Identification of Arylphorin interacting with the insecticidal protein PirAB from Xenorhabdus nematophila by yeast two-hybrid system.

PirAB toxin was initially found in the Photorhabdus luminescens TT01 strain and is a demonstrated binary toxin with high insecticidal activity. In this paper, we co-expressed the pirAB gene of Xenorhabdus nematophila HB310 in a prokaryotic expression system, and we found that the PirAB protein showed high hemocoel insecticidal activity against Galleria mellonella, Helicoverpa armigera and Spodoptera exigua. LD50 values were 1.562, 2.003 and 2.17 µg/larvae for G. mellonella, H. armigera, and S. exigua, respectively (p > 0.05). Additionally, PirAB-interaction proteins were identified from G. mellonella by 6 × His Protein Pulldown combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Of which, arylphorin of G. mellonella showed the highest matching rate. A protein domain conservative structure analysis indicated that arylphorin has three domains including Hemocyanin-N, Hemocyanin-M, and Hemocyanin-C. Among these protein domains, Hemocyanin-C has immune and recognition functions. Further, Hemocyanin-C domain of arylphorin was identified to interact with PirA but not PirB by Yeast two-hybrid system. These findings reveal, for the first time, new host protein interacting with PirAB. The identification of interaction protein may serve as the foundation for further study on the function and insecticidal mechanism of this binary toxin from Xenorhabdus.