A hybrid, broad-spectrum inhibitor of Colorado potato beetle aspartate and cysteine digestive proteinases

Abstract

Protein engineering approaches are currently being devised to improve the inhibitory properties of plant proteinase inhibitors against digestive proteinases of herbivorous insects. Here we engineered a potent hybrid inhibitor of aspartate and cysteine digestive proteinases found in the Colorado potato beetle, Leptinotarsa decemlineata Say. Three cathepsin D inhibitors (CDIs) from stressed potato and tomato were first compared in their potency to inhibit digestive cathepsin D-like activity of the insect. After showing the high inhibitory potency of tomato CDI (M(r) approximately 21 kDa), an approximately 33-kDa hybrid inhibitor was generated by fusing this inhibitor to the N terminus of corn cystatin II (CCII), a potent inhibitor of cysteine proteinases. Inhibitory assays with recombinant forms of CDI, CCII, and CDI-CCII expressed in Escherichia coli showed the CDI-CCII fusion to exhibit a dual inhibitory effect against cystatin-sensitive and cathepsin D-like enzymes of the potato beetle, resulting in detrimental effects against 3rd-instar larvae fed the hybrid inhibitor. The inhibitory potency of CDI and CCII was not altered after their fusion, as suggested by IC(50) values for the interaction of CDI-CCII with target proteinases similar to those measured for each inhibitor. These observations suggest the potential of plant CDIs and cystatins as functional inhibitory modules for the design of effective broad-spectrum, hybrid inhibitors of herbivorous insect cysteine and aspartate digestive proteinases.

Tailoring the Specificity of a Plant Cystatin toward Herbivorous Insect Digestive Cysteine Proteases by Single Mutations at Positively Selected Amino Acid Sites

“…In this perspective, SlCYS8 variants such as P2F and P2L, which exhibit IC 50 values decreased by 2-to 3-fold for the Colorado potato beetle cystatin-sensitive proteases (Table II), could provide improved antidigestive effects at given expression rates in planta and thus help avoid effective compensatory processes in this target herbivore (Cloutier et al, , 2000Gruden et al, 2003Gruden et al, , 2004Brunelle et al, 2004). An inhibitory spectrum extended to the so-called cystatininsensitive proteases for these same two variants also could make them more effective in vivo, as suggested by a recent study reporting increased deleterious effects for a hybrid corn cystatin II bearing an extra protease inhibitory function (Brunelle et al, 2005). SlCYS8 variants such as P2F and P2L, which combine an improved inhibitory potency against cystatin-sensitive proteases of Colorado potato beetle with a broader inhibitory spectrum against the overall digestive protease complement of this same insect, would thus likely represent promising candidates for the design of insect-resistant transgenic potato lines.…”

“…Work is also under way to estimate the actual antidigestive effects of these same variants against the Colorado potato beetle, keeping in mind the well-documented ability of this insect to overcome the inhibitory effects of plant protease inhibitors by various compensatory and hydrolytic processes. Studies documented the deleterious effects of broad-spectrum low-molecular-weight, nonprotein protease inhibitors (Bolter and Latoszek-Green, 1997) or the significant antidigestive effects of a hybrid, dual cystatin with an extended inhibitory spectrum (Brunelle et al, 2005) against Colorado potato beetle larvae, but effective compensatory processes following cystatin ingestion cannot be ruled out at this stage.…”

Herbivore exploits orally secreted bacteria to suppress plant defenses

“…To investigate if microbial symbionts present in OSs affect induced defenses, we measured the expression of selected defense genes and enzymatic activities in plants damaged by AB-treated or untreated larvae. The cysteine proteinase inhibitor (CysPI) and polyphenol oxidase F/B (PPOF/B) were selected as JA-marker genes because they are well-established JA-inducible proteins that function against CPBs (19)(20)(21). For an SA-marker gene, pathogenesis-related 1 (PR-1(P4)) was selected (22).…”

Review: The future of cystatin engineering

“…Fusing amino acids of a potato carboxypeptidase A inhibitor to the rice OC-I C-terminus decreased OC-I potency against papain [52]. In contrast, a hybrid protease inhibitor targeted against aspartate and cysteine proteases and generated by fusing a cathepsin D inhibitor to the N-terminus of corn cystatin II inhibited both cystatin-sensitive and cathepsin D-like enzymes in the Colorado potato beetle [53].…”