A plant defensive cystatin (soyacystatin) targets cathepsin L-like digestive cysteine proteinases (DvCALs) in the larval midgut of western corn rootworm (Diabrotica virgifera virgifera)

Feeding bioassay results established that the soybean cysteine: proteinase inhibitor N (soyacystatin N, scN) substantially,; inhibits growth and devel opment of western corn rootworm (WCR), bg. attenuating digestive proteolysi s [Zhao, Y. et al, (1996) Plant Physiol. 111, 1299-1306], Recombinant scN w as more inhibitory than the potent and broad specificity cysteine proteinas e inhibitor E-64. WCR digestive proteolytic activity was separated by mildl y denaturing SDS-P,ICE into two fractions and in-gel assays confirmed that the proteinase activities of each were largely scN-sensitive. Since binding affinity to the target proteinase [Koiwal H. et al. (1998) Plant J. 14, 37 1-380] governs the effectiveness of scN as a proteinase inhibitor and an in secticide, five peptides (28-33 kDa) were isolated from WCR gut extracts by scN affinity chromatographic separation. Analysis of the N-terminal sequen ce of these peptides revealed similarity to a cathepsin L-like cysteine pro teinase (DvCAL1, Diabrotica virgifera virgifera cathepsin L) encoded by a W CR cDNA. Our results indicate that cathepsin L orthologs are pivotal digest ive proteinases of WCR larvae, and are targets of plant defensive cystatins (phytocystatins), like scN. (C) 2000 Federation of European Biochemical So cieties.