Skip to main content
ARS Home » Research » Publications at this Location » Publication #104637
Title: INHIBITION OF ASPARTYL AND SERINE PROTEINASES IN THE MIDGUT OF SUGARBEET ROOT MAGGOT WITH BIOCHEMICAL AND PLANT-DERIVED PROTEINASES

Author
item WILHITE, STEPHEN - GENBANK (INDUSTRY)
item ELDEN, THOMAS - USDA, ARS (RETIRED)
item PUIZDAR, VIDA - DEPT BIOCHM, SLOVENIA
item ARMSTRONG, SCOTT - TEX. TECH., LUBBOCK, TX
item Smigocki, Anna

Submitted to: Entomologia Experimentalis et Applicata
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/30/2000
Publication Date: N/A
Citation: N/A

Interpretive Summary: This study characterized the major digestive enzymes (proteinases) in the gut of the sugarbeet root maggot (Tetanops myopaeformis). These larvae are major pests of sugarbeet for which traditional breeding techniques for resistance have not been successful. Insects depend on protainases to break down ingested dietary proteins necessary for their normal growth and development. Numerous inhibitors of proteinases have been found in plants and their genes have been cloned and introduced to crop plants for imparting resistance to insect pests. Significant variations exist among the types and properties of the digestive proteinases, and numerous proteinase inhibitors are available that have a broad range of specificities. It is necessary, therefore, to characterize the gut proteolytic activities of each individual species of insects in order to devise a rational and effective insect control strategy that utilizes proteinase inhibitor genes. This study has identified two major proteinas forms in the sugarbeet root maggot midgut (a serine and an aspartyl proteinase). Thus any rational approach of insect control in transgenic plants expressing proteinase inhibitor genes should target each of these digestive proteinases. This research will be used by scientists to devise environmentally friendly approaches for the control of sugarbeet root maggot, leading to higher crop yields and productivity.

Technical Abstract: The primary aim of this work was to identify the major mechanistic classes of digestive proteinases in midguts of the sugarbeet root maggot (SBRM) Tetanops myopaeformis von Roder (Diptera: Otitidae). Proteolytic activity in extracts prepared from SBRM larvae peaked at pH 2.5 and 9.5. Addition of low-molecular weight biochemical inhibitors targeting the three major classes of insect digestive endoproteinases revealed that Pepstatin A, an aspartyl proteinase inhibitor, was by far the most effective inhibitor at pH 3.0 (83.9% inhibition). E-64, which has high potency toward virtually all known cysteine proteinases had only minor inhibitory activity (6.5%). At pH 8.5, PMSF treatment resulted in a sizable decrease in proteolysis (47.3% inhibition) suggesting that serine proteinases are major contributors to proteolysis at the higher pH. Squash aspartyl proteinase inhibitor (SQAPI) blocked virtually all the proteolytic activity at pH 3.0, ,thus confirming the importance of the aspartyl class at acidic pH. Soybea trypsin-chymotrypsin inhibitor (Bowman-Birk inhibitor I, or BBI) blocked nearly all proteolysis at pH 8.5, suggesting the presence of trypsin and/or chymotrypsin-like serine proteinases in the extract. The potential of successfully implementing these plant proteinase inhibitors in transgenic applications is discussed.