Submitted to: Journal of Insect Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/27/2006
Publication Date: 1/1/2006
Citation: Mohan, S., Ma, W.K., Pechan, T., Bassford, E., Williams, W.P., Luthe, D.S. 2006. Degradation of the S. frugiperda peritrophic matrix by an inducible maize cysteine protease. Journal of Insect Physiology. 52:21-28. Interpretive Summary: Feeding by fall armyworm and southwestern corn borer on leaves of young corn plants can markedly reduce yields and quality of grain. The development and release of corn germplasm lines with genetic resistance to these pests provides a desirable and cost effective way to reduce losses. An understanding of the factors responsible for resistance will expedite incorporation of insect resistance into commercial corn hybrids through conventional breeding methods or molecular marker assisted selection. Fall armyworm or southwestern corn borer larvae that feed on leaves of resistant plants are smaller than those that feed on susceptible plants. Scanning electron micrographs of the peritrophic membrane, a membrane lining of the insect’s gut, excised from insects that were fed on resistant corn genotypes revealed extensive damage. A unique protein that accumulates at the sites where larvae feed on leaves within the plant whorls has been identified. Treating membranes that had been removed from laboratory-reared insects with a purified form of the unique protein, a cysteine protease, indicated that this protein directly affects the peritrophic membrane. These results indicate that transferring the gene responsible for production of this protein into susceptible corn genotypes lacking the gene should increase their resistance to fall armyworm and, thereby, reduce losses to this insect.
Technical Abstract: A unique 33-kD cysteine protease (Mir1-CP) rapidly accumulates at the feeding site in the whorls of maize (Zea mays L.) lines that are resistant to herbivory by Spodoptera frugiperda and other lepidopteran species. When larvae were reared on resistant plants, larval growth was reduced due to impaired nutrient utilization. Scanning electron microscopy (SEM) indicated that the peritrophic matrix (PM) was damaged when larvae fed on resistant plants or transgenic maize callus expressing Mir1-CP. To directly determine the effects of Mirl-CP on the PM, a baculovirus-based system was used to express the protease in S. frugiperda larvae. The expressed protein was collected from the hemolymph of infected larvae and purified by size exclusion and reverse phase chromatography. The effect of purified Mir1-CP on PM permeability was determined by measuring the movement of Blue Dextran 2000 across the PM. Mirl-CP completely permeabilized the PM and the time required to reach full permeability was inversely proportional to the concentration of Mir1-CP. Inclusion of E64, a specific cysteine protease inhibitor prevented the damage. The lumen side of the PM was more vulnerable to Mir1-CP attack than the epithelial side. Mir1-CP damaged the PM at pH values as high as 8.5 and was more active than equivalent concentrations of the cysteine proteases papain, bromelain and ficin. The effect of Mir1-CP on the PMs of Helicoverpa zea, Danaus plexippus, Ostrina nubilalis, Periplaneta americana and Tenebrio molitor also was tested, but the greatest effect was on the S. frugiperda PM. These results demonstrate that the insect-inducible Mir1-CP directly damages the PM in vitro.