Identification of two Bacillus thuringiensis Cry3Aa toxin-binding aminopeptidase N from Rhynchophorus ferrugineus (Coleoptera: Curculionidae).

ABSTRACT

Rhynchophorus ferrugineus is a quarantine pest that mainly damages plants in tropical regions, which are essential economic resources. Cry3Aa has been used to control coleopteran pests and is known to be toxic to R. ferrugineus. The binding of the Cry toxin to specific receptors on the target insect plays a crucial role in the toxicological mechanism of Cry toxins. However, in the case of R. ferrugineus, the nature and identity of the receptor proteins involved remain unknown. In the present study, pull-down assays and mass spectrometry were used to identify two proteins of aminopeptidase N proteins (RfAPN2a and RfAPN2b) in the larval midguts of R. ferrugineus. Cry3Aa was able to bind to RfAPN2a (Kd = 108.5 nM) and RfAPN2b (Kd = 68.2 nM), as well as midgut brush border membrane vesicles (Kd = 482.5 nM). In silico analysis of both RfAPN proteins included the signal peptide and anchored sites for glycosyl phosphatidyl inositol. In addition, RfAPN2a and RfAPN2b were expressed in the human embryonic kidney 293T cell line, and cytotoxicity assays showed that the transgenic cells were not susceptible to activated Cry3Aa. Our results show that RfAPN2a and RfAPN2b are Cry3Aa-binding proteins involved in the Cry3Aa toxicity of R. ferrugineus. This study deepens our understanding of the action mechanism of Cry3Aa in R. ferrugineus larvae.