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

Koiwa, Hisashi; Shade, Richard E.; Zhu-Salzman, Keyan; D’Urzo, Matilde Paino; Murdock, Larry L.; Bressan, Ray A.; Hasegawa, Paul M.

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Volume 471, Issue 1 , Pages 67-70, 7 April 2000

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

Edited by Maurice Montal

Hisashi Koiwa
- Center for Plant Environmental Stress Physiology, Purdue University, West Lafayette, IN 47907-1165, USA
Richard E. Shade
- Department of Entomology, Purdue University, West Lafayette, IN 47907-1165, USA
Keyan Zhu-Salzman
- Department of Entomology, Texas A&M University, College Station, TX 77843-2475, USA
Matilde Paino D’Urzo
- Center for Plant Environmental Stress Physiology, Purdue University, West Lafayette, IN 47907-1165, USA
Larry L. Murdock
- Department of Entomology, Purdue University, West Lafayette, IN 47907-1165, USA
Ray A. Bressan
- Center for Plant Environmental Stress Physiology, Purdue University, West Lafayette, IN 47907-1165, USA
Paul M. Hasegawa
- Center for Plant Environmental Stress Physiology, Purdue University, West Lafayette, IN 47907-1165, USA
- Corresponding author. Fax: (1)-765-494 0391

Received 31 January 2000; received in revised form 8 March 2000

Abstract

Feeding bioassay results established that the soybean cysteine proteinase inhibitor N (soyacystatin N, scN) substantially inhibits growth and development of western corn rootworm (WCR), by attenuating digestive proteolysis [Zhao, Y. et al. (1996) Plant Physiol. 111, 1299–1306]. Recombinant scN was more inhibitory than the potent and broad specificity cysteine proteinase inhibitor E-64. WCR digestive proteolytic activity was separated by mildly denaturing SDS–PAGE 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 [Koiwa, H. et al. (1998) Plant J. 14, 371–380] governs the effectiveness of scN as a proteinase inhibitor and an insecticide, five peptides (28–33 kDa) were isolated from WCR gut extracts by scN affinity chromatographic separation. Analysis of the N-terminal sequence of these peptides revealed similarity to a cathepsin L-like cysteine proteinase (DvCAL1, Diabrotica virgifera virgifera cathepsin L) encoded by a WCR cDNA. Our results indicate that cathepsin L orthologs are pivotal digestive proteinases of WCR larvae, and are targets of plant defensive cystatins (phytocystatins), like scN.

Keywords: Cysteine proteinase inhibitor, Digestive proteolysis, Insect