AG-BIO: How Budworms Resist Bt

Applied Genetics News,  August, 2001  

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The August 3 issue of Science contains two papers on the mechanisms that insects use to develop resistance to the Bt toxins used in crop plants. Linda J. Gahan, an assistant professor of biological sciences at Clemson, and coauthors Fred Gould from North Carolina State University and David G. Heckel of the University of Melbourne, Australia, have found an efficient method to genetically track the tobacco budworm's efforts to become resistant to biotechnological pest controls. Also, Joel S. Griffits and a team of scientists from Raffi Aroian's lab at UC San Diego (San Diego, CA) report on the cloning of a mutant gene from the laboratory roundworm C. elegans that confers Bt resistance.

The tobacco budworm is a major pest of cotton and other field crops in the Americas, which over the years, developed resistance to chemical pesticides. That problem was solved through biotechnology when researchers added a gene to cotton bacteria, Bacillus thuriengis (Bt) that naturally produces an insect toxin. Bt cotton resists the tobacco budworm and other insects.

Under laboratory conditions, however, strains of tobacco budworms could be made Bt-resistant by feeding the insects a high-dose diet of the toxin. Therefore, federal regulators required non-Bt cotton to be planted alongside of Bt cotton, diminishing the chances of budworms taking a Bt-rich diet in the fields. The question was how to monitor the situation, catching Bt resistance before it ended the usefulness of Bt cotton.

Through DNA analysis, the researchers found that disruption of a gene could be linked to Bt-resistance.

"We have identified a gene in the budworm that binds Bt-toxin produced in genetically modified plants like cotton," Gahan says. "If the gene is altered or defective in the insect, the insect will not bind the toxin and effectively becomes resistant to it. Knowing the gene involved in resistance allows us to develop a DNA test for monitoring resistance in field population of insects."

In the UCSD study, the researchers concluded that the roundworm's resistance to a particular Bt toxin known as Cry5B. resistance resulted from the loss of a gene, called bre for Bt resistance, which encodes galactosyltransferase, an enzyme that adds carbohydrates to proteins and lipids. Their discovery prompted the scientists to hypothesize that crystalline Bt toxins-which act by attacking and dissolving the intestines of their hosts-normally recognize the outer surface of intestinal cells by means of carbohydrates or sugars. When the galactosyltransferase gene is missing, these sugars are not made and the toxin fails to recognize its host.

Using toxins labeled with fluorescent dyes fed to normal and resistant forms of C. elegans, the USCS researchers found that the Bt toxin is taken up into the gut cells of a normal roundworm but not a resistant roundworm. If the toxin is not recognized, as is the case in resistant animals, it simply passes through the lumen of the gut and is defecated without entering the gut cells.

Whether galactosyltransferase is essential for many other Bt toxins remains to be determined. But the UCSD scientists discovered that their mutant roundworms were also resistant to a Bt toxin that is lethal to beetles, suggesting that the development of resistance by the loss of carbohydrate-modifying enzyme is relevant to insects as well. Furthermore, the 3-D structure of diverse insecticidal Bt toxins contains a fold that is predicted to bind precisely the sugar modification made by the galactosyltransferase, raising the possibility that this mechanism of resistance could be widespread.

"For people using Bt toxins to control insects, this is a particularly threatening scenario," says Aroian. "The reason is that with one swoop, you can knock out the binding of multiple toxins to multiple receptors. But now that we know this mechanism of resistance, we can devise strategies to cope with this." One possible strategy, he suggested, is for scientists to modify the toxins such that they can bind to the inner lining of the insects' or roundworms' guts independent of this carbohydrate modification.

The UCSD study may help scientists employ Bt toxins in the growing problem of roundworm, or nematode, infestations in plants, animals, and humans.

"Even if Bt toxins weren't used to fight insects, nematodes are a huge problem," says Aroian. "At last estimate, which was 13 years ago, they caused $80 billion worth of crop damage per year. And the damages will become worse, because the main chemical now used to control them in agriculture, methyl bromide, has been banned by the Montreal protocol. They are also a human health problem-a quarter of the world's population are infected with animal parasitic nematodes."

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