TRANSGENIC POTATOES RESISTANT TO COLEOPTERAN AND LEPIDOPTERAN PESTS THROUGH THE USE OF SINGLE HYBRID BACILLUS THURINGIENSIS CRY GENE
Ruud A. de Maagd and Samir Naimov

Bacillus thuringiensis (Bt) is a bacterium commonly found in soil and on plant surfaces. What makes the bacterium interesting for agricultural research is that many of its strains kill insect pests. It does so primarily because during the formation of its spores, which help the bacterium to survive adverse conditions, it forms one or more insecticidal proteins enclosed in a crystal. The crystal (Cry) proteins, when ingested by insect larvae, dissolve in the insect's midgut fluid, undergo a process of activation by gut enzymes, and subsequently bind to receptors on the cells lining the gut. This is followed by the formation of pores in the membranes of those cells, killing them, which eventually leads to the death of the insect¹. Sprays based on Bt crystals and spores have a long history of safe use in agriculture. More recently, transgenic crops such as corn, cotton, and potato expressing a Cry protein (Bt crops), rendering them resistant to one or several pests, have been commercialized.

One of the big advantages of Cry proteins is sometimes also a drawback: specificity. Cry proteins form a large family of proteins, similar in overall structure, but differing in details that determine their activity for particular insect species. Each Cry protein is active against only one or a few pest species, usually of the same insect order (Lepidoptera: caterpillars; Diptera: larvae of flies and mosquitoes; Coleoptera: larvae of beetles and weevils). While this helps to reduce non-target effects of sprays or Bt crops, it limits the utility of such an application. Additionally, the use of a single yet effective toxin in a transgenic crop leads to a high selection pressure on the insect population that, it is feared, may then rapidly develop resistance to the toxin. Although this resistance has not yet been seen in the field since the introduction of Bt crops, strategies for delaying or preventing the occurrence of resistance (resistance management) are deemed necessary. One of these strategies is the simultaneous expression of two toxins that recognize different receptors, so that the insect population would have to lose two, not one, receptors to become resistant to both toxins. In our laboratories, we are studying the mode of action of Cry proteins and the factors that determine specificity, and applying this knowledge to the production of toxins that are more effective or have a broader spectrum, or may be used for resistance management strategies.

Essential for the understanding of specificity is the relation between structure and function of the Cry proteins. The Cry proteins used in our study probably all have the same overall three-dimensional structure shown in Fig. 1.

Figure 1. Schematical representation of the three-dimensional structure of a typical Bacillus thuringiensis Cry protein, showing its three structural domains (I-III).

The proteins consist of three structural domains, of which the first domain is thought to be responsible for forming the pores in insect cell membranes. The second and third domains function in receptor recognition and are thereby in a large part responsible for determining specificity. How these two domains act together in receptor recognition is not well understood, but several groups, including ourselves, recognize that "swapping" single domains between toxins having different activity or specificity may result in "hybrid" or "chimeric" toxins with improved activity or target spectrum. As screening of Bt strains for new Cry proteins proceeds and different toxins are found, it is becoming increasingly clear that recombination between cry genes resulting in a domain swap is in fact likely to be one of the mechanisms that generated the diversity of toxins in Nature. In the laboratory, we are mimicking this process by inducing recombination between genes of our choice and screening for hybrid toxins with interesting properties.

In more recent research, we focused our attention on toxins that are active against Colorado potato beetle (CPB), a coleopteran pest of potatoes in both North America and (particularly Eastern) Europe. The most active known Cry protein for CPB is Cry3Aa, which has been utilized against this pest in sprays as well as in transgenic plants (e.g., NewLeaf potatoes from Monsanto Company). Two proteins of the Cry1 subfamily, Cry1Ba and Cry1Ia, which are relatively unrelated to Cry3Aa, are primarily active against Lepidopterans and have been shown to have some, albeit low, activity against CPB (see Fig. 2).

Figure 2. Domain compositions and activity against neonate CPB larvae of wild type and hybrid toxins, with Cry3Aa shown for comparison. The three structural domains of the active toxin as shown in Fig. 1 are depicted in color. The gray areas are parts of the so-called protoxin but are removed during activation. LC50 is the concentration (µg per ml of leaf dipping solution) giving 50% mortality. Relative toxicity is on per mol basis, with Cry3Aa activity set at 100.

