Post‐ingestive stability of a mulberry Kunitz‐type protease inhibitor MnKTI‐1 in the digestive lumen of silkworm: dual inhibition towards α‐amylase and serine protease

Abstract BACKGROUND Adaptation of specialist insects to their host plants and defense responses of plants to phytophagous insects have been extensively recognized while the dynamic interaction between these two events has been largely underestimated. Here, we provide evidence for characterization of an unrevealed dynamic interaction mode of digestive enzymes of specialist insect silkworm and inhibitor of its host plant mulberry tree. RESULTS MnKTI‐1, a mulberry Kunitz‐type protease inhibitor, whose messenger RNA (mRNA) transcription and protein expression in mulberry leaf were severely triggered and up‐regulated by tens of times in a matter of hours in response to silkworm, Bombyx mori , and other mulberry pest insects, suggesting a quick response and broad spectrum to insect herbivory. MnKTI‐1 proteins were detected in gut content and frass of specialist B. mori , and exhibited significant post‐ingestive stability. Recombinant refolded MnKTI‐1 (rMnKTI‐1) displayed binding affinity to digestive enzymes and a dual inhibitory activity to α‐amylase BmAmy and serine protease BmSP2956 in digestive juice of silkworm. Moreover, data from in vitro assays proved that the inhibition of recombinant rMnKTI‐1 to BmAmy can be reverted by pre‐incubation with BmSP15920, an inactivated silkworm digestive protease that lack of complete catalytic triad. CONCLUSION These findings demonstrate that mulberry MnKTI‐1 has the potential to inhibit the digestive enzyme activities of its specialist insect herbivore silkworm, whereas this insect may employ inactivated proteases to block protease inhibitors to accomplish food digestion. The current work provides an insight to better understand the interacting mode between host plant Kunitz protease inhibitors and herbivorous insect digestive enzymes. © 2024 Society of Chemical Industry.