Molecular modeling and inhibitory activity of cowpea cystatin against bean bruchid pests

Abstract

Plant cystatins show great potential as tools to genetically engineer resistance of crop plants against pests. Two important potential targets are the bean weevils Acanthoscelides obtectus and Zabrotes subfasciatus, which display major activities of digestive cysteine proteinases in midguts. In this study a cowpea cystatin, a cysteine proteinase inhibitor found in cowpea (Vigna unguiculata) seeds, was expressed in Escherichia coli and purified with a Ni-NTA agarose column. It strongly inhibited papain and proteinases from midguts of both A. obtectus and Z. subfasciatus bruchids, as seen by in vitro assays. When the protein was incorporated into artificial seeds at concentrations as low as 0.025%, and seeds were consumed by the bruchids larva, dramatic reductions in larval weight, and increases in insect mortality were observed. Molecular modeling studies of cowpea cystatin in complex with papain revealed that five N-terminal residues responsible for a large proportion of the hydrophobic interactions involved in the stabilization of the enzyme-inhibitor complex are absent in the partial N-terminal amino acid sequencing of soybean cystatin. We suggest that this structural difference could be the reason for the much higher effectiveness of cowpea cystatin when compared to that previously tested phytocystatin. The application of this knowledge in plant protein mutation programs aiming at enhancement of plant defenses to pests is discussed.

Crystal structure of tarocystatin–papain complex: implications for the inhibition property of group-2 phytocystatins

“…Recently, the inhibitory ability of cysteine protease inhibitors in plants was used to enhance the antipest and antifungus abilities of plants (Martinez et al 2003, 2005; Aguiar et al 2006; Christova et al 2006; Goulet et al 2008; Senthilkumar et al 2010). Several lines of evidence support that cystatins in plants regulate the activity of cysteine protease for physiological and developmental processes in seed germination, organogenesis, and programed cell death (Kumar et al 1999; Arai et al 2002; Rivard et al 2007; Valdes-Rodriguez et al 2007; Martinez et al 2009) and are involved in the complicated stress response to salt, drought, and oxidation (Diop et al 2004; Zhang et al 2008; Megdiche et al 2009).…”

Biochemical and molecular characterization of senescence‐related cysteine protease–cystatin complex from spinach leaf

“…The W residue in the C‐terminal region is at the second inhibitory loop, with one or two glycine residues placed at the N‐terminal region (Margis et al 1998). The G residue in the N‐terminal region does not interact directly with the active site but is required for activity (Bode et al 1988, Turk et al 1997), and surrounding residues stabilize the protease–cystatin complex (Aguiar et al 2006). Most of them consist of a single domain with a molecular mass of 11–16 kDa, show meaningful amino acid sequence homology and contain no disulfide bonds, although some are multicystatins, and an eight‐unit cystatin has been found (Girard et al 2007, Waldron et al 1993).…”

Resistance of important bean genotypes to the Mexican bean beetle [Zabrotes subfasciatus (Bohemann)] during storage and its control with chemical synthetic and botanical insecticides

“…Despite this fact, Z. subfasciatus is recognized as being one of the few Bruchids able to feed on a variety of beans, including those belonging to the genera Phaseolus (common beans), Vigna (cowpea) (Sales et al, 2005;Bifano et al, 2010) and Vicia (broad beans) (Pacheco and Paula, 1995;Toledo et al, 2013). Differences in resistance of bean species to Z. subfasciatus have been well documented and are attributed to many causes, including the presence of trypsin and proteinase inhibitors, lecithins and tannins, all of which are deleterious to the development of Z. subfasciatus (Osborn et al, 1986;Posso et al, 1992;Pereira et al, 1995;Guzmán-Maldonado et al, 1996;Acosta-Gallegos et al, 1998;Barbosa et al, 2000;Aguiar et al, 2006;Moraes et al, 2011). However, many of these defenses do not seem to affect Z. subfasciatus, as its larvae are able to secrete α-amylases that are insensitive to the α-amylase inhibitor found in seeds of P. vulgaris (Bifano et al, 2010).…”

“…Despite the relative plasticity shown by Z. subfasciatus during host selection, slight differences may exist in Z. subfasciatus preference for or performance on different beans because it is well accepted that inhibitors can be very efficient in blocking enzymes from organisms that are not specialized on that plant (Aguiar et al, 2006).…”