To test our hypothesis that a combination of domains from two weakly active toxins, in this case Cry1Ba and Cry1Ia, may form a more active toxin, we swapped restriction enzyme fragments of the two genes corresponding to the parts encoding domain III of the active toxins². Toxins were expressed in Escherichia coli, which is easier to handle in the laboratory. Typical of such experiments, some of the hybrid genes did not express much protein or gave proteins that were unstable, yet some could be purified in sufficient amounts for bioassays on CPB larvae using potato leaves dipped in toxin solution. We determined the concentrations of Cry3Aa, the parental Cry1 toxins, and the hybrid toxins giving 50% mortality (LC₅₀) in newly hatched CPB larvae. One of the hybrid proteins, SN15, consisting of domains I and II of Cry1Ia and domain III of Cry1Ba, had considerably higher activity (lower LC₅₀) than both parent toxins (Fig. 2). SN15 was not expressed very efficiently, suggesting that its stability is not optimal. In an attempt to improve this, we replaced the first domain of SN15 with that of Cry1Ba as well. The resulting mosaic toxin SN19 not only was expressed at higher levels, but also had even higher activity against CPB than SN15. On a molar basis, SN19 activity against CPB larvae is near (42%) to that of the most active known toxin, Cry3Aa. This result showed that domain swapping might improve toxin activity, not only for lepidopterans as shown before, but also for coleopterans like CPB.

To test the actual utility of SN19 for CPB control, we transferred the encoding gene back to Bt cells as well as used it for transformation of potato plants. Results of the latter experiments were reported earlier this year³. Cry genes, of obviously bacterial origin, are not expressed very well in transgenic plants without extensive modifications both in the transcription regulation sequences (promoter and terminator) as well as in the protein coding sequence. Starting from a synthetic, plant-optimized cry1Ba gene, we replaced the domain II encoding region with that of Cry1Ia, either with or without extra modification for expression in plants. For transcription signals we used a chrysanthemum Rubisco small subunit-promoter and terminator isolated in our laboratory, which had been shown previously to give high expression levels in green tissues of various plant species⁴. Potato cultivar Desiree plants were transformed with one of the two gene constructs, or an empty vector as a negative control, via Agrobacterium tumefaciens-mediated gene transfer. Not surprisingly, without modification of the domain II encoding part, there was no detectable expression of SN19 in transgenic plants. With the modifications, several transgenic plants with expression levels up to 0.25% of total soluble leaf proteins were obtained. Detached leaves of transgenic plants were infested with 10 neonate CPB larvae to test their resistance. All plants expressing SN19 at 0.2% or higher were fully resistant to CPB larvae, giving 100% mortality and no detectable damage on the leaves. Not only CPB larvae, but also the adults are voracious pests on potato, and older larvae or adults usually require much higher doses of Bt toxins to be killed. Our transgenic SN19-potato leaves with the high expression levels were also fully resistant to attack by adult CPB; however, not all CPB adults were killed during the timeframe of the experiment. The insect simply would barely feed on the leaves, resulting in minimal damage, and not grow as a result.

As mentioned earlier, the parental toxins of SN19, Cry1Ba and Cry1Ia, are known more for their activity against lepidopterans (caterpillars, larvae of moths and butterflies) including, for both toxins, European corn borer (ECB) and potato tuber moth larvae (PTM). Although the name implies differently, ECB can be an occasional pest on potato in North America, where it damages the plant by boring into the stems. PTM is a more tropical pest, infesting and spoiling stored potato tubers as well as infesting plants in the fields where the larvae make tunnels inside the leaf tissue. We speculated that a hybrid of these proteins might well have retained these properties and tested SN19-potato and control leaves with ECB and PTM larvae. SN19 in transgenic leaves gave full protection and 100% mortality for both species. Thus, we have produced the first transgenic Bt crop with resistance to insects from two different orders, Lepidoptera and Coleoptera, conferred by a single gene.

Potato may rarely, if ever, encounter all three mentioned pests in the same field, but two out of three of these are not uncommon. SN19, with good activity against CPB as shown in our studies, may well be a good alternative for (or beside) Cry3Aa against CPB in resistance management strategies. This presumption, however, awaits further experimental confirmation that SN19 and Cry3Aa recognize different receptors in this insect. Similarly, SN19 could be an alternative for Cry1Ab in corn, currently in use for ECB control. The use of SN19 in a Bt spray, preferably with other active toxins like Cry1Ab and Cry3Aa, could yield a product with a broad activity spectrum as well as built-in resistance management. We are currently testing the utility of SN19 in Bt spore/crystal-mixtures. We conclude from this and previous work that, in the future, domain swapping may be an effective approach to increase the utility of Bt toxins in agriculture, whether in transgenic crops or in sprays.

