TIBTECH - JUNE 1'~J91 [Vol. 9] 197

Insect control with genetically engineered

crops Karen J. Brunke and Ronald L. Meeusen

The ability to move bacterial genes encoding 'na tural ' insecticidal proteins into plants is permitt ing the development of crops intrin- sically resistant to insect attack, with advantages over conventional insect-control agents (i.e. externally appl ied synthetic chemicals). These advantages include absence of residues in soil or ground- water, lack of toxicity to non-target organisms, and protection of plant parts which are difficult or impossible to spray (e.g. roots). Continued elucidation of the mechanisms by which such agents act, coupled with genetic engineering techniques, should lead to an increasing variety of insect-resistant crops in the coming years. Agricul ture will thus gain inexpensive, effective and environ-

mentally safe alternatives to current insect-control methods.

In this century, control of insects in crop plants has depended largely on synthetic chemistry. This de- pendence will probably continue. However, an alternative has been available for more than 30 years: a biolog.ical insecticide from the bac- terium, Bacillus thuringiensis (Bt). This biological insecticide remains the product of choice for environ- mentally sensitive applications (e.g. insect control just prior to harvest, forestry applications and home garden use). Despite its remarkable safety and efficacy, Bt has not garnered a great proportion of the market for insecticides because of the low cost and longer persistence of synthetic organic insecticides. With the arrival of methods to engin- eer crops genetically we now have the ability to make broader use of 'natural' insecticides. Moving Bt insecticidal genes from the original bacterium to plants allows crop pro- tection from insect attack without the aid of externally applied chemi- cals. In fact, within two years of the first demonstration of plant trans- formation in 1984, control of insect pests in the field was demonstrated with plants containing a Bt insecti- cidal protein.

K. J. Brunke is at the Sandoz Crop P,-otec- tion Corporation, Palo Alto, CA 941304, USA. R. L. Meeusen is at Northrup King Co., Stanton, MN 55081, USA.

Plant transformation Methods developed over the past

15 years enable cell- and molecular biologists to Insert genes into crop plants (for an overview, see Ref. 1). The earliest plant-transformation experiments used the soil bacterium, Agrobacterium tumefaciens, as vec- tor; transfer of foreign DNA along with a portion of its own plasmid DNA into a plant cell occurs during infection. Small plant explants in- fected with the genetically altered bacterium can then be regenerated to form whole plants that carry the newly inscrted genes in their genomes, and the traits are heritable. This technology works for many dicotyledonous plants but not for most of the monocotyledonous species (e.g. grasses and cereals).

With the exception of reports of rice transformation in late 1989, cereals did not appear to be readily amenable to transformation (for review, see Ref. 2). Transformation of maize protoplasts by electro- poration had been reported several years earlier 3, but the regenerated corn plants were not fertile (prob- ably due to the age of the cell cultures that were electroporated). However, in early 1990, a report appeared in the press that corn trans- formation had been achieved by Biotechnica International using the ballistic gun. In the following months, stable transformation of

~) 1991, Elsevier Science Publishers Ltd (UK) 0167 - 9430/91/$2.00

maize wa~ reported at scientific meatings 4 and in the literature s'6. This progress has centered around the ability to recover fertile trans- formants which transmit the intro- duced genes to their progeny.

The ability to isolate genes from any source and insert these genes into a variety of plants opens the door for rapid crop improvement. The first application of plant genetic engineering was the protection of crops against insect attack.

Insect-control agents Two types of insect-control agents

have been developed a:~d proven effective following introduction into plants: (1) the protein delta endo- toxins from Bt have been most widely studied in transformed plants; and (2) the class of proteins known as proteinase inhibitors, when present at relatively high levels in the diet, has been shown to be effective against certain insects. In addition, the combination of Bt endotoxins with proteinase inhibi- tors is being studied 7.

