BmTudor-sn Is a Binding Protein of Destruxin A in Silkworm Bm12 Cells.

Destruxin A (DA), a hexa-cyclodepsipeptidic mycotoxin secreted by the entomopathogenic fungus , was reported to have an insecticidal effect and anti-immunity activity. However, its molecular mechanism of action remains unclear. Previously, we isolated several potential DA-affinity (binding) proteins in the Bm12 cell line. By docking score using MOE2015, we selected three proteins-BmTudor-sn, BmPiwi, and BmAGO2-for further validation. First, using Bio-Layer Interferometry in vitro, we found that BmTudor-sn had an affinity interaction with DA at 125, 250, and 500 µM, while BmPiwi and BmAGO2 had no interaction signal with DA. Second, we employed standard immunoblotting to verify that BmTudor-sn is susceptible to DA, but BmPiwi and BmAGO2 are not. Third, to verify these findings in vivo, we used a target engagement strategy based on shifts in protein thermal stability following ligand binding termed the cellular thermal shift assay and found no thermal stability shift in BmPiwi and BmAGO2, whereas a shift was found for BmTudor-sn. In addition, in BmTudor-sn knockdown Bm12 cells, we observed that cell viability increased under DA treatment. Furthermore, insect two-hybrid system results indicated that the key site involved in DA binding to BmTudor-sn was Leu704. In conclusion, in vivo and in vitro experimental evidence indicated that BmTudor-sn is a binding protein of DA in silkworm Bm12 cells at the 100 µM level, and the key site of this interaction is Leu704. Our results provide new perspectives to aid in elucidating the molecular mechanism of action of DA in insects and developing new biopesticide.