References

1. de Maagd RA, Bravo A, and Crickmore N. (2001) How Bacillus thuringiensis has evolved specific toxins to colonize the insect world. Trends Genet 17: 193-199.

2. Naimov S, Weemen-Hendriks M, Dukiandjiev S, and de Maagd RA. (2001) Bacillus thuringiensis delta-endotoxin Cry1 hybrid proteins with increased activity against the Colorado potato beetle. Appl Environ Microbiol 67: 5328-5330.

3. Naimov S, Dukiandjiev S, and de Maagd RA. (2003) A hybrid Bacillus thuringiensis delta-endotoxin gene gives resistance against a coleopteran and a lepidopteran pest in transgenic potato. Plant Biotechnol J 1: 51-57.

4. Outchkourov NS, Peters J, de Jong J, Rademakers W, and Jongsma MA. (2003) Novel rubisco small subunit promoter from chrysanthemum (Dendranthema grandiflora L) yields very high foreign gene expression levels in plants. Planta 21: 1003-1012.

Ruud A. de Maagd Business Unit Bioscience Plant Research Intl The Netherlands ruud.demaagd@wur.nl

Samir Naimov Dept of Plant Phys and Mol Biol Univ of Plovdiv Bulgaria naimov0@pu.acad.bg

ANTIBIOSIS-TYPE INSECT RESISTANCE IN TRANSGENIC PLANTS EXPRESSING A GENE FROM A HYMENOPTERAN ENDOPARASITE
Douglas L. Dahlman, Indu B. Maiti and Bruce A. Webb

Several species of the lepidopteran genera Heliothis and Helicoverpa cause annual crop losses of over $1 billion. Thus, crop protection from insect damage is of essential and worldwide concern. Use of chemical pesticides raises environmental concerns, and their application may result in the generation of insect resistance. Therefore, it is important to develop alternative insect control measures. Limited success using native or introduced parasitoids and predators has been realized. More recently, greater success has been achieved using microbials, particularly Bacillus thuringiensis (Bt), either via application of formulated Bt, or expression of insecticidal Bt proteins in transgenic crops. Other insecticidal genes have been introduced into plants using transgenic approaches (e.g., inhibitors of digestive enzymes, resistance genes, lectin genes, and chitinases). Recently, the first development of transgenic plants expressing a parasitoid gene (TSP14) that inhibits the growth and development of pest insects has been described¹. These results are similar to early reports related to the transgenic expression of Bt toxins in plants.

Many parasitoids inhibit insect growth and immune systems. Reported causative factors include venoms, accessory gland products, polydnaviruses, and teratocytes. Teratocytes are formed when the eggs of some braconid endoparasitic wasps hatch and the cells of the serosal membrane, which surround the developing parasitoid embryo, dissociate to give rise to this unique group of cells. Teratocytes may have trophic, immunosuppressive, or secretory functions². Teratocytes circulating in the host hemolymph may secrete proteins (teratocyte secretory proteins or TSP) that produce responses similar to parasitization, including arrested insect growth and development. We isolated from Microplitis croceipes, a braconid parasitoid of Heliothis virescens (tobacco budworm), a single TSP (TSP14) that caused arrested growth and development in its host³. TSP14, produced either by expression in a baculovirus recombinant system or in yeast, inhibited protein synthesis in the rabbit reticuloctye lysate assay³ but did not inhibit protein synthesis in intact mammalian cells and insect cell lines⁴. However, in vitro translation of mammalian and insect cellular RNA was arrested⁴. It seems that TSP14 requires a specific protein receptor in order to enter into target cells. Thus, TSP14 may not be toxic to mammalian cells even though the mechanism of translation inhibition is unknown.

Microplitis croceipes teratocytes are the sole natural source of TSP14, which is encoded by the parasitoid wasp genome as a single copy gene. The expression of the TSP14 gene in transgenic plants renders antibiosis-type resistance to insect-feeding and potentially provides a novel and powerful method for controlling insect populations that cause crop damage¹.

Chimeric TSP14 genes, both with and without the signal peptide, were introduced into tobacco plants by typical Agrobacterium-mediated transformation procedures. Gene integration and expression in the transgenic plants derived from each primary transformant were assayed by PCR, real-time quantitative RT-PCR, Northern blot, and immunoblot analyses. Transgenes were stably integrated as shown by PCR analysis of 2^nd and 3^rd generation seedlings. The independent transgenic lines showed different levels of expression of TSP14 transcripts. Immunoblot analysis of plant extracts showed the presence of a polypeptide of expected size that cross-reacted with TSP14-specific antibodies. Control plants, obtained from transformed seedlings developed with pKLP36GUS⁵, were morphologically indistinguishable from the TSP14 plants (Fig. 1D).

