Abstract
Understanding how Bacillus thuringiensis (Bt) toxins interact with proteins in the midgut of susceptible coleopteran insects is crucial to fully explain the molecular bases of Bt specificity and insecticidal action. In this work, aminopeptidase N (TcAPN-I), E-cadherin (TcCad1), and sodium solute symporter (TcSSS) have been identified by ligand blot as putative Cry3Ba toxin-binding proteins in Tribolium castaneum (Tc) larvae. RNA interference knockdown of TcCad1 or TcSSS proteins resulted in decreased susceptibility to Cry3Ba toxin, demonstrating the Cry toxin receptor functionality for these proteins. In contrast, TcAPN-I silencing had no effect on Cry3Ba larval toxicity, suggesting that this protein is not relevant in the Cry3Ba toxin mode of action in Tc. Remarkable features of TcSSS protein were the presence of cadherin repeats in its amino acid sequence and that a TcSSS peptide fragment containing a sequence homologous to a binding epitope found in Manduca sexta and Tenebrio molitor Bt cadherin functional receptors enhanced Cry3Ba toxicity. This is the first time that the involvement of a sodium solute symporter protein as a Bt functional receptor has been demonstrated. The role of this novel receptor in Bt toxicity against coleopteran insects together with the lack of receptor functionality of aminopeptidase N proteins might account for some of the differences in toxin specificity between Lepidoptera and Coleoptera insect orders.
Highlights
Interaction with insect midgut receptors is required for Bacillus thuringienesis (Bt) toxicity
Using LC-MS/MS spectrometry, among other proteins, we have identified as putative Cry3Ba toxin receptors an APN (NP_001164285, in this work denoted as TcAPN-I), an E-cadherin protein (XP_971388, denoted as TcCad1), and a sodium solute symporter protein (EFA03129, denoted as TcSSS) containing cadherin repeats
Binding experiments showed that Cry3Ba toxin was able to bind to Tribolium castaneum (Tc) Brush Border Membrane Vesicle (BBMV), and this interaction was specific because it was competed with 1000-fold excess of unlabeled Cry3Ba toxin but not with 1000-fold unlabeled Cry3Aa
Summary
Interaction with insect midgut receptors is required for Bacillus thuringienesis (Bt) toxicity. Remarkable features of TcSSS protein were the presence of cadherin repeats in its amino acid sequence and that a TcSSS peptide fragment containing a sequence homologous to a binding epitope found in Manduca sexta and Tenebrio molitor Bt cadherin functional receptors enhanced Cry3Ba toxicity This is the first time that the involvement of a sodium solute symporter protein as a Bt functional receptor has been demonstrated. V-ATP-synthase, and prohibitin have been identified as Cry binding partners in Coleoptera through targeted mass spectrometry protein analysis.4 Whether these molecules act facilitating the toxic process or contributing to the insect response against Cry toxins remains to be investigated and constitutes an important issue to reveal the entire picture of the Bt mode of action. RNA interference experiments with dsRNA of the corresponding genes demonstrated that TcCad and TcSSS proteins were Cry3Ba toxin functional receptors in Tc, whereas TcAPN-I was not related to Cry3Ba susceptibility
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