Tuesday, August 1, 2000

Karl J. Kramer

What human food proteins might be candidate biopesticides in transgenic plants? Trying to answer that question was the motivation for a collaborative research project between biochemists, molecular biologists, and entomologists at the Agricultural Research Service, USDA`s Grain Marketing and Production Research Center in Manhattan, Kansas and two agricultural biotechnology companies, Pioneer Hi-Bred International Inc., Johnston, Iowa and ProdiGene Inc., College Station, Texas. Some of the results were published recently in the journal Nature Biotechnology(1).

With all of the controversy surrounding transgenes and the questionable safety of their encoded proteins, we thought that it would be beneficial to develop as biopesticides in transgenic plants some proteins that were already being consumed in the human diet. This type of advancement might result in fewer objections to the use of transgenes in foods or feeds for humans and animals. The end result of the project was the development of a transgenic maize that contains the hen`s egg white protein avidin, which makes the grain resistant to stored-product insect pests.

The project began about ten years ago when Thomas Czapla, a graduate student at Kansas State University who subsequently worked as a molecular entomologist at Pioneer, expressed an interest in determining whether some of the proteins we were evaluating for biocidal activity against stored-product insect pests might also be tested against field crop pests such as corn borers and rootworms. At that time, as now, there were very few biopesticides other than the endotoxins from the bacterium, Bacillus thuringiensis, that have been commercially developed in transgenic plants for insect pest control. Pioneer was interested in prospecting for proteins that would have a broader spectrum of activity than Bt-type proteins.

Czapla, who unexpectedly passed away last January at the young age of 38, and members of my laboratory discovered that chicken avidin and a related protein, bacterial streptavidin, when administered in semi-artificial diets, caused a deficiency of the vitamin biotin in the corn borer and several species of stored-product insects. This, in turn, led to stunted growth and mortality of those species(2). What makes avidin particularly unique as a biopesticide is that not only is it a common dietary protein, it also has an antidote, biotin, which may be used as a supplement to prevent toxicity or to rescue potential victims from adverse effects. Biopesticides such as the Bt toxins do not have any kind of antidote.

Those encouraging results led to the next phase of the project, which was the creation of transgenic avidin maize by scientists at Pioneer and ProdiGene. John Howard, who at that time was the manager of the Pioneer`s Protein Products Group, which later was merged with Terramed Inc., a Texas pharmaceutical biotechnology firm, to form ProdiGene, and his group at Pioneer were developing maize for commercial production of industrial proteins. The first transgenic maize product commercialized by ProdiGene was avidin, which also is used as a research chemical and diagnostic reagent(3). Avidin is produced in corn at a fraction of the cost that it can be produced in chicken eggs.

ProdiGene and Sigma Chemical Co., St. Louis, Missouri, began marketing avidin produced in maize in 1997. At about the same time, the Kansas research group began evaluating avidin maize for host plant resistance to stored-product insect pests. When present in maize at levels of ~100 ppm, avidin was toxic to and prevented development of many internally and externally feeding insect pests that damage grains during storage, including the rice weevil, lesser grain borer, Angoumois grain moth, warehouse beetle, sawtoothed grain beetle, flat grain beetle, red and confused flour beetles, Indianmeal moth, and Mediterranean flour moth. Other species reported to be susceptible to avidin toxicity include the house fly, hide beetle, fruit fly, olive fruit fly, flour mite, tobacco hornworm, tobacco budworm, black cutworm, sunflower moth, beet armyworm, and cotton bollworm. The only species tested to date that was not susceptible to avidin toxicity was the larger grain borer. This species is occasionally found in southern Texas but is not a significant pest in the US. It has been a pest, however, in Mexico, Central America, northern South America, and Africa. How the larger grain borer tolerates high levels of avidin is unknown but the question will be addressed in a future study. The existence of an avidin-tolerant species, however, may indicate the possibility of the development of insect resistance to avidin if resistance management is not conducted properly. Nevertheless, avidin acted as a biopesticide in transgenic maize with a specific toxicity comparable to Bt toxins, and the spectrum of activity of the former protein was much broader than the latter.

Because expression of avidin in maize was under the control of the ubiquitin promoter, expression of the protein in the anthers occurred, which caused male sterility and non-uniform protein expression. About half of the individual kernels had inadequate insect resistance because those kernels contained little or no avidin. That result led us to develop a nondestructive method, utilizing near infrared spectroscopy, to screen for avidin content and separate low- from high-avidin kernels prior to conducting bioassays of intact kernels. A future goal is to increase the proportion of avidin-containing, insect-resistant kernels by performing a new transformation event using promoters designed to avoid expression in the anthers and to yield kernels containing a more uniform and effective level of avidin.

Avidin maize was demonstrated to have excellent resistance to storage insect pests after the kernels were ground into a meal, because an average concentration of ~100 ppm avidin in the meal was sufficient for protection, a level substantially lower than that present in chicken egg white. Humans can suffer "egg white injury" but only after consuming exceedingly large quantities of avidin, such as eating a couple dozen raw eggs a day for several months. Avidin maize was not toxic to mice when administered as the sole component of their diet for three weeks.

Following a thorough and satisfactory safety risk assessment of avidin corn, its utilization as a food or feed grain would be an exciting development that could impact on post-harvest losses caused by stored-product insect pests. Avidin maize might be processed to include supplementation with biotin or by treatment with heat. The latter process would denature the avidin as well as the avidin-biotin complex and release most of the biotin for use by the consumer. Another use of avidin maize could be as an insect-resistant background host plant germplasm for farming of other valuable bio-pharmaceutical or industrial proteins. Whatever utilization does occur, avidin maize is at least a proof of concept for the efficacy of a common human food protein as a biopesticide in transgenic plants.

Sources

1. Kramer KJ, Morgan TD, Throne JE, Bailey M, and Howard JA. 2000. Transgenic maize expressing avidin is resistant to storage pests. Nature Biotechnology 18:670-674.
2. Morgan TD, Oppert B, Czapla TH, and Kramer KJ. 1993. Avidin and streptavidin as insecticidal and growth inhibiting dietary proteins. Entomologia experimentalis applicata 69:97-108.
3. Hood EE, et al. 1997. Commercial production of avidin from transgenic maize: Characterization of transformant, production, processing, extraction and purification. Molecular Breeding 3:291-306.