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 Fall/Winter
1997 |
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Searching for a Sticky Gene
The discovery of a single gene can have far-reaching implications. Such a discovery was made this year by researchers at Penn State and at Brookhaven National Laboratory, a facility operated for the U.S. Department of Energy by Associated Universities, Inc. in Upton, New York. Building on decades of research, the scientists collaborated to identify a gene that is directly linked with a mechanism of plant resistance to insect pests. The discovery could have an important impact on agriculture as well as on other industries. When more is learned about this mechanism, plant geneticists may be able to transfer it to important crops such as tomatoes and potatoes. David Schultz, who received his Ph.D. in genetics from Penn State last spring and now is a postdoctoral researcher at Michigan State University, identified the gene in the laboratory of June Medford, assistant professor of biology and biotechnology in Penn State's Eberly College of Science. The university currently is in the process of patenting the use of the gene. Isolation and identification of the gene is the culmination of years of interdisciplinary study. Since the early 1960s, Penn State scientists, including horticulturists, plant morphologists, entomologists, geneticists, molecular biologists, and biochemists, have looked at thousands of plants to learn what makes some geraniums resistant and others susceptible to pests. The researchers determined that resistant geraniums produce a viscous material from hairlike structures, called trichomes, on their stems and leaves. This material impedes insects and mites in several ways–by trapping them in the viscous liquid, by killing them outright, and by inhibiting the ability of females to lay eggs. The viscous liquid contains compounds known as unsaturated anacardic acids, which are synthesized from unsaturated fatty acids in plants. "Trichomes in both resistant and susceptible plants produce anacardic acids, but only the resistant plants produce unsaturated anacardic acids," says Schultz, who did his research with Richard Craig, J. Franklin Styer professor of horticultural botany, and Ralph Mumma, distinguished professor of environmental quality. "Unsaturated anacardic acids form the viscous liquid, which is like vegetable oil, so they stick to the insects," he explains. "You can feel these sticky substances on the stems of the resistant geraniums. The saturated anacardic acids found on susceptible plants are more solid and do not stick to insects." Before Schultz began his doctoral studies at Penn State in 1992, a group of students and postdoctoral scholars working with Craig and Mumma had identified the two unsaturated fatty acids that were the precursors to the unsaturated anacardic acids in the resistant geraniums. They also knew that a single gene was responsible for the formation of the unsaturated fatty acids. "My role was to find that gene," Schultz says. The researchers knew that the sticky compounds could be found only in the trichomes, but they needed a method to obtain large quantities of them in order to find the gene. Postdoctoral scholar Ellen Yerger, working with Diana Cox-Foster, associate professor of entomology, developed a method of removing the tiny trichomes from the plants so that their genetic makeup could be examined. By freezing the parts of the plant covered with trichomes in liquid nitrogen and then vigorously shaking them, she could get the trichomes to break away from the supporting plant tissue. "This was crucial for my research because the gene I was looking for could be found only in these trichomes," Schultz explains. "It took months to collect enough trichomes so that I could begin the research. Without Ellen's technique, my work would have been impossible." The type of gene Schultz was looking for encodes a desaturase. "It desaturates the fatty acid, changing its molecular structure by adding a double bond at a specific point along the fatty acid's carbon chain," Schultz explains. "I was looking for the desaturase gene that leads to the production of two fatty acids known as 16:1 D11 and 18:1 D13. What we were hunting for was a gene that looked like it had the characteristics to convert fatty acids in precisely the way necessary to create the unsaturated anacardic acids. We also knew from previous research that this gene would be expressed only in the trichomes of the resistant plants and nowhere else–not in other parts of the plants and not in the trichomes of susceptible plants." Next, Schultz and Medford did RNA assays to analyze the expression of genes in the trichomes of resistant geraniums, in those of susceptible geraniums, and in tissue from other parts of the plants. They narrowed the search to two genes that they thought might encode the desaturase responsible for the production of unsaturated fatty acids. "One of the genes was a perfect match," says Medford. "We found it only in the trichomes of the resistant plants and nowhere else. The gene was present in all resistant plants regardless of whether they were parents, hybrids, or progeny from our genetic experiments. We were pretty sure this was our gene." Before they could be certain this was the desaturase gene they were looking for, however, the researchers needed more evidence. "We had to follow up with a biochemical evaluation," says Schultz. "That means we had to insert the gene into some type of living tissue and see if it actually would convert the saturated fatty acids to unsaturated fatty acids, leading to the production of the sticky material in the resistant geraniums." The researchers had planned to insert the gene into plant tissue and look for the formation of these fatty acids, a time-consuming study involving growing the plants to maturity. But in December 1994, at a conference in Annapolis, Maryland, Medford met biochemist John Shanklin, who worked at Brookhaven National Laboratory. "This was extremely lucky for us, because it just so happened that he was working with a method of desaturase gene expression using the bacterium E. coli," Medford says. "E. coli very quickly expresses genes that are inserted into it, so using this method can speed up some genetic research," explains plant biochemist Edgar Cahoon, who works in Shanklin's lab. "Our research fit perfectly with the Penn State study." Teaming up with Shanklin and Cahoon enabled the Penn State researchers to insert the gene into E. coli, allowing it to be expressed in the bacterium. This resulted in the production of two unsaturated fatty acids new to the E. coli. The researchers then isolated enzymes from the E. coli and placed them in contact with various saturated fatty acid substrates. The interaction of the enzymes with the proper substrate showed whether the gene encoded a functional desaturase and identified the preferred substrate. "We expected the desaturase encoded by our gene to act on the saturated fatty acids known as palmitic acid (16:0) and stearic acid (18:0) , but we discovered that the pathway actually was more complex," says Schultz. "Instead of desaturating these two fatty acids, the gene encoded a protein that acted on a different fatty acid, known as myristic acid (14:0) and converted it to myristoleic acid (14:1  9).
This fatty acid was then converted into the 16:1 D11 and 18:1
D13 fatty acids by the elongation of 14:1D9. The
behavior of the desaturase was somewhat different from what we expected,
but the outcome showed that we definitely had identified the desaturase
gene associated with plant resistance. I see this as the first step in
defining one pathway of pest resistance in plants at the molecular level."
Along with enhancing plant breeding and integrated pest management programs, the research may have other important applications. "Discovery of this gene could benefit both agriculture and industries that require specialized oils," Medford says. "These oils are expensive to manufacture, but they might be produced far more cheaply in plants or in microorganisms. Perhaps farmers could grow crops that produce these specialty oils."
"I could not
have accomplished this research alone," Schultz says. "No
one scientist could have all the skills or knowledge necessary, not
to mention the time–there are decades of research leading up to
this discovery. It was truly a collaborative effort, and being part
of it was a great learning experience."
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