Submitted to: Journal of Insect Biochemistry and Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/15/2001
Publication Date: 1/31/2002
Citation: FABRICK, J., BEHNKE, C., CZAPLA, T., BALA, K., RAO, A.G., KRAMER, K.J., REECK, G.R. EFFECTS OF A POTATO CYSTEINE PROTEINASE INHIBITOR ON MIDGUT PROTEOLYTIC ENZYME ACTIVITY AND GROWTH OF THE SOUTHERN CORN ROOTWORM, DIABROTICA UNDECIMPUNCTATA HOWARDI (COLEOPTERA: CHYSOMELIDAE). JOURNAL OF INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY 32: 405-415. 2002. Interpretive Summary: The southern corn rootworm causes extensive damage to many grass crops. Cultivars resistant to rootworms can be developed using biotechnology. We identified and characterized a protein from potato that selectively inhibits the major proteinase in rootworm gut extracts and also demonstrated that this protein retards insect growth when administered orally. The results indicate that the gene for this inhibitor could be manipulated through genetic engineering to develop insect-resistant plants.
Technical Abstract: The major proteinase activity in extracts of larval midguts from the southern corn rootworm (SCR), Diabrotica undecimpunctata howardi, was identified as a cysteine proteinase that prefers substrates containing an arginine residue in the P1 position. Gelatin-zymogram analysis of midgut proteinases indicated that diet-fed SCR, root-fed SCR, and root-fed western ncorn rootworms (Diabrotica virgifera virgifera) possess a single major proteinase with an apparent molecular mass of 25 kDa and several minor proteinases. Similar proteinase activity pH profiles also were exhibited by root-fed and diet-fed rootworms, with the optimal activity being slightly acidic. Rootworm larvae reared on corn roots exhibited significantly less caseinolytic activity than those reared on an artificial diet. Midgut proteolytic activity from SCR was most sensitive to inhibition by inhibitors of cysteine proteinases. Furthermore, rootworm proteinase activity was particularly sensitive to inhibition by a commercial protein preparation from potato tubers (PIN-II). One of the proteins, potato cysteine proteinase inhibitor-10', PCPI-10', obtained from PIN-II by ion-exchange chromatography, was the major source of inhibitory activity against rootworm proteinase activity. PCPI-10' and E-64 were of comparable potency as inhibitors of southern corn rootworm proteinase activity (IC50 = 31 and 35nM, respectively) and substantially more effective than chicken egg white cystatin (IC50 = 121 nM). Incorporation of PCPI-10' into the diet of SCR larvae in feeding trials resulted in a significant increase in mortality and growth inhibition. We suggest that expression of inhibitors such as PCPI-10' by transgenic corn plants in the field is a potentially attractive method of controlling larvae of these Diabrotica species.