Bt Research 2024, Vol.15, No.1, 42-52 http://microbescipublisher.com/index.php/bt 46 5.3 Biochemical and biophysical assays Biochemical and biophysical assays have provided empirical evidence to support the findings from structural and computational studies. Techniques such as enzyme-linked immunosorbent assay (ELISA), ligand blotting, and site-directed mutagenesis have been used to validate the binding interactions and identify key residues involved (Zhang et al., 2014). For instance, binding assays using Cry1Ab toxin and a fluorescently labeled EC12 fragment of the BT-R1 receptor demonstrated high specificity and affinity, confirming the critical role of conserved sequence motifs in toxin binding (Liu et al., 2018). Additionally, site-directed mutagenesis and binding assays have identified hot spot residues in both Cry1Ac and its receptor, further elucidating the molecular basis of their interaction (Hu et al., 2018). 6 Case Studies of Toxin-Receptor Interactions 6.1 Interaction studies in lepidoptera Lepidopteran insects, such as Spodoptera litura and Spodoptera frugiperda, have been extensively studied to understand the interactions between Bt toxins and their receptors. Cry toxins, particularly Cry1A and Cry2A, bind to specific receptors in the midgut of these insects, leading to cell lysis and insect death (Figure 2). Key receptors include cadherin, aminopeptidase N (APN), and alkaline phosphatase (ALP) (Bravo et al., 2011; Li et al., 2021; Dutta et al., 2022). For instance, ALP2 in Spodoptera exigua has been identified as a functional receptor for Cry2Aa, playing a crucial role in the insect's susceptibility to this toxin (Yuan et al., 2017). Additionally, RNAi-mediated silencing of receptor genes such as CAD, ABCC2, ALP1, and APN in S. frugiperda and S. litura has shown that these receptors are essential for Cry1AcF toxicity (Dutta et al., 2023). These studies highlight the importance of specific receptor interactions in determining the efficacy of Bt toxins against lepidopteran pests. Figure 2 Insecticidal activity of Cry1AcF toxin inS. frugiperdaandS. litura(Adopted from Dutta et al., 2023) Image cation: (A) Oral ingestion of the toxin using hypodermic needle in starved S. frugiperda fourth-instar larvae. (B) At 24 h after inoculation. Dose-response curves depict the percent survival of S. frugiperda (C) and S. litura (D) at 24 h after toxin ingestion (Adopted from Dutta et al., 2023) Dutta et al. (2023) demonstrated the potent insecticidal activity of Cry1AcF toxin against larvae of Spodoptera frugiperda and Spodoptera litura. A precise method of delivering the toxin to hungry fourth-instar S. frugiperda
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