The Synergistic Mechanism Of Bacillus Thuringiensis Insecticidal Proteins Against Lepidopteran Pests

Bacillus thuringiensis(Bt)produces different insecticidal proteins such as the vegetative insecticidal proteins(Vip)during vegetative phase and the crystal proteins(Cry)during the sporulation phase of growth.Due to excellent insecticidal activity against lepidopteran,coleopteran and dipteran pests specifically,both Bt toxins have been expressed in genetically modified crops,resulting in extraordinary activity to control insect pests.However,evolution of insect resistance to these toxins in the field has been reported,representing a major threat for the use of these toxins in insect control.While,the insect resistance could be overcame or delayed by the synergistic combination of Bt proteins with high insecticaidal activity and wide insecticidal spectrum.A novel synergistic interaction between Cry9Aa3 and Vip3Aa11 proteins against the rice pest Chilo suppressalis when applied together.However,from now on,the study about mechanism of synergistic effect between Cry and Vip proteins is still empty in worldwide.This study focuses on the synergistic mechanism of Cry9 Aa and Vip3 Aa proteins against the Chilo suppressalis and the screening of Bt proteins with synergistic activity against corn pests subsequently,the progress is as followed:1.Cry9 Aa and Vip3 Aa proteins bound specifically to midgut brush border membrane vesicles of Chilo suppressalis,and did not share binding sites since no-binding competition between them was observed.2.Different binding assays showed that Cry9 Aa and Vip3 Aa proteins interacted with each other with high affinity.3.The specific interacting regions between Cry9 Aa and Vip3 Aa were mapped in domain?and?of Cry9 Aa.The binding site of Cry9 Aa with Vip3 Aa protein was narrowed into 495RRS497 of domain?-loop3 by Site-direct-mutagenesis.The result of membrane bound peptides confirmed that these binding regions of Cry9 Aa participate in binding with Vip3 Aa protein.On the other hand,the specific interacting regions of Vip3 Aa with Cry9 Aa were mapped into Vip3Aa-Lys352-Asp451.Two overlapping regions,356-ALIGFEISNDS-366 and 428-TKKMKTL-434,were involved in binding with Cry9 Aa.Site-direct-mutagenesis confirmed that Vip3Aa-KTL432-434 participate in binding and that binding between these two proteins directly correlates with their synergism by synergistic bioassay between Vip3Aa-432KTL434 AAA and Cry9 Aa protein.Finally,bioassay against Chilo suppressalis showed the synergy activity were decreased with Cry9Aa-495RRS497 AAA and Vip3Aa-432KTL434 AAA mutant proteins.The results indicate that Bt Cry9 Aa and Vip3 Aa display a potent synergistic activity based in a specific interaction among them.4.On the basis of clarification of synergistic mechanism above,further screening of other synergy combinations were performed against corn pests.Cry9 Aa and Vip3 Aa proteins were found synergistic effect on Ostrinia furnacalis,as well as Cry1 Bb and Cry9 Ee proteins against Mythimna separata.Also,no cross-resistance of Ostrinia furnacalis towards these two proteins was found andthis phenomenon was probably caused by no competitive binding between Cry9 Ee and Cry1 Ab proteins on the BBMV.The study clarified that premised synergistic effect on the specific binding of Cry9 Aa and Vip3 Aa proteins with BBMV and no competitive binding,subsequently,the interaction of Cry9 Aa and Vip3 Aa proteins directly triggered synergism.The discovery optimized insecticidal mechanism of multiple Bt proteins,and settled the theoretical basis in application of “Pyramids” genetically modified crops.Otherwise,the clarification of binding epitopes between two proteins,supplied theoretical support to directional modification of Bt proteins.