November, 2002

From: ISB News Report* November 2002
Emerging Biotechnologies: Upgrading the Terminator
by Zac Hanley and Kieran Elborough

Terminator technology has been a lightning rod for debate and confrontation on the issue of genetic modification. Most of the discussion, and all of the hypothesising over potential consequences, has taken place while Terminator and similar technologies were largely theoretical concepts. While it is proper to discuss the ethical ramifications at the early stages, in this case it has often led to the technical advances being relegated to the introduction or the appendix. We have previously described the benefits, risks, impacts, and opportunities of Terminator (more properly known as Genetic Use Restriction Technologies, or GURTs1). Now, here is the appendix.

Traitors and Terminators

Two types of GURTs have been described.2 The first GURT patent (USP# 5,723,765 - see footnote 3) outlined the concept later described as Variety-restriction GURT, or V-GURT. This type is the emotively named `Terminator', as it causes the production of sterile seeds. The reproductive viability of the plant is under the proprietary control of the owning company, ensuring that viable seed is not available for the farmer to harvest. This is achieved via triggering a disrupter gene prior to the sale of seed, which has a delayed effect, rendering the next generation non-viable. In `Terminator', this triggering is by commission, that is, treatment prior to seed sale with an activating stimulus. In the technology dubbed `Traitor' by critics, this is by omission; a suppressing stimulus is withdrawn and the disrupter is then able to act.

The concept of a V-GURT is not tied to any particular disrupter gene; there are many possibilities. Examples are genes encoding proteins which break down essential cellular components such as RNA or the cell wall; or which activate the apoptosis-like programmed cell death pathway in plants; or which perturb fundamental aspects of metabolism such as osmolyte balances or proton gradients. The aim is to disrupt the creation of the next generation or to render that generation incompetent to grow and
survive. This is where real ingenuity is required. It is vital to arrange the genetic elements so that the disruptor has its effect in a timely manner.

The essential component is the promoter, which must respond to an exogenous compound or some triggering stimulus. One interesting example is the oestrogen receptor transactivation system described by Zuo and co-workers.4 Their paper discusses the application of this system to marker excision; marker excision systems are but one application of GURTs.5 In the oestrogen receptor transactivation system, application of ß-oestradiol causes the promoter to express strongly; this promoter could be used to drive a suitable disruptor gene. Without ß-oestradiol, expression is extremely low and disruption does not occur. However there are several obstacles to the successful use of exogenous chemical regulators in GURTs. Firstly, the regulatable promoter must remain physically linked in the genome to the disrupter gene, else they may on rare occasions separate during the development of the next generation. This would lead to viable and fertile trait- and modification-bearing offspring. Secondly, it is not clear how a chemical treatment can be 100% effective, as some cells or even whole seeds may not be penetrated.

Trait Transfer Termination

The second GURT concept is the Trait-restriction GURT, or T-GURT, where it is the inheritance of the elite trait differentiating the plant from other germplasm that is under control, rather than the plant's reproductive ability. T-GURTs can be described as less crude than V-GURTs since their effects are localized to one area of the genome, and such finesse is increasingly desirable given public concerns over the process of germplasm enhancement. Control over the inheritance of a trait may occur via regulating the expression of genes conferring the trait, or ensuring the disruption or loss of these genes in the next generation (as deployed in marker excision systems such as described in footnote 4).

To achieve such relative finesse, a different range of disrupter genes is required. Genes encoding essential components of flowering or embryogenesis are suitable leverage points, and the inducible promoter here must drive the expression of an antisense construct. Alternatively, T-GURTs can employ marker excision-type mechanisms to remove the elite trait and dispense with disrupter genes altogether. This may be an important consideration if the plant is part of the human food chain. The site of action for T-GURTs is the reproductive tissues of the first generation, so tissue- or developmental stage-specific promoters are used. The triggering may be by omission or commission, as for V-GURTs (above).

The next generation of inducible promoters useful for GURTs are likely to respond to more complex, multi-factorial signals involving environmental conditions, such as those which initiate flowering in overwintering plants. Or they may be switched on and off during certain developmental stages with great precision. Control via such triggers would circumvent some of the problems inherent in activation via exogenous chemicals. Early studies on such promoters can be found in the literature.6

The Third Type?

Tobacco transgene containment has been demonstrated by Kuvshinov and coworkers,7 devisors of the `Recoverable Block of Function' (RBF) GURT concept. They attempt to differentiate it from previous GURTs, although it is conceptually identical to mechanisms described by the Food and Agriculture Organization working party.2 RBF can be used in T-GURTs or V-GURTs, depending on the DNA constructs and target genes chosen, and is applicable to vegetatively propagated plants as well as seed. Kuvshinov and co-workers give a V-GURT example in their publication.