Figure 1. Effect of H. virescens larvae on tobacco plants expressing the TSP14 gene. (A) The plant on the left is the representative control (transformed control) and on the right is the representative TSP14 line. Control plants showed extensive feeding damage. (B) Leaves on the top are from a control plant; leaves at the bottom are from a TSP14 plant. Leaves from the TSP14 plant showed less feeding damage compared to control leaves. (C) After 18 days of feeding, larvae removed from the control plants and from the TSP14 plants are shown at the top and bottom, respectively. Larvae from the control plants were on average much larger compared to those from TSP14 plants. (D) The plant on the left is a transformed control (3 months old) and on the right is a TSP14 expressing line (3 months old). Control and TSP14 plants with seed pods are morphologically indistinguishable.

Relative to controls, nine of the 28 transformed lines tested were associated with significantly reduced growth and/or development in H. virescens bioassays in one or more of the parameters measured. For example, after a 7-day feeding trial initiated with neonate larvae, the larvae feeding on TSP14 transgenic plant leaf disks were smaller and developmentally delayed relative to controls. We found lines transformed with truncated TSP14 (without the native signal peptide) to be more effective.

A pilot assay that placed neonate H. virescens on small whole plants was terminated after six days when the larvae on control plants had consumed most of the leaf material. The control larvae, which had reached the 4^th instar, had a mean weight of 74.2 ± 13.7 mg. In contrast, larvae recovered from the TSP14 transgenic plants were mostly in the 3^rd instar and smaller (53.2 ± 15.2 mg). The transgenic plants had visibly more leaf tissue remaining than the control plants.

Whole plants (two months old, average height 30-35 cm) were used for longer feeding experiments. A visual examination of the control plants at the end of 18 days clearly showed extensive leaf loss and numerous entrance and exit holes in the stems. Much less damage was apparent on plants containing TSP14 (Fig. 1A, B). Fifty-five percent of the larvae survived on control plants while only 30% were recovered from the TSP14 transgenic plants. Surviving larvae from control plants were more developmentally advanced and larger than those recovered from plants containing TSP14 (Fig. 1C). For example, 63.6% of the larvae on the control plants were in the final (5^th) instar compared to only 33.3% on the TSP14 plants. The mean weight of all the larvae from the control plants was 224.3 ± 40.3 mg compared to 85.9 ± 16.9 mg for all larvae collected from the TSP14 plants.

A single feeding experiment with neonate Manduca sexta (tobacco hornworm) larvae, which are not normally exposed to TSP14 via parasitization, suggests that this insect was actually more sensitive to TSP14 than H. virescens, with significant mortality and inhibition of growth evident within five days of exposure to several transgenic lines.

Because TSP14 is normally delivered directly to the hemolymph via expression from teratocytes, we examined the mode of action of TSP14 following ingestion. We conducted droplet-feeding assays with neonate H. virescens larvae and in vitro labeling of midgets from 4^th and 5^th instar H. virescens larvae in the presence or absence of TSP14. The fact that starved neonate larvae would only consume TSP14-containing fluids when sucrose was added at concentrations >30% indicate the TSP14 has a previously unsuspected anti-feedant activity. Our observation that in vitro labeling of midgut in the presence or absence of TSP14 showing that protein synthesis was inhibited by TSP14 relative to controls (Fig. 2) was consistent with the activity of TSP14 in the hemocoel and other in vitro tissue assays³. Thus, it is likely that TSP14 contributes to the mortality and growth inhibition whether or not the protein enters the hemocoel and affects other target tissues.

Genes from parasitic insects and their associated viruses that either disable insect immunity or inhibit growth and development might be employed to improve methods for insect control. Both crude TSP preparations and the TSP14 gene product from recombinant expression systems inhibit the growth and development of larval H. virescens and impair protein synthesis in some insect tissues^3,4. Translation of all tested sources of mRNA used in in vitro rabbit reticulocyte lysate assays was inhibited by TSP³. We found that plants that expressed genes encoding TSP14 either with or without its signal peptide showed antibiosis activity against lepidopteran pests (e.g., tobacco budworm and tobacco hornworm)¹. Larvae fed transgenic plants experienced a higher mortality and grew more slowly than did larvae ingesting leaf tissue from control plants. These observations were consistent with those previously noted in insects injected with either teratocytes or crude TSP.

