Applying Biology to Protecting the World’s Food Supply
In America, most of us think of bananas as a tasty snack. In east Africa, however, bananas are a major food source, providing over a quarter of the daily intake of carbohydrates. In fact, bananas are the fourth most important food item in the world, surpassed only by rice, wheat, and milk products. In countries such as Burundi, Kenya, and Uganda, a large portion of the population survives through subsistence farming of bananas. Raffi Aroian, a professor of biology at UC San Diego, is involved in a project to genetically engineer banana plants to resist parasitic worms.
Like all crops, bananas are subject to infestation by pests. In particular, parasitic worms, known as nematodes, destroy anywhere from 25-70% of banana crops each year, depending on the region. The most common parasites are Radopholus similis, the burrowing nematode, and Pratylenchus goodeyi, the root-lesion nematode. Both of these worms live inside the roots of banana plants, devouring them from within and severely weakening the root structure. Often, the roots are so severely damaged that a light wind causes the plant to tip over. Since a banana plant typically lives for 15 to 30 years, the loss of a single plant has a long-lasting effect on crop yield, which is compounded by the fact that new plants must grow for 1 to 2 years before producing fruit. Crop loss due to nematodes is a major reason why banana production has not been able to keep pace with population growth in east Africa.
Preventing banana plant loss from nematodes presents several challenges. The first challenge stems from the unusual genetic make-up of banana plants. In the words of Aroian, “Banana genetics are a mess, it’s not an easy plant to work with.” The fruit-bearing plants are hybrids of two other species, meaning they have three or four sets of chromosomes instead of two, which causes sterility. Because the fruit-bearing plants are sterile, it is more difficult and time consuming to breed resistance traits into bananas than other crops such as wheat.
The second challenge is the lack of acceptable pesticides. Chemical pesticides, such as methyl bromide, are a very common treatment for plant-parasitic nematodes. However, these compounds are extremely toxic to people and damaging to the ozone layer; the use of methyl bromide as a pesticide was actually banned by the Montreal Protocol, an international agreement designed to limit ozone depletion. In Africa, these pesticides are also out of reach of the typical subsistence farmer due to their cost. As a result, “You can’t control the nematodes with chemicals, so right now, the farmers basically have to accept the losses,” says Aroian.
As a potential solution to the nematode problem in banana plants, Aroian has been exploring genetic engineering as a means to introduce nematode resistance into banana plants. A precedent exists in other crops, such as cotton and corn, which have been successfully modified to resist insects through the introduction of Crystal (Cry) proteins. Cry proteins have an interesting history. They are produced by a common soil bacteria, Bacillus thuringiensis (Bt), and were first discovered in 1901 by a Japanese researcher named Ishiwata Shigetane, who studied their toxic effect on silk worms. These bacteria were further characterized and named by a German researcher, Ernst Berliner, in 1911. Cry proteins were then made into commercial pesticides in 1938 by a French company and have been used successfully as an insecticide for many years.
These Cry proteins are an ideal pesticide because they are very specific, only affecting insects. Exhaustive testing, including tests on humans performed in the 1950’s, has demonstrated that Cry proteins are harmless to mammals and other vertebrates, unlike chemical pesticides such as DDT. Organic farmers make extensive use of Cry proteins because they are non-toxic and a natural product of bacteria. Cry proteins have also been used by the World Health Organization to control mosquito and black fly populations that spread various diseases in Africa.
In the mid-1990’s, crops genetically engineered to express Cry proteins, called Bt crops (in reference to Bacillus thuringiensis), were made commercially available, further increasing the utility of Cry proteins in preventing crop loss to insects. In 2005, Bt corn accounted for 12% of all corn planted in the world, and Bt cotton comprised 24% of all cotton. Those figures are continuing to increase. In China, the use of Bt crops has reduced the need for toxic chemical pesticides, benefiting both the environment and the health of the farmers.
For a long time, the prevailing opinion concerning the specificity of Cry proteins was that only insects were affected and that all other invertebrates and vertebrates were impervious. The Aroian lab was the first academic laboratory to show that some strains of B. thuringiensis produced Cry proteins that were toxic to nematodes as well. For a time, his was the only lab in the world that studied the effects of Cry proteins on nematodes. Recently, a few more labs have begun their own research programs.
