Coevolution of Colorado potato beetle digestive Cys proteases and potato multicystatin inhibitory domains: An example of mutual functional diversification?

Herbivorous Coleoptera, such as the Colorado potato beetle (CPB) (Leptinotarsa decemlineata Say), compensate to dietary protease inhibitors in plant tissues via a multicomponent defensive strategy involving the overexpression of inhibitor-insensitive proteases. Nearly 40 digestive Cys digestive proteases have been identified in the CPB, belonging to six structural, functionally distinct subgroups. In potato, the eight inhibitory domains of multicystatin (PMC), a wound inducible inhibitor of Cys proteases, show structure/function heterogeneity thought to be the result of a functional diversification process to match CPB midgut proteases diversity. Here we show that primary structure variations among PMC subunits indeed resulted in functional diversification towards CPB Cys proteases. As shown with diet bioassays, both growth and the midgut protease complement are differentially impacted in CPB larvae supplied with individual PMC inhibitory domains. Growth data also suggest that the most potent domains of PMC, as inferred by in vitro enzyme assays, significantly delay larval growth, unlike less efficient domains having no impact. We are currently comparing the inhibitory efficiency of PMC inhibitory domains with the efficiency of the inhibitory domains of tomato multicystatin, a close structural homologue from a non-host plant. The aim is to test further the hypothesis of a mutual coevolutionary link between the inhibitory potency of PMC inhibitory domains against CPB midgut Cys proteases and functional diversity of these digestive enzymes in the insect herbivore.