Cadherin CDH-8 Is The Potential Receptor For Parasporal Crystal Cry5Ba In Caenorhabditis Elegans

Root-Knot Nematodes (RKNs) have become an important class of plant pathogens, and caused tremendous economic losse. RKNs live in plant roots after its second instar, so there are different degrees of limitations for traditional methods to control RKNs. Bacillus thuringiensis (Bt) produces insecticidal crystal proteins during the sporulation phase. Cry5Ba is high toxicity to nematode. By far, the nematicidal mechanism of Cry5Ba is not clear. As RKNs are plant parasitic nematodes specifically, it is not conducive to start the research work. Among the nematode, Caenorhabditis elegans is an ideal material for biological research, because of its relatively simple structure, body translucent, short growth cycle, keeping a simple, fast breeding and its completed genome sequencing. Meanwhile, it has been widely used in molecular biology, developmental biology and neurobiology as an ideal model organism. So we take C.elegans as the object. The study of nematicidal mechanism of Cry5Ba may provide an important theoretical guidance for the biological control of RKNs, and we carried out the following studies.1. It has been reported that ICPs, such as Cry1, Cry3 which play in perforation mechanism contain five conserved blocks and with a three-domain structure. Cry5Ba has been predicted the spatial structure with the three classical domains.The former studies in our laboratory found Cry5Ba can form oligomers in C. elegans, and there is a large cadherin superfamily in C. elegans containing 13 different cadherins. Cry5Ba has similar structure with insecticidal crystal proteins, suggesting that nematodes may have similar receptors as insects. Analyzed with bioinformatics, we found that cadherins CDH-8 and CDH-7 in C. elegans have the highest similarity with the cadherin receptors in insects. Therefore, we predicted that they may be the receptor for Cry5Ba.2. This study also processed LC50 bioassay experiments of Cry5Ba to C. elegans cdh-8 mutant RB815, that RB815 had a very obvious resistance on Cry5Ba.3. The toxin binding regions (TBR) of cadherin in insects were reported mainly in repeat 7 to membrane-proximal extracellular domain (CR7-MPED). The CR7-MPED of CDH-8 is high homologous with the TBR of Cadherin in insects. Then the CR7-MPED coding gene fragments in CDH-8 were cloned. The peptides containing CR7, CR9-CR10, and CR10- MPED were then expressed and purified from E. coli.4. Through ligand bolt, we found that CR7, CR9-CR10, and CR10-MPED are able to combine with Cry5Ba.5. Through competitive binding experiments, we found that only CR9-CR10 is able to bind with Cry5Ba specifically.This study provided valuable research materials for further determine whether CDH-8 is the receptor and toxin binding region for Cry5Ba in C. elegans. it will further enrich the research of nematicidal mechanism of Bt, and provide an important theoretical guidance for the biological control of PPNs.