"The enzyme is found in insect-resistant strains of corn, and it breaks down proteins and peptides in the insects’ gut. It is a unique active defense against herbivory," says Dawn Luthe, professor of plant stress biology at Penn State.
Luthe and researchers at Mississippi State University have since developed several lines of corn resistant to multiple pests, using conventional plant breeding and insect-resistant strains of corn from Antigua.
Researchers have found that when caterpillars fed on the insect-resistant plants, one enzyme -- Mir1-CP or maize insect resistance cysteine protease, in particular --accumulated at the feeding site within an hour of the caterpillar’s feeding and continued to accumulate at the site for several days.
"Upon isolation and purification of the enzyme, we found that Mir1-CP binds to chitin, a major component of insects and fungi," says Luthe. "Physiological tests show that caterpillars have impaired nutrient utilization when they eat the enzyme. They just can’t convert what they eat into body mass."
Luthe presented the findings at the annual meeting of the American Chemical Society today (March 30) in Atlanta.
With the help of antibodies specific to the enzyme, the researchers were able to determine that Mir1-CP is made in the vascular bundles, or strands of conducting vessels in the stem and leaves of a plant. Luthe thinks that when an insect starts feeding, the enzyme is probably transported to vascular tissue that conducts sugars and other metabolic products upward from the leaves, as well as to the soft tissue found in leaves and stem.
Though it is still unclear whether the transport of Mir1-CP is a specific response to the insect feeding, studies show that maize tissue that naturally expresses Mir1-CP causes a 50 percent inhibition in caterpillar growth. Transgenic black Mexican sweet corn cells that express Mir1-CP inhibit caterpillar growth by 70 percent, Luthe says.
Mir1-CP is harmful to caterpillars mainly because of its damaging effect on their peritrophic matrix. This is a membrane that lines the gut of most insects and aids digestion. It also protects the insects from being invaded by microorganisms and parasites through the food they eat.
At the heart of the matrix is a protein called the insect intestinal mucin, or IIM. It is very similar to the mucus layer in animals and is vital for nutrient utilization because it helps the flow of nutrients into the food gut.
The researchers tested the permeability of the matrix using blue dextran, a fermented sugar solution commonly used as a molecular size marker. Results showed that Mir1-CP created holes in the matrix.
To replicate the test in vivo, the researchers fed caterpillars with plants susceptible to the insects and those resistant to them. Results indicate that after seven days, the level of both IIM and IIM messenger RNA in insects that were feeding on the resistant plants had fallen significantly.
"If the IIM is being degraded by the enzyme, pieces of it should not appear in the fecal pellets of the insect," notes the Penn State researcher.
When used in conjunction with the Bt-toxin, a low dose of Mir1-CP was able to achieve a very high mortality rate in the insects, as well as an extremely low growth rate.
"In the long run, the enzyme degrades the insect’s peritrophic matrix and retards the caterpillar’s ability to generate a new one," says Luthe.
The research has potential global implications in generating a cheap and highly effective way of controlling crop pests.
Other authors of the paper include Tibor Pechan, Srinidi Mohan, Renuka Shivaji, Lorena Lopez, Alberto Camas, Erin Bassford, Seval Ozkan, Peter Ma, all at Mississippi State University; and W. Paul Williams, U.S.D.A.
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