Bacillus thuringiensis is the most widely used biopesticide, and its Cry toxin genes are essential transgenes for the generation of insect‐resistant transgenic crops. Recent reports have suggested that ATP‐binding cassette transporter subfamily C2 (ABCC2) proteins are implicated in Cry intoxication, and that a single amino acid insertion results in high levels of resistance to Cry1 toxins. However, there is currently no available direct evidence of functional interactions between ABCC2 and Cry toxins. To address this important knowledge gap, we investigated the role of Bombyx mori ABCC2 (BmABCC2) or its mutant from a Cry1Ab‐resistant B. mori strain on Cry1A toxin action. When we expressed BmABCC2 ectopically on Sf9 cells, it served as a functional receptor, and the single amino acid insertion found in BmABCC2 from Cry1Ab‐resistant larvae resulted in lack of susceptibility to Cry1Ab and Cry1Ac. Using the same expression system, we found that Bo. mori cadherin‐like receptor (BtR175) conferred susceptibility to Cry1A toxins, albeit to a lower degree than BmABCC2. Coexpression of BtR175 and BmABCC2 resulted in the highest cell susceptibility to Cry1A, Cry1F, and even the phylogenetically distant Cry8Ca toxin, when compared with expression of either receptor alone. The susceptibility observed in the coexpressing cells and that in Bo. mori larvae are likely to be correlated, suggesting that BtR175 and BmABCC2 are important factors determining larval susceptibility. Our study demonstrates, for the first time, Cry toxin receptor functionality for ABCC2, and highlights the crucial role of this protein and cadherin in the mechanism of action of Cry toxin.

Hemocytin, a multidomain protein from Bombyx mori L. (Lepidoptera: Bombycidae), is an ortholog of von Willebrand factor and is expected to be a major mediator of hemocyte aggregation. Antiserum was generated against hemocytin, and immune staining of hemocytes, hemolymph, and nodules was performed. Hemocytin was observed in steady-state hemocytes but not in plasma, even after bacterial injection. When hemolymph was smeared on glass slides, hemocytin-containing fibrous structures formed a cellular network mainly consisting of granulocytes and oenocytoids. Hemocytin was stained only in the granules of the granulocytes. When nodule-like aggregates formed 30 sec after bacterial injection, both granulocytes and bacterial cells were observed binding to hemocytin-containing fibrous structures. When nodule sections were stained with antiserum, hemocytin was seen in the matrix of the nodules surrounding the hemocytes. These data suggest that hemocytin plays a major role in nodule formation as a component of the sticky fibrous structure exocytosed from granulocytes.

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The determination of the receptor‐binding region of Cry toxins produced by Bacillus thuringiensis is expected to facilitate an improvement in their insecticidal ability through protein engineering. We analyzed the region on Cry1Aa molecules involved in interactions with the cadherin‐like protein receptor BtR175 using cysteine‐substituted mutant toxins and several synthetic peptides corresponding to the loops in domain 2. In addition, the region necessary to trigger oligomerization was analyzed using these mutant toxins. The mutant toxins were modified by two types of molecule, i.e. digested fragments of the Cry1Aa precursor with an average molecular mass of 2 kDa and 5‐iodoacetamidofluorescein, which has a molecular mass of 515 kDa. We examined whether these modifications interfere with the toxin–BtR175 interaction as a result of steric hindrance. 5‐Iodoacetamidofluorescein modification of R311C, N376C and G442C revealed steric hindrance effects, indicating that R311 on loop 1, N376 on loop 2 and G442 on loop 3 are on the contact face of the toxin–BtR175 interface when Cry1Aa binds to BtR175. Loop 2 is thought to interact with BtR175 directly, as a peptide corresponding to the N‐terminal half of loop 2, (365)LYRRIILG(372), has the potential to bind to BtR175 fragments. Meanwhile, mutant toxins with cysteine substitutions in loops 1 and 2 were oligomerized by the binding of digested fragments in the activation process without receptor interaction, and the wild‐type toxin formed oligomers by interaction with BtR175 fragments. These observations suggest that loops 1 and 2 form both a binding region and a sensor region, which triggers toxin oligomer formation. Structured digital abstract http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7259673, http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7259722, http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7259737, http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7259757, http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7259774, http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7259791, http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7259808, http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7259685, http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7259707, http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7259830: btr175 (uniprotkb:http://www.ebi.uniprot.org/entry/Q9XY09) binds (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407) to cry1Aa (uniprotkb:http://www.ebi.uniprot.org/entry/P0A366) by surface plasmon resonance (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0107)