Bacillus thuringiensi~ endotoxins The choice of a Bt endotoxin as the

first insecticidal protein for intro- duction into plants was based on the extensive knowledge about this class of crystal proteins (for review, see Ref. 8). The entomocidal bacterium Bacillus thuringiensis, upon sporu- lation, normally produces a para- sporal (from the Greek 'beside the spore') crystalline toxin. When in- gested by a susceptible insect, a combination of the high pH and proteinases of the insect's midgut is believed to be responsible for the solubilization of the crystals and the rapid cleavage of protoxin to yield an active toxin. The effects of the toxin occur within m~nutes of in- gestion, beginning wit]~ midgut par- alysis and ending with disruption of midgut cells.

Strains of Bacillus thuringiensis have been registered as commercial insecticides for over 30 years begin- ning with 'Thuricide' (Sandoz label) in 1957. The crystals within the bacteria can contain one or more types of toxin proteins, each of which can have its own specificity to a class of insects. Although the majority of the Bt strains and toxin proteins are active against the larvae of caterpillars (lepidopteran insects),

198 TIBTECH-JUNE 1991 [Vol. 9]

--Box I

Possible s t ra tegies for m a n a g i n g insect res is tance*

High dose: Market crops with very high levels of Bt protein expression to kill marginally resistant insects and prevent stepwise development of resist- ance through multiple mutations.

Low dose: Sublethal levels of Bt protein in plants retard growth of insects and increase susceptibility to predation by beneficial insects. This approach precludes combination with broad spectrum insecticides which would reduce populations of beneficial insects.

Rotation: Alternate use of chemical pesticides and crops containing Bt insecticidal proteins. Intermittent selection pressure would favor wild-type insect population if resistant insects are found to be less competitive, or if resistance is unstable and rapidly lost when selection stops.

Stacked genes: Multiple types of insecticidal genes added to crops (equiv- alent to mixtures of chemical insecticides, a successful resistance manage- ment strategy). Current paucity of alternative genes (such as proteinase inhibitors) makes this a future strategy. A variant of this approach combines crops containing Bt insecticidal protein with selective sPraying to eliminate resistant insects.

Replacement: If resistance to Bt protein arises more slowly than new Bt insecticidal genes can be introduced, then it may be possible to 'outrun' resistance. This strategy will be limited to the degree to which cross- resistance to different Bt insecticidal proteins is found to occur.

Refugia: Combination of seed products containing Bt insecticidal proteins with a small proportion of seed that lacks the insecticidal protein. Wild-type insects would then have refuge in the field and presumably would out- compete less fit resistant insects each season.

* Options based on the June 1990 'Symposia on Management Strategies for Bacillus thuringiensis Based Product' hosted by the Monsanto Agricultural ~ompany.

others have been found to be active against dipteran or coleopteran larvae s,9.

The long history of safe use of Bt endotoxins as insecticides, the rela- tively narrow range of target insects affected by a single protein toxin, the strong insecticidal activity of this toxin against the larvae of the sus- ceptible insect, and the single-gene nature of the protein are clear advan- tages for transferring this insecti- cidal protein into plants. In addition, the presence of the Bt endotoxin inside the plant should provide a cost-effective strategy for the grower by the preclusion of the need for repeated spraying of a field with insecticides, and should kill the in- sect before any significant crop damage occurs. Another potential advantage would be the expression of insecticidal proteins in internal and underground regions of the plant that are inaccessible by traditional spray regimes. The potential dis- advantages of transgenic Bt plants are: (1) that the bacterial gene might be difficult to express at effective

concentrations in the plant, and {2) that insect resistance might develop with time.

Evidence is accumulating both in the laboratory 1°-1z and in the field ls'~4 that the widespread use of Bt-protected crops may select for resistant insects. Experience with resistance to chemical insecticides has led academic and industrial groups to recognize at least six options for delaying development of resistant insects {Box 1}.

