Immunolocalization and ultrastructure shows ingestion of Cry protein expressed in Glycine max by Heterodera glycines and its mode-of-action.

Abstract

Great interest exists in developing a transgenic trait that controls the economically important soybean (Glycine max) pest, soybean cyst nematode (SCN, Heterodera glycines), due to its adaptation to native resistance. Soybean plants expressing the Bacillus thuringiensis delta-endotoxin, Cry14Ab, were recently demonstrated to control SCN in both growth chamber and field testing. In that communication, ingestion of the Cry14Ab toxin by SCN second stage juveniles (J2) was demonstrated using fluorescently labeled Cry14Ab in an in vitro assay. Here, we show that consistent with expectations for a Cry toxin, Cry14Ab has a mode-of-action unique from the native resistance sources Peking and PI 88788. Further, we demonstrate in planta the ingestion and localization of the Cry14Ab toxin in the midgut of nematodes feeding on roots expressing Cry14Ab, using immunogold labeling and transmission electron microscopy. We observed immunolocalization of the toxin and resulting intestinal damage primarily in the microvillus-like (MvL)-containing region of the midgut intestine, but not in nematodes feeding on roots lacking toxin. This demonstrated that Cry14Ab was taken up by the J2 SCN, presumably through the feeding tube within the plant root cell that serves as its feeding site. This suggests that relatively large proteins can be taken up through the feeding tube. Electron microscopy showed that Cry14Ab caused lysis of the midgut MvL membrane, and eventual degradation of the MvL and the lysate, forming particulate aggregates. The accumulated electron dense aggregate in the posterior midgut intestine was not observed in SCN in non-Cry14Ab expressing plants.