The RBF is a gene construct that includes a disrupter gene (the `blocker' of reproductive `function') and a rescue gene, which disrupts the disrupter and is under the control of an inducible promoter. In Kuvshinov et al. (2001), the BARNASE protein is expressed during embryogenesis and destroys most cellular RNA at this critical developmental stage, leading to the production of sterile seed. The barstar gene is also present in the same plant and can be triggered by a particular treatment. The authors
used a heat-shock-specific promoter. On production of the BARSTAR protein, BARNASE is inhibited and viable seed are formed. However, barnase is reset to be expressed during the next embryogenesis, so the block is `recovered' and the heat treatment would be required in the next generation, if it is not to be the last. A different `blocker' and rescuer combination could control a T-GURT; for example, an antisense construct could `block' the trait-conferring gene and be rescued by a gene, which specifically targets the promoter of the `blocker'.

Trends for Tomorrow

Today's GURTs have been criticized on a number of fronts, and most commentators have been concerned over their economic, social, and environmental impacts. These were addressed in our previous article.1 But it is also important to say that ongoing research and development of new and improved GURTs continues, is exciting, and suggests remedies for many of the technical objections. However, GURTs do not have quite the necessary specifications and mostly remain interesting concepts or elegant creatures residing in the protective environment of the laboratory or glasshouse. This is the best reason for continuing research, not inhibiting it. Widespread application in different species and extensive field testing are required. Government funding should be placed into programs developing GURTs. Publication in peer-reviewed journals of new developments, as with the RBF concept, is to be encouraged and should be recognized by proponents and critics as laudable. As an aside, the current public mistrust over patent protection often extends to the misunderstanding that patents are secrets; the current revolution in public access to patent databases (as in footnote 3) should help the public image of companies who wish to protect their investment by publishing their discoveries at the patent offices. At the toolkit level, more and different promoters are needed for T- and V-GURT systems, and there are many research groups in academic institutions that could apply their discoveries here. Lastly, a demonstration of the RBF system in a T-GURT application would be most helpful.

Taking insurance and mitigating risk through `belt and braces' approaches are the best practises where public perceptions of safety, however distorted for political ends, are a factor. For this reason, further investment and research into GURTs are needed but emphatically do not imply that genetic modification is dangerous. GURTs developed as described herein are one component of several in a portfolio of technologies that can and should be employed (others include apomixis, transplastomics, male sterility, and enforced cleistogamy). Such technologies could be shared between companies. Some customers seek assurances; GURTs and the biotechnology industry are well on the way to providing them.

References

1. See for example our article "Re-emerging Biotechnologies: Rehabilitating the Terminator," in ISB News Report, June 2002. http://www.isb.vt.edu/news/2002/news02.jun.html#jun0203. 

2. Most articles acknowledge the report of the FAO Working Group on Plant Genetic Resources for Food and Agriculture available online at http://www.fao.org/waicent/FaoInfo/Agricult/AGP/AGPS/pgr/itwg/pdf/P1W7E.pdf. 

3. US Patent No. 5,723,765 entitled `Control of Plant Gene Expression' granted to Delta and Pine Land Corporation and the USDA. Search at http://patents.uspto.gov for the full text.

4. The marker excision application of this system can be found in: Zuo J, Niu Q-W, Moller SG, Chua N-H. 2001. Chemical-regulated, site-specific DNA excision in transgenic plants. Nature Biotechnology 19(2): 157-61.

5. A recent review: Hare PD and Chua N-H. 2002. Excision of selectable marker genes from transgenic plants. Nature Biotechnology 20(6): 575-580.

6. Day-length-responsive promoter elements: Kiyosue T and Wada M. 2000. LKP1 (LOV kelch protein 1): a factor involved in the regulation of flowering time in Arabidopsis. Plant Journal 23(6): 807-15. Tissue-specific promoter: Rossak, Smith, and Kunst. 2001. Expression of the FAE1 gene and FAE1 promoter activity in developing seeds of Arabidopsis thaliana. Plant Molecular Biology 4(6): 717-25.

7. Kuvshinov V, Koivu K, Kanerva A, and Pehu E. 2001. Molecular control of transgene escape from genetically modified plants. Plant Science 160: 517-22.

Zac Hanley and Kieran Elborough
Consultants in Plant Biotechnology
New Zealand
biotech@greengenz.com

* Information Systems for Biotechnology (ISB) was established in 1988 as part of the National Biological Impact Assessment Program (NBIAP), a program administered by USDA's Cooperative State Research, Education, and Extension Service (CSREES). ISB is funded on an annual basis through a grant to the Agricultural Experiment Station at Virginia Tech.
ISB is funded through a grant from USDA's Cooperative State Research, Education, and Extension Service to Virginia Tech in Blacksburg, Virginia.