Mechanism Of Action Of Vip3AcAa Insecticidal Protein Against Several Lepidopteran Pests And Interaction With Cry1Ac

Wide planting of transgenic Bt cotton in China since 1997 to control cotton bollworm(Helicoverpa armigera)has increased yields and decreased insecticide use,but the evolution of resistance to Bt cotton by H.armigera remains a challenge,and many secondary lepidopteran pests that are tolerant or nonsusceptible to Cry1Ac are now reported to cause considerable economic damage.To develop a new generation of insect-resistant transgenic crops,prevent the evolution of insect resistance to the Bt cotton,expand the insecticidal spectrum and complementing current integrated pest management strategies in China.In a previous study,we used an overlap method to develop a Vip3Ac and Vip3Aa chimeric protein,Vip3AcAa(consists of the N-terminal 600 amino acid residues of Vip3Acl and the C-terminal 189 amino acid residues of Vip3Aal).This chimeric protein is insecticidal to Ostrinia nubilalis(O.nubilalis is tolerant to almost all known Vip3A proteins),exerted high toxicity to the fall armyworm(Spodoptera frugiperda)and was highly insecticidal to a Cry1Ac-resistant strain of cabbage looper(Trichoplusia ni).In the present study,we observed the pathologic characteristics of Vip3AcAa protein on the intestinal tissue of the target pests by transmission electron microscopy and hematoxylin-eosin staining fluoroscopy microscope;analysed the proteolysis of Vip3AcAa protoxin by midgut extract juice from the target pests;demonstrated that Vip3AcAa specifically bound to BBMV from target pests(H.armigera,Spodoptera litura,Spodoptera exigua,and Agrotis ipsilon)and that it does not share the same binding sites with Cry1Ac by homologous and heterologous competition binding experiments and identified the putative receptor for this chimeric protein in BBMV of the four target pests;tested insecticidal activities of Vip3AcAa against H.arnigera and the secondary lepidopteran pests:S.litura,S.exigua,and A.ipsilon,which are relatively tolerant to Cry1Ac proteins;investigated cross resistance and interactions between Vip3AeAa and Cry1Ac aganist H.armigera;tested the insect resistance of the pryramid Vip3AcAa and Cry1Ac cotton in laboratory experiment.The main research results are as follows:1.Transmission electron microscope and hematoxylin-eosin staining fluoroscope microscope observation found that Vip3AeAa protein mainly effected on target pests(H.armigera,S.litura,S.exigua,and A.ipsilon)of midgut tissue.Histopathological characteristics of the midgut tissue included basement membrane isolated from the columnar cells,a strong vacuolization of columnar cells,the goblet cells swollen and elongated,apical microvilli dropped off.2.The midgut extract juice from H.armigera,S.litura,S.exigua and A.ipsilon proteolysis of the Vip3AcAa protoxin produced a major 70 kDa fragment.In the proteolysis assays,the Cry 1 Ac-resistant H.armigera mediated by changes in intestinal proteases or a mutation in the ABCC2 gene did not differ in proteolytic products or in the activation rate of the Vip3AcAa protein,indicating that differences among the midgut proteases in these strains of H.armigera do not seem to be a critical factor in determining Vip3AcAa insecticidal toxicity.The proteolysis rate of Vip3AcAa protoxin was different in different species.3.In the homologous and heterologous compete binding assays,the activated Vip3AcAa bound specifically to BBMV from H.armigera,S.litura,S.exigua and A.ipsilon and did not compete with Cry1Ac.In vitro binding of Vip3AcAa protein to the BBMV separated by SDS-PAGE shown that Vip3AcAa bound to three putative receptors of apprpxomately 40,80 and 100 kDa on the BBMV from H.armigera larvae,approximately 36,39,68,and 100 kDa receptors on BBMV from S.litura larvae;approximately 34 and 100 kDa receptors on BBMV from S.exigua larvae;approximately 35,55,and 110 kDa receptors on BBMV from A.ipsilon larvae;suggested that the Vip3AcAa has similar size receptors(34-36 kDa)in these three pests,and they are probably the same type of binding receptors.4.The susceptibility of Cry1Ac-resistant and susceptible strains of H.armigera to Vip3AcAa protoxin was similar indicating no cross resistance with Cry1Ac.The observed mortality was equivalent to the expected mortality in all combinations of Vip3AcAa and Cry 1 Ac proteins tested against H.armigera,based on the responses to each toxin tested individually,reflecting independent effects of Vip3AcAa and Cry1Ac toxins.Besides,CrylAc had very low toxicity against S.exigua larvae and negligible toxicity against S.litura and A.ipsilon larvae,but the Vip3AeAa had high insecticidal activity against all three secondary lepidopterans.5.Pyramid Vip3AcAa+CrylAc cotton has good control effect on the susceptible and Cry1Ac-resistant H.armigera.Moreover,this pyramid cotton is one single-trait cotton for these secondary pests(S.litura,S.exigua and A.ipsilon),it also could effectively control these three secondary pests in our laboratory experiment.Vip3AcAa is a kind of new type of man-modified insecticidal protein.It has good insecticidal activity against H.armigera,S.litura,S.exigua and A.ipsilon larvae,and Vip3AcAa and Cry1Ac do not have cross resistance and independent activity when combination of this two toxins.Cotton pyramided Vip3AcAa and CrylAc can effectively protect cotton against the major pest H.armigera and secaodary pest S.litura,S.exigua and A.ipsilon.Our results indicate that Vip3AcAa can be used as a new toxin against lepidopteran pests in transgenic plant breeding for preventing the evolution of insect resistance to the Bt cotton,expanding the insecticidal spectrum and complementing current integrated pest management strategies in China.