Proteinase inhibitors Many plants have evolved natural

defense mechanisms against her- bivorous insects. One such mech- anism is the synthesis of proteinase inhibitors {for review, see Ref. 15). In contrast to the Bt endotoxins, these proteins have anti-metabolic activity against a wide range of insects. They are present in the tissues of some plants at relatively high concen- trations where they participate in a complex defense response with other molecules produced by the plant. Clearly, the introduction of

specific proteinase inhibitors into plants which do not produce these molecules is an alternative approach for obtaining crops that are resistant to insect attack.

Four classes, serine, thio!, metallo- and aspartyl-proteinase inhibitors, have been identified. The class of serine proteinase inhibitors {active against trypsin and/or chymotrypsin} is further divided into 13 families, each of which shows no homology with any of the other families. How- ever, they all inhibit serine protein- ages using the same competitive mechanism. Presence of trypsin in- hibitor in the diet of insects reduces the effective concentration of trypsin available for digestion. This triggers a series of events including the in- ability to obtain amino acids from ingested food {for review, see Ref. 15).

Thiol {or cysteine} proteinase in- hibitors have a major role in the plant-defense repertoire because cer- tain insects use thiol proteinases as primary digestive enzymes. Plants containing high levels of serine proteinase inhibitors can still be consumed by these insects. Colorado Potato Beetles, for example, readily consume potato tubers which con- tain serine proteinase inhibitors. Transformation of plants lacking thiol proteinase inhibitors with genes encoding these proteins could protect them against such insects.

The advantages of using protein- age inhibitors as insect-control agents include: {1} their activity against a wide range of insects, {2} their use as a second mechanism to help prevent development of insects that are resistant to the Bt endotoxin, {3} their inactivation with cooking, and {4} the common nature of such inhibitors in the foods of humans and animals. The major disadvan- tages are {1} the high levels of protein required for insect killing, and (2) the potential need to regulate protein expression to specific plant organs.

Introduction of insect control agents into plants Bt endotoxin genes

A Bt endotoxin gene with insecti- cidal activity against lepidopteran larvae was reported to be introduced successfully into tobacco plants in July 198716 by Plant Genetic Systems {PGS}, a Belgian biotechnology com- pany. Truncation of the full-length

TIBTECH- JUNE 1991 [Vol. 9] 199

gene to the size encoding the toxic core did not reduce insecticidal ac- tivity. Their success w~s attributed to their choice of promoter (wound- inducible upon insect feeding) and to fusion of the Bt endotoxin with the gene for kanamycin resis- tance, which allowed them to use resistance to kanamycin to select plants expressing high levels of the fusion gene product. The levels of Bt endotoxin produced were sufficient to kill first-instar Manduca sexta larvae, and this insecticidal property was heritable.

A second report of successful transformation with Bt followed in August 1987, with the expression of Bt in tomato by researchers at Monsanto Co. ~7. A 35S CaMV promoter was used to direct ex- pression of the insecticidal protein and sufficient protein was produced to kill Manduca sexta larvae. The best insecticidal activity was found in plants expressing a truncated Bt endotoxin. Later in 1987, Agracetus Co. reported expression of the Bt endotoxin in tobacco ~s, with the 35S CaMV promoter linked to an AMV (alfalfa mosaic virus) leader se- quence giving enough Bt mRNA to be easily detectable on Northern blot analysis. Using immunoblot tech- niques, they identified a peptide in resistant plants corresponding in size to that expected for a truncated Bt endotoxin. In these three initial reports (above), the Bt endotoxin genes chosen generated insecticidal activity against lepidopteran insects.

Since 1987, Bt endotoxin genes have been reported to be introduced into additional plant species in- cluding potato ~9 and cotton z°. Ef- forts are under way to engineer a variety of other crops (such as sun- flower and vegetables] to resist lepi- dopteran and some coleopteran in- sects. Modifications to the bacterial gene sequence of the Bt endotoxin to make it more readily expressed in plants have been reported 2~ and have been shown to increase concen- tration (up to 500-fold) and, con- sequently, host range to include less sensitive pests such as the tomato fruitworm (Heliothis zea), and tomato pinworm (Keiferia lycopersicella).