The TSP14 protein levels in the transgenic plants were too low for quantitative immunoassay. However, all lines producing detectable TSP14-specific mRNA had an observed impact on plant-feeding insects. Modifying the coding sequence to plant-preferred codon usage and increasing the GC content of the TSP14 gene in a way similar to that used with Bt toxin genes⁶ could have a marked impact on the toxicity and utility of the TSP14 transgenic plants and thereby potentiate its application in insect control.

References

1. Maiti IB et al. (2003) Antibiosis-type insect resistance in transgenic plants expressing a teratocyte secretory protein TSP14 gene from a hymenopterous endoparasite. Plant Biotech J 1: 209-219.

2. Dahlman DL and Vinson SB. (1993) IN: Parasites and Pathogens of Insects, vol. 1, pp 145-165, Academic Press.

3. Dahlman DL et al. (2003) Insect Mol Biol (in press).

4. Rana RL et al. (2002) Expression and characterization of a novel teratocyte protein of the braconid, Microplitis croceipes (cresson). Insect Biochem Mol Biol 32: 1507-1516.

5. Maiti IB and Shepherd RJ. (1998) Isolation and expression analysis of peanut chlorotic streak caulimovirus (PCISV) full-length transcript (FLt) promoter in transgenic plants. Biochem Biophys Res Comm 244: 440-444.

6. Perlak FJ et al. (1991) Modification of the coding sequence enhances plant expression of insect control protein genes. Proc Natl Acad Sci USA 88: 3324-3328.

Agricultural biotechnology holds a great promise for Africa. Tissue culture and marker-assisted selection are already in widespread use across the continent while for most countries genetic transformation is still in the developing stages. The safe application of these technologies requires functioning biosafety systems throughout Africa. This article focuses on the special issues related to biosafety in Africa and describes the current status of biosafety in the continent, with specific examples of current progress given for Egypt, South Africa, and Zimbabwe.

Agricultural and social systems in Africa differ considerably with those in the West; therefore, some differences between Africa and the West are encountered in both the approach to and emphasis placed on biosafety issues. First, while hybrid seed adoption by smallholder farmers is now considerably high, some farmers still save seed from the previous harvest to plant in the next growing season. The right of farmers to save seed is probably one of the biggest issues in risk management, since seed-saving makes it almost impossible to specify and monitor the conditions of use¹. In some sectors, genetically modified organisms (GMOs) are still being identified with the terminator technology (which has never been commercialized), leading some people to fear that these technologies could create a kind of dependency on large seed companies, driving farmers into a technological fix. While the potential role of the terminator technology in biosafety has been suggested, it cannot be recommended under these circumstances. Second, many of the most important crops in Africa, such as banana and the root and tuber crops (cassava, sweet potato, potato, etc.), are not normally supplied through seed companies. Currently, there is some systematic distribution of tissue culture (virus eliminated) material in countries such as Zimbabwe and Kenya, but informal propagation will always occur. Such a scenario creates a challenge for the biosafety framework to be adopted. Once a genetically modified cultivar of sweet potato is released into the market, it will spread to other areas through this informal propagation.

The third issue relates to food aid. Food security and food safety offer regulatory challenges in Africa. Africa frequently runs into food shortages compounded by drought and unstable political systems. In such situations, there is provision of food aid from other countries. A biosafety protocol may need to address how to deal with this food aid. Zambia made headlines when it rejected GM food aid from America, a decision that was made against a background of starvation in some parts of the country. The urgent need for food will put pressure on the biosafety issues to be considered when dealing with food aid. Some consider it a luxury to debate biosafety while people are starving, when others argue that safety comes first. Other countries have accepted GM food aid on the condition that the grain is milled to prevent it from being propagated in the fields.

The obvious differences in molecular capacity between western countries and developing nations in Africa are also an issue. In designing a biosafety system, a national assessment should be made of the existing scientific and technical capacity². A weak scientific and technical capacity impacts negatively on the biosafety framework. Capacity building in these countries is required not only to enable the development of biotechnologies, but also to assist the regulatory authorities in critically assessing draft models and deriving functional biosafety frameworks.

FAO-BiotechNews is an e-mail list containing news and events items that are relevant to applications of biotechnology in food and agriculture in developing countries. Its aim is to inform policy makers and technical decision-makers about current developments and issues in agricultural biotechnology, with a particular emphasis on developing countries, as well as to inform scientists of the wider policy/regulatory/agricultural development aspects of their work. The news and events items focus on FAO's work and the work of its main United Nations (UN) and non-UN partners. The items cover crops, fish, livestock, forest trees and agro-industry. It was launched in January 2002 and there are currently 2,700 subscribers. Updates are sent roughly every three weeks, containing on average eight short items.

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