The eight Cys protease inhibitory domains of potato multicystatin exhibit strong functional variability against digestive Cys proteases of the Colorado potato beetle

Herbivorous Coleoptera, including the Colorado potato beetle, compensate to dietary protease inhibitors in plant tissues through a multicomponent defensive strategy involving the overexpression of inhibitor-sensitive digestive proteases, the expression of proteases insensitive to the inhibitors, and proteolytic inactivation of the inhibitory proteins using nontarget proteases. More than 30 digestive Cys proteases have been identified so far in the potato beetle, in line with the observed ability of this insect to elude the detrimental effects of Cys protease inhibitors -or cystatins- induced in wounded potato leaves. Our goal, in this study, was to determine whether functional diversity of the potato beetle digestive Cys protease complement was matched with some functional variability of the potato host cystatin complement, using the wound-inducible eight-domain cystatin, potato multicystatin (PMC), as a model. Computational modelling of the PMC domains interacting with Cys proteases of the beetle suggested the onset of variable interaction strengths for the PMC domains, then confirmed empirically with protease inhibitory data showing complementary protease inhibitory spectra among the eight domains. Some domains of PMC showed strong activity against potato beetle cathepsin L-like proteases (e.g. domains 2 and 6), in contrast with other domains targeting cathepsin B-like proteases (domains 1 and 3) or with domains showing no particular target specificity (domains 4 and 5). These data suggest, overall, that functional variability among PMC inhibitory domains has evolved in response to predatory challenges by the Colorado beetle relying on Cys proteases for potato leaf protein digestion.