ARS | Publication request: Cloning eleven midgut trypsin cDNAs and evaluating the interaction of proteinase inhibitors with Cry1Ac against the tobacco budworm Heliothis virescens (F.) (Lepidoptera: Noctuidae)

Submitted to: Journal of Invertebrate Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 10, 2012
Publication Date: N/A

Interpretive Summary: In addition to participating in the digestion of dietary proteins, trypsins are associated with Bt protoxin activation and toxin degradation. Proteinase inhibitors have potential insecticidal toxicity against a wide range of insect species. Using proteinase inhibitors to synergize Bt performance may delay Bt resistance development and increase the durability of Bt plant technology. Intensive cloning and sequencing of midgut proteinase cDNAs may facilitate future gene knockdown or other functional studies to examine the individual role of proteinases in Bt toxicity and resistance development. This study focused on sequencing of larval midgut proteinase cDNAs from the tobacco budworm and evaluation of proteinase inhibitors for synergistic interaction with Bt toxin, with an ultimate goal to make transgenic cotton versatile and durable. Eleven trypsin-like cDNAs were cloned and sequenced from the midgut of the tobacco budworm. All of the predicted trypsins contained the conserved N-termini motif IVGG, three catalytic center residues, substrate specificity determinant residues, and 6 cysteines for disulfide bridges. These putative trypsins were separated into 3 distinct groups, indicating the diverse proteinases that have evolved in this polyphagous insect. A sublethal dose (15 ppb) of Bt Cry1Ac, 0.75% soybean trypsin inhibitor (STI), and 0.1% and 0.2% N-a-tosyl-L-lysine chloromethyl ketone (TLCK) significantly suppressed midgut azocaseinase, tryptic, and chymotryptic activities, and resulted in reductions in larval and pupal size and mass. The treatment with inhibitor+Bt suppressed approximately 65% more larval body mass and 21% more enzymatic activities than the inhibitor-only or Bt-only. These results indicated that proteinase inhibitors have potential insecticidal activity and the interaction of Bt with proteinase inhibitors is desirable for enhancing Bt toxicity and delaying resistance development in targeted insects.

Technical Abstract: Midgut trypsins are associated with Bt protoxin activation and toxin degradation. Proteinase inhibitors have potential insecticidal toxicity against a wide range of insect species. Proactive action to examine trypsin gene profiles and proteinase inhibitors for interaction with Bt toxin is necessary for making transgenic cotton versatile and durable. Eleven trypsin-like cDNAs were sequenced from the midgut of Heliothis virescens. All trypsins contained three catalytic center residues (H73, D153, and S231), substrate specificity determinant residues (D225, G250, and G261), and 6 cysteines for disulfide bridges. These putative trypsins were separated into 3 distinct groups, indicating the diverse proteinases evolved in this polyphagous insect. A sublethal dose (15 ppb) of Cry1Ac, 0.75% soybean trypsin inhibitor, and 0.1% and 0.2% N-a-tosyl-L-lysine chloromethyl ketone significantly suppressed midgut proteinase activities, and resulted in reductions in larval and pupal size and mass. The treatment with inhibitor+Bt suppressed approximately 65% more larval body mass and 21% more enzymatic activities than the inhibitor-only or Bt-only. These results indicated that intensive sequencing of trypsin cDNAs will facilitate future functional examination of individual role in Bt toxicity and resistance development and facilitate target control with RNAi and/or proteinase inhibitors. Proteinase inhibitors have potential insecticidal activity and the interaction of Bt with proteinase inhibitors is desirable for enhancing Bt toxicity and delaying resistance development.