Proteinase inhibitor genes In 1987, in addition to reports of

insect control using Bt endotoxin genes in plants, a group in England

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funded by Agricultural Genetics Company claimed to have produced insect-resistant tobacco plants through introduction of a serine proteinase inhibitor gene derived from cowpea 22. The tobacco bud- worm (Heliothis virescens), a pest of tobacco, was used in the bioassay. Plants expressing very high levels of cowpea trypsin inhibitor showed decreased insect damage. These were the first direct results demon- strating that a proteinase inhibitor had the potential to act as an insect- control agent in plants.

Subsequently, other reports from the same laboratory 23 have extended the original findings to demonstrate the broad spectrum of activity of cowpea trypsin inhibitor. In artificial diets where the amount of protein could be varied, cowpea trypsin in- hibitor was found to kill a wide variety of lepidopteran and coleop- teran insects including army worm (Spodoptera littoralis), the corn ear- worm (Heliothis zea), and corn root- worm { Diabrotica undecirnpunctata). However, the relatively higher levels of protein required for insect control with proteinase inhibitors compared to Bt proteins remains a hurdle for effective insecticidal use in plants.

Introduction of genes encoding other proteinase inhibitors into tobacco plants and subsequent in- secticidal activity has been reported by another laboratory z4. As we learn more about these natural plant de- fense mechanisms, we can use this knowledge for crop improvement.

Prospects for insect control in plants The grower gains unique advan-

tages for insect control following

introduction of genes encoding in- secticidal proteins into crop plants. Since the proteins are produced con- tinuously inside the plant, season- long control of the insect pest is possible, as is protection of plant parts such as roots, shaded lower leaves, or new growth emerging between spray applications, none of which are accessible through con- ventional spray-application of in- secticides. Since Bt insecticidal proteins are contained within the plant, only insects feeding on the plant will be exposed to the toxic effects, thus sparing non-pest and beneficial insects. Coupled with the lack of toxicity of these proteins to other organisms, and their rapid bio- degradation in the environment, these characteristics promise that the wide- spread use of these insecticides should be safe and compatible with environ- mental-protection considerations.

The insect-control agents currently available for genetic engineering into plants are, however, limited. In the next few years, much effort will be devoted to expanding our present knowledge and devising new and better strategies for insect control. An example of one such avenue is the search for Bt endotoxins with different insect toxicity spectra. From information on the receptor interactions in the insect midgut with the Bt endotoxin, 'designer' proteins might be made to target a currently unsusceptible class of insects. In addition, completely new classes of proteins may be found which complement the Bt insecti- cidal protein and delay the develop- ment of insects resistant to these control agents.

200 TIBTECH- JUNE 1991 [Vol. 9]

Over the next ten years we will go from field tests of genetically engin- eered crops to agricultural use of such crops for insect control. Pest management strategies will be devel- oped to incorporate a new insect- control agent, th~ 'ant itself.

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5 Gordon-Kamm, W. J., Spencer, T. M., Mangano, M. L., Adams, T. R., Daines, R. J., Start, W. G., O'Brien, J. V., Chambers, S. A., Adams, W. R., Jr, Willetts, N. G., Rice, T. B., Mackey, C. J., Krueger, R. W., Kausch, A. P. and Lemaux, P. G. (1990) The Plant Cell 2, 603-618

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J. and Klein, T. M. {1990) Bio/ Technology 8, 833-840

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16 Vaeck, M., Reynaerts, A., H6fte, H., Jansens, S., De Beuckeleer, M., Dean, C., Zabeau, M., Van Montagu, M. and Leemans, J. (1987) Nature 328, 33-37

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