Targeting delta-endotoxin (Cry1Ac) of Bacillus thuringiensis to subcellular compartments increases the protein expression, stability, and biological activity

Evolving insect resistance to delta-endotoxins can be delayed by using a few strategies like high dosage, refugia, and gene stacking which require the expression of delta-endotoxins at sufficiently high levels to kill the resistant insects. In this study, we comparatively analyzed the efficacy of targeting truncated cry1Ac protein to the cytoplasm, endoplasmic reticulum (ER), and chloroplast to obtain high protein expression. mRNA and protein profiling of cry1Ac showed that both ER and chloroplast are efficient targets for expressing high levels of truncated cry1Ac. A maximum of 0.8, 1.6, and 2.0% cry1Ac of total soluble protein were obtained when the truncated cry1Ac was expressed in the cytoplasm, routed through ER, and targeted to the chloroplast. We further showed that not only the protein content but also the biological activity of truncated cry1Ac increases by sub-cellular targeting and the biological activity is slightly greater in the ER routed transgenic lines by conducting different bioassays on Helicoverpa armigera. Using native Western analysis, we demonstrated that the truncated cry1Ac protein could exist as oligomers in plant cells and this oligomerization capability is low in the cytoplasm. In conclusion, routing of delta endotoxins through ER is the first choice to obtain high protein expression and bioactivity.