Bug-Free Gene

Rose Pruyne
May 01, 1997

Why do some geraniums have sticky stems?

One answer is to fend off bugs. The stuff that exudes from their hairlike trichomes impedes insects and mites in several ways – by trapping them, by killing them outright, and by keeping females from laying eggs. The liquid contains compounds called unsaturated anacardic acids, which plants synthesize from fatty acids.

bug spikes

These sticky spikes grow on the unassuming garden geranium – at least on some of them – to keep pesky insects at bay. A team of Penn State researchers has cloned the gene responsible for this effective defense.

"Trichomes in both resistant and susceptible plants produce anacardic acids, but the difference is that anacardic acids in resistant plants are unsaturated," says plant geneticist David Schultz. "Unsaturated anacardic acids form a viscous liquid, like vegetable oil, so they stick to the insects. 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."

So why, if it's so clearly beneficial, do only some geraniums have sticky stems?

Since the early 1960s, Penn State horticulturists, plant morphologists, entomologists, geneticists, molecular biologists, and biochemists have studied thousands of plants to learn what makes some garden geraniums pest-resistant and others not. By 1992, graduate students working with horticulturist Richard Craig and entomologist Ralph 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," says Schultz, "was to find that gene."

Schultz, who received his Ph.D. in genetics from Penn State last May, explains, "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 the susceptible plants."

Postdoctoral fellow Ellen Yerger, working in entomologist Diana Cox-Foster's laboratory, developed a way to remove the tiny trichomes from the plants so that their genetic makeup could be examined. By freezing parts of the plants in liquid nitrogen and then vigorously shaking them, she could get the trichomes to break away from the supporting plant tissue.

"It took months to collect enough trichomes so that I could begin my research," Schultz says. "Without Ellen's technique, my work would have been impossible."

Next, Schultz and June Medford, then an assistant professor of biology and biotechnology, did RNA assays to analyze the expression of genes in the trichomes and from other parts of both susceptible and resistant plants. "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."

To be certain, however, the researchers had planned to insert the gene into plant tissue and grow the plants to maturity. But at a conference in December 1994, Medford met biochemist John Shanklin of 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.

"Because E. coli very quickly expresses genes that are inserted into it, using this method can considerably speed up some genetic research," says plant biochemist Edgar Cahoon, who works in Shanklin's lab. "Our research fit perfectly with the study being conducted at Penn State."

With Shanklin and Cahoon, the Penn State researchers inserted their gene into the bacterium and allowed it to be expressed, resulting in the production of two unsaturated fatty acids new to E. coli. The researchers then isolated enzymes from E. coli and placed them in contact with various saturated fatty acids.

"We expected the enzyme encoded by our gene to act on the saturated fatty acids known as palmitic (16:0) acid and stearic (18:0) acid," says Schultz. "We discovered that the pathway actually was more complex. 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."

Penn State is currently in the process of patenting use of this gene, which could have applications in both agriculture and industry. For instance, when more genes are identified, geneticists may be able to transfer this mechanism of pest resistance to important crops such as tomatoes and potatoes, which have similar trichomes.

David Schultz, Ph.D., received his degree from the College of Agricultural Sciences in May 1996; he is currently a postdoctoral researcher at Michigan State University. June Medford, Ph.D., is now at Colorado State University. Other collaborators were: Richard Craig, Ph.D., the J. Franklin Styer professor of horticultural botany (16 Tyson Building, University Park, PA 16802; 814-863-2191; xza@psuvm.psu.edu); Diana Cox-Foster, Ph.D., associate professor of entomology; postdoctoral fellow Ellen Yerger; and Ralph Mumma, Ph.D., distinguished professor of environmental quality, all in the College of Agricultural Sciences. Their work was funded by the USDA, the Commonwealth of Pennsylvania, and the University, and published in the August 6 Proceedings of the National Academy of Sciences.

Last Updated May 01, 1997