The induced knockdown of GmCAD receptor protein encoding gene in Galleria mellonella decreased the insect susceptibility to a Photorhabdus akhurstii oral toxin

Abstract

Photorhabdus bacteria secrete a repertoire of protein toxins that can kill the host insect. Among them, toxin complex (Tc) proteins have gained significant attention due to their wider conservation across the different bacterial genera. In our laboratory, a C-terminal domain of TcaB protein was characterized from P. akhurstii bacterium that conferred the potent oral insecticidal effect on Galleria mellonella . However, the role of insect gut receptors in the TcaB intoxication process was yet to be investigated. In the current study, we examined the transcription of candidate midgut receptors in TcaB-infected larvae and subsequently cloned a cadherin-like gene, GmCAD , from G. mellonella. GmCAD was highly transcribed in the fourth-instar larval stage and specifically in the midgut tissues. Our ligand blot and binding ELISA assays indicated that TcaB binds to the truncated peptides from the GmCAD transmembrane-proximal region with greater affinity than that from the transmembrane-distal region. Oral administration of bacterially expressed GmCAD dsRNA in G. mellonella severely attenuated the expression of target mRNA, which in turn alleviated the negative effect of TcaB on insect survival (TcaB-induced mortality in CAD dsRNA pretreated larvae reduced by 72–83% compared to control), implying the association of GmCAD in the TcaB intoxication process. Present findings form a basis of future research related to the insect gut receptor interactions with Photorhabdus toxins.

Molecular characterization and functional analysis of Cry toxin receptor‐like genes from the model insect Galleria mellonella

“…CAD contains 34 calcium‐binding sites in its extracellular domain. CAD sequences are evolutionarily highly conserved in their corresponding insect orders as lepidopteran clade branches away from hemipteran, coleopteran, isopteran and hymenopteran clades (Dutta et al, 2021). Seventeen conserved motifs were localized in the majority of CAD homologues.…”

“…(c) A photomicrograph of larval alimentary canal at 1 h after oral administration of the toxin admixed with 0.4% trypan blue, indicating the onward dispersion of dye across the gastrointestinal tract. (d) Dead or moribund toxin ingested larvae (unresponsive to prodding) with intense hemolymph melanization compared to no melanization in control larvae at 24 h. Scale bar = 0.5 cm conserved in their corresponding insect orders as lepidopteran clade branches away from hemipteran, coleopteran, isopteran and hymenopteran clades (Dutta et al, 2021). Seventeen conserved motifs were localized in the majority of CAD homologues.…”

RNAi-mediated knockdown of gut receptor-like genes prohibitin and α-amylase altered the susceptibility of Galleria mellonella to Cry1AcF toxin

“…The three-dimensional structures of Cry1AcF and different receptor proteins were modelled via homology modeling using SWISS-MODEL server. The obtained models were manually curated for precise sequence alignment with the query by employing the graphics program in Discovery Studio v. 14 (Biovia) [30][31][32]. Ligand-receptor docking analysis was carried out in "Macromolecules" module of Discovery Studio v. 14.…”