificity toward the RNA that they replicate. Even if there was amplification of a host RNA, there is no evidence that it would result in any visible symptoms, that it could move from cell-to-cell, or that it could move from plant to plant.
Question: All plants have an endogenous RNA-dependent RNA polymerase. What is likelihood that this enzyme could make the complementary minus strand of replicase transgene? If the minus strand is produced, then the sequences that encode subgenomic promoter sequences would be produced. Allen Miller has suggested that these sequences have been important in recombinational events between subgroup I and II luteoviruses.
Based on the studies of this plant enzyme and viral replicases, there is no evidence to support a hypothesis that the complementary minus strand of the transgene mRNA could be synthesized by the host plant RNA polymerase.
Question: Assume that replicase transgene is expressed in cells where the virus does not replicate. Compared to natural infections, the number of cells that contain replicase sequences has increased. In your opinion does this increase the likelihood of a recombination occurring? Of recombinant virus occurring?
This topic generated the most discussion (both here and when Item 2 of Issue 1 was discussed). It is generally accepted that the amount of viral RNA (either viral or viral-derived transgene) is a factor in recombination. However, it is not clear on how one compares the amounts of viral-derived transgene and challenge virus RNA to the amount of RNA in cells when two viruses naturally infect the same host. As stated in number 4 in Issue 1, it is prudent to reduce the amount of transgene when possible to the lowest level that provides resistance. It was noted that generally infected plants have higher levels of RNA than transgenic plants, however, whether these levels affect recombination rates are not known.
However, it is clear that unrelated viruses do replicate in the same cell at the same time. Examples, potato (potex)virus X (PVX) and potato (poty)virus Y (PVY) (and the many other potyviral synergisms) where a potyviral gene can increase the amount of PVX RNA synthesized. Special note was made that many tobacco necrosis necrovirus (TNV) strains fail to move systemically in naturally infected hosts. These viral strains co-infect plants with several viruses that have functional movement proteins and a recombinant TNV strain that systemically infects has not been observed.
Several points were mentioned regarding recombination and risk. First, there is the possibility that recombination between a transgene and virus could occur but the recombinant virus would not be viable or that the recombinant virus was viable but not competitive with the wild type virus. Second, although much of the discussion has focused on the risk of recombination/recombinant virus, there is no persuasive evidence that recombination or recombinant viruses are definitively a risk.
In concluding this discussion, the participants were polled for their opinion to above listed questions. With respect to both questions, the majority of attendees chose to abstain or answered, "I don't know." A slim majority stated that this scenario would raise the rate of recombination, however, no one stated that they believed this raised the likelihood of a viable recombinant virus arising.
ISSUE III. STACKING OF RESISTANCE TRANSGENES FROM A SINGLE VIRUS
The combining of two or more resistance genes against a single pathogen in a single plant is called stacking. Plant pathologists generally believe that stacking of resistance genes delays the selection of resistance-breaking strains because two mutations must occur in the pathogen to overcome the two resistance genes. Since several different viral genes have been effective as sources of transgene resistance, the stacking of two or more viral derived transgenes is strategy that is likely to be developed. No specific issues were raised as a result of potential stacking of viral genes based on the following caveats. First, that in the designing of transgenes the strategies discussed above (Issue I) be given serious consideration. Second, that little has been published on transgenic plants with stacked viral derived transgenes with respect to their effectiveness in the field and whether this stacked resistance approach results in immunity phenotype rather than a resistant phenotype. (Some commercialized transgenic virus resistant plants do allow limited amount of virus replication. Transgenes may also be stacked in a plant using traditionally bred resistance or tolerance genes. The stacking of transgene with a tolerance gene may allow the use of the latter gene which when used singly was not sufficient to provide control.
With respect to specifically addressed questions (see next document) on approaches to stacked genes, no specific recommendations were stated. With respect to the designing of defective viral movement proteins as resistance strategy, deletion mutants would be much harder to restore to functionality than point mutations.
ISSUE IV. GEMINIVIRUSES
Most of the public discussions regarding the risk of using transgenes derived from plant viruses have been focused on RNA viruses. Single stranded DNA viruses, the geminiviruses, are a group of viruses that can have devastating impact on plant production. Several small scale field tests of geminivirus resistant plants have taken place in the U.S. This discussion opened by a presentation by Virginia Ursen, from Calgene, who gave a brief overview of the genome and replication strategy of the three geminivirus groups and discussed what is known about pseudorecombinants/recombinants. The discussion in this session was limited possibly because only three of the attending virologists work on geminivirus. However, it was clear that the genetic approaches to engineer geminivirus resistance into plants will likely be quite different from the approaches used for RNA viruses. First, an effective resistance strategy, comparable to coat protein mediated transgenic resistance in RNA viruses, has not been effective under field conditions. Second, the replication of DNA viruses are more dependent than are RNA viruses on factors that are involved host DNA synthesis. Thus, expression of geminivirus-derived transgene in plants may affect host DNA synthesis. Although no specific issues were identified, it was apparent that issues arising from the use of geminiviruses genes as sources of resistance are likely to be different from those described for RNA viruses.
Attendees often mentioned unpublished data on certain topics. Although this information was discussed, we have not included this information.
List of Participants
Gus de Zoeten
Last Modified: October 23, 2007