Identification Of Cry-binding Proteins In The Midgut Juice From Plutella Xylostella And Spodoptera Exigua

Cry toxins are insecticidal toxins produced by a spore-forming Gram-positive bacterium Bacillus thuringiensis(Bt)during sporulation.These B.thuringiensis Cry proteins have been widely used in biopesticide formulations and transgenic crops for insect pest control.However,certain insect species have developed resistance to Cry toxins,which might hinder further application of Cry toxins.Interactions between the Cry toxins and their midgut brush border membrane vesicles(BBMVs)receptors are key steps for the specificity and insecticidal activity of Cry toxins.There are three major modes of actions of Cry toxins:(1)Sequential binding mode suggested that after ingestion by insect larvae,Cry toxin will bind sequentially to its BBMVs receptors and form ion-permeable pores in midgut epithelial cells,leading to larval death;(2)Signal transduction mode suggested that ingested Cry toxins will bind to certain receptors that trigger apoptosis that leads to larval death.Much attention had been drawn on the discovery and identification of the BBMVs receptors,including Cadherin(CAD),Alkaline phosphatase(ALP),Aminopeptidase(APN),ABC transporter C2(ABCC2)and the recently identified V-ATPase subunit B.However,whether there exist Cry-binding proteins in the midgut juice of insects that could affect the insecticidal process of Cry toxins remained elusive.Thus,we hypothesize that there exist Cry-binding proteins in the midgut juice that could affect Cry insecticidal process.In order to validate the hypothesis,we used Cry1Ab1-coupled Sepharose beads to isolate Cry-binding proteins from midgut juice of Plutella xylostella and Spodoptera exigua by pull-down experiment.Dorsal and Peroxidase C(POXC)were found to be the Cry-binding proteins in the midgut juice of P.xylostella and S.exigua,respectively.Dorsal is a nuclear factor that plays vital role in the insect immune response(Toll-pathway)to bacteria and fungi infections,thus the interaction of Cry1Ab1 with Dorsal can alter its original functions.Based on our results,we proposed possible insecticidal mechanisms in which,Cry1Ab1 interferes with the Toll-pathway by binding to Dorsal,thus leading to an over-firing immune response that speeds up the killing process of Cry1Ab1.POXC belongs to the typical 2-Cysperoxiredoxins(Prx)family whose major functions are the clearance of reactive oxygen species(ROS)and the repairing of cell damages.Molecular docking between Cry1Ab1 and the two peroxiredoxins indicated that Cry binding to PRX-4 and POXC blocked the two conserved Cys residues that usually participate in the clearance of ROS;this can alter the peroxidase behavior of the peroxiredoxins.On the other hand,the increased peroxiredoxin level post-infections might lead to the sequestration of Cry1Ab1,thus weakening the toxicity of the toxin.We further constructed Pxdorsal and Sepoxc and recombinant plasmids and prepared the two recombinant proteins: Dorsal and POXC for further usage.Also we successfully expressed the toxin core of Cry7Ab4;the identification of its binding proteins is currently in processing.In conclusion,our results suggested that there exist,in the midgut juice,Cry-binding proteins,which are different from BBMVs' receptors and can interfere with the Cry insecticidal process.To fully understand the influence of Cry-binding proteins on the Cry insecticidal process,further research is warranted.