Plant cystatin-mushroom macrocypin fusions for the broad-spectrum inhibition of Leptinotarsa decemlineata digestive Cys proteases

Numerous papers have discussed the potential of protease inhibitors to engineer insect resistance in transgenic plants but the general usefulness of this approach in crop protection still remains equivocal. Herbivorous insects have evolved various strategies to cope with dietary protease inhibitors, such as the use of digestive proteases re­calcitrant to inhibition, that often make the selection of effective inhibitors very challenging. In this study, we assessed the potential of mushroom macrocypins, a recently described family of Cys protease inhibitors from edible mushrooms, as a complement to tomato cystatin SlCYS8 and different cystatin hybrids for the inhibition of Colorado potato beetle (CPB) (Lep­tinotarsa decemlineata) digestive Cys proteases. Plant cystatins, in general, show good inhibitory potency against CPB cathepsin L-like proteases but weak affinity for cathepsin B-like enzymes, a family of proteases known to be recalcitrant to inhibition. We here used an activity-based functional proteomics approach to capture and iden­tify CPB midgut Cys proteases sensitive to macrocypins Mcp1 and Mcp4. We show the mushroom inhibitors to exhibit affinity for CPB cathepsins B but weak affinity for CPB cathepsins L compared to SlCYS8. We also show translational fusion inhibitors composed of plant cystatins and Mcp1 (or Mcp4) to retain the original affinity spectrum of the two inhibi­tor partners, and thus to represent broad-spectrum inhibitors for the whole set of CPB midgut Cys proteases. These data confirm the potential of plant cystatin–mushroom macrocypin fusions to reproduce the broad inhibitory effect of E-64, a low-molecular-weight Cys protease inhibitor of fungal origin toxic to CPB larvae.