Enhanced apoptosis contributes to the synergistic interaction between Cry1Ac and xanthotoxin

Bt crops are bound to ingest plant defensive allelochemicals and Bt toxins, how the two types of toxins interact has been rarely studied. To address this gap, we studied the interaction type and mechanism of the plant allelochemical xanthotoxin and the Bt toxin Cry1Ac in Helicoverpa armigera, a major target pest of Bt crops. A binary mixture of xanthotoxin and Cry1Ac at LC25 effected a mortality significantly greater than that of either toxin and their additive mortality. Transcriptomic and RT-qPCR analyses revealed that the genes up-regulated by this mixture were mainly enriched in the pathways leading to mitophagy, apoptosis, and proteolysis. Staining of H. zea midgut cells with reactive oxygen species (ROS)-sensitive probe DCFH-DA, apoptotic cell probe FITC-12-dUTP or necrotic cell probe 7-AAD showed that the ROS burst and the rates of apoptotic and necrotic cells caused by the mixture were significantly greater than those of each toxin alone and the expected values. In parallel, H. armigera larvae exposed to the mixture also displayed a greater ROS burst and apoptosis rate in the midgut tissue. Section and hematoxylin-eosin staining of 48h-old H. armigera larvae showed that the mixture caused more dramatic cell shrinkage, a hall mark of apoptosis, than either toxin in the larval midgut tissue. These results indicate that concomitant intake of xanthotoxin and Cry1Ac synergistically induced a larger ROS burst, which in turn "triggered" more severe apoptosis, resulting in massive death of larval midgut cells and synergistic killing of H. armigera larvae.