Before undertaking the project of modifying plants to produce Cry proteins, Aroian’s lab studied several aspects of Cry protein function in nematodes using the model organism C. elegans, including the mechanism through which Cry proteins kill nematodes and how nematodes develop resistance to Cry proteins. In addition to developing Bt crops that are less vulnerable to nematodes, these ongoing studies are providing crucial insights that will enhance the insect-resistant Bt crops already in use.
Research in Aroian’s lab has led to a working model for the method of action of Cry proteins. A genetic screen identified five genes necessary for the toxic effects of Cry proteins on C. elegans, named bre-1, bre-2, bre-3, bre-4, and bre-5. Four of these genes make proteins known as glycosyltransferases, which are involved in the synthesis of long sugar molecules. Further studies revealed that the activity of BRE proteins in the intestine is critical for the toxicity of Cry proteins. These data demonstrate that the BRE proteins create specific sugar molecules that serve as receptors for the Cry proteins on the surface of the worm intestine. After binding to these receptors, it is thought that the Cry proteins then cause holes to form at the surface of intestinal cells, which then leads to death of the animal. Current studies are confirming this model.
This research from Aroian’s lab also suggests possible reasons why Cry toxins only affect insects and nematodes. Mammals do not have any genes similar to bre-3, and only have genes that vaguely resemble bre-5. It seems that bre-3 and bre-5 make specific markers that are recognized by Cry proteins in insects and nematodes. Because mammals lack these genes, they are naturally resistant.
The discovery of Cry proteins that could target nematodes inspired Aroian to think of potential applications. Says Aroian, “There is actually a need for good anti-nematicides, and here we have one that’s a natural product with an excellent safety track record.” This need led Aroian to explore the possibility of genetically engineering nematode-resistant plants using Cry proteins.
The research began using tomato plants as a model. Researchers in Aroian’s lab set about the task of introducing Cry proteins into the genome of tomato plants in order to make them resistant to a common parasite, the root-knot nematode. To introduce new genes into the tomato genome, another type of bacteria, Agrobacterium rhizogenes, was used as a carrier. This bacteria infects plant roots and injects pieces of its DNA into the host plant’s genome. When pieces of the Agrobacterium’s DNA are replaced with other genes, it injects those instead. By substituting the genes that make Cry proteins into the Agrobacterium, the researchers were able to insert Cry proteins into the tomato plant roots.
Coaxing the tomato roots into actually producing high amounts of Cry proteins, however, turned out to be quite a challenge because plants are not equipped with the right machinery to produce bacterial proteins such as Cry proteins. More than three years of experimentation were required to modify the Cry protein genes to enable them to be produced efficiently by the tomato plants. The result was worth the effort. According to Aroian, “We found that at least one of the crystal proteins we work with can control the root-knot nematode in tomato plants. It inhibits their ability to reproduce four-fold, which is comparable to what commercial nematicides will do.”
This work represents the first time anyone has used a specific Cry protein to protect plants from nematodes. Having demonstrated that Bt-plants can resist nematodes, Aroian sent the re-designed Cry genes to collaborators in Uganda, who are currently engineering the proteins into banana plants. Since banana plants are slow to work with, it will be some time before success can be determined, but the outlook is promising.
“It’s really ground-breaking research that could have major impacts,” says Aroian. “Nematodes cause 100 billion dollars in crop damage each year, all over the world, not just in Africa.”
Aroian has now set his sites on animal parasites. According to the World Health Organization, roughly one out of every three people throughout the world is infected with a parasitic nematode. In terms of the disease burden, parasitic nematodes rank right along side HIV, malaria, and tuberculosis. Aroian hopes to use his expertise with nematicidal Cry proteins to relieve the human suffering caused by parasitic nematodes.
Professor Raffi Aorian Aorian Lab More about the Aroian lab’s research
Contributing Writer: Matthew Busse
From BioSphere Magazine, 2007-2008 issue, page 14.
