Study On The Insecticidal Mechanism Of Vip3A Against Spodoptera Exiguas

Vegetative insecticidal proteins(VIPs),the novel insecticidal proteins of Bacillus thuringensis(Bt),are synthesized and secreted into extracellular by Bt during the vegetative growth stage.Vip3 A proteins show low similarity with Cry toxins in amino acid sequences and its insecticidal spectrum and target insects also differ from Cry toxins.That means that Vip3A has a wide application prospect in pest control and resistance control.However,the insecticidal mechanism of Vip3A was not clear and remained to be further studied.In previous work,we cloned and identified two novel Bt insecticidal genes which encoded two Vip3A proteins with different toxicity against Spodoptera exigua.Vip3Aa60 was highly toxic against S.exigua,while Vip3Ad5 showed no insecticidal activity.In this study,we explored the insecticidal mechanism of Vip3 A against S.exigua using Vip3 Aa60 and Vip3 Ad5 proteins.The main results were as follows.(1)Bioinformatics analysis showed that 195KVKK198 was the potential trypsin cleavage sites of Vip3Aa60,which was associated with the production of activated toxin.The cleavage site of Vip3Aa60