title: Effects of a potato cysteine proteinase inhibitor on midgut proteolytic enzyme activity and growth of the southern corn rootworm, Diabrotica undecimpunctata howardi (Coleoptera: Chrysomelidae).
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 the midgut proteinases indicated that the artificial diet-fed SCR, corn root-fed SCR, and root-fed western corn rootworms (Diabrotica virgifera virgifera) possess a single major proteinase with an apparent molecular mass of 25kDa and several minor proteinases. Similar proteinase activity pH profiles 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 the 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 (IC(50) =31 and 35nM, respectively) and substantially more effective than chicken egg white cystatin (IC(50) =121nM). 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 host plant resistance to these Diabrotica species.
authors.affiliation: Department of Biochemistry, Willard Hall, Kansas State University, Manhattan, KS 66506, USA.
authors.forename: J
authors.initials: J
authors.lastname: Fabrick
authors.forename: C
authors.initials: C
authors.lastname: Behnke
authors.forename: T
authors.initials: T
authors.lastname: Czapla
authors.forename: K
authors.initials: K
authors.lastname: Bala
authors.forename: A G
authors.initials: AG
authors.lastname: Rao
authors.forename: K J
authors.initials: KJ
authors.lastname: Kramer
authors.forename: G R
authors.initials: GR
authors.lastname: Reeck
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002364:Caseins / Q000378:metabolism; D001517:Coleoptera / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development; D003546:Cysteine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D004064:Digestive System / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005247:Feeding Behavior; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000494:pharmacology*; D011480:Protease Inhibitors / Q000494:pharmacology; D011198:Solanum tuberosum; D013379:Substrate Specificity; D014020:Tissue Extracts; D017186:Titrimetry
chemical_list: D002364:Caseins; D015853:Cysteine Proteinase Inhibitors; D010940:Plant Proteins; D011480:Protease Inhibitors; D014020:Tissue Extracts; D003546:Cysteine Endopeptidases