The focus of this research is a unique protein-degrading enzyme, a cysteine protease called Mir1-CP that rapidly accumulates in insect resistant corn lines in response to caterpillar attack. The insect resistant corn lines were developed by conventional plant breeding from plant material that originated in Antigua. Mir1-CP is unique because the sequence of the enzyme's last 25 amino acids has no matches in the currently available databases. Mir1-CP appears to be mobilized within one hour of caterpillar feeding and accumulates at the wound site. When caterpillars feed on plant tissues containing Mir1-CP, their growth is reduced approximately 50% to 80%. This growth reduction is due to impaired nutrient utilization.
To investigate the effect of Mir1-CP on caterpillars, the investigators examined the insect's first line of defense against potentially toxic components in food, the peritrophic matrix (PM). The PM is a membrane-like structure that consists of proteins and chitin. It surrounds the food bolus, protects the insect midgut from chemical and physical damage and plays an active role in digestion and nutrient absorption. Scanning electron microscopy (SEM) revealed that PMs of insects that feed on plant material containing Mir1-CP were severely damaged with numerous perforations, cracks, and holes. Damage to the PM is likely to disrupt the digestive system and stunt caterpillar growth. Consequently, the smaller caterpillars have longer life-cycles and are more susceptible to parasites and predators in the environment.
The major goal of this research project is to determine how Mir1-CP damages the caterpillar midgut structure. Does it degrade PM proteins? Does it attack specific PM proteins? Does it prevent PM formation by binding to chitin or other PM components? The investigators will use two different strategies for testing Mir1-CP's effect on the PM. First, caterpillars will be fed transgenic plant material that expresses either normal Mir1-CP, Mir1-CP that has been mutated and has no enzyme activity, or Mir1-CP that is missing the last 25 amino acids. The appearance and permeability of the midgut and PM structure of caterpillars that eat these transgenic lines will be examined. The results will reveal whether protease activity or the presence of the last 25 amino acids is needed to damage the caterpillar midgut and PM. Second, the investigators will express Mir1-CP and the mutated forms in a heterologous system that will permit purification of large amounts of each Mir1-CP form. The purified proteins will be fed to caterpillars to see if they result in the same effects as those expressed in plants. In addition, PMs that have been dissected from caterpillars will be incubated with the three Mir1-CP forms to determine if they have a direct effect on PM structure, or degrade PM proteins.
Ultimately, this research will permit determination of whether Mir1-CP can be used as an environmentally friendly insect control agent. Insect herbivory is responsible for 15% of the world's crop losses each year. These losses are major economic and ecological problems that decrease the supply of food to a growing world population. US farmers have been controlling caterpillar pests by planting transgenic crops that express Bacillus thuringiensis (Bt) toxin. Currently, Bt-toxin is highly effective, but scientists predict that insects will eventually become resistant to the toxin. By understanding how Mir1-CP stunts caterpillar growth, scientists may be able to use Mir1-CP as an alternative to Bt-toxin in the future.
