Phage display selection can differentiate insecticidal activity of soybean cystatins

Abstract

SummaryPlant cysteine proteinase inhibitors (phytocystatins) have been implicated as defensive molecules against Coleopteran and Hemipteran insect pests. Two soybean cystatins, soyacystatin N (scN) and soyacystatin L (scL), have 70% sequence identity but scN is a much more potent inhibitor of papain, vicilin peptidohydrolase and insect gut proteinases. When these cystatins were displayed on phage particles, papain-binding affinity and CPI activity of scN were substantially greater than those of scL, in direct correlation with their relative CPI activity as soluble recombinant proteins. Furthermore, scN substantially delayed cowpea weevil (Callosobruchus maculatus (F.)) growth and development in insect feeding bioassays, whereas scL was essentially inactive as an insecticide. Papain biopanning selection of phage-displayed soyacystatins resulted in a 200-1000-fold greater enrichment for scN relative to scL. These results establish that binding affinity of cystatins can be used in phage display biopanning procedures to select variants with greater insecticidal activity, illustrating the potential of phage display and biopanning selection for directed molecular evolution of biological activity of these plant defensive proteins.

Purification and Biochemical Characterization of a Cystatin-Like Thiol Proteinase Inhibitor from Cicer arietinum (Chickpea)

“…The unique feature of phytocystatin family is a highly conserved G-58 residue in the QVVAG motif (102-106) and the PW motif (132-133) [18]. Cystatin has been successfully isolated in the plants as Rice [8,19,20], soybean [21,22], sugarcane [23], Maize [24] and Kiwi fruit [25]. Phytocystatins have also been isolated from plants such as cowpea, potato, and carrot [26][27][28].…”

Purification of trypsin inhibitor from seeds of Cicer arietinum (L.) and its insecticidal potential against Helicoverpa armigera (Hübner)

“…Plants synthesize proteinase inhibitors, alpha-amylase inhibitors, lectins and chitin binding proteins to resist herbivorous insects, pathogens and wounding (De Leo et al 2001). Proteinase inhibitors have also been shown to act as a defensive compound against phytophagous insects by the direct assay or expression in transgenic crop plants (Koiwa et al 1998, Vain et al 1998. Studies have shown that a major part of Helicoverpa armigera gut proteinase activity can be blocked by soybean kunitz trypsin inhibitor ( Johnston et al 1991, Harsulkar et al 1999.…”

“…Studies have shown that a major part of Helicoverpa armigera gut proteinase activity can be blocked by soybean kunitz trypsin inhibitor ( Johnston et al 1991, Harsulkar et al 1999. Numerous insect-feeding bioassays and experiments with transgenic plants have also shown the delayed growth and development of the insect (Koiwa et al 1998, Parde et al 2010.…”

EVOLUTIONAL AND FUNCTIONAL ANALYSIS OF A SERINE PROTEASE IN Spodoptera litura

“…SPs act in insect growth and development, which raises the possibility that SP inhibitors may be the bases of a new insect pest management technology (Girard et al, 1998;Koiwa et al, 1998). The effect of these inhibitors is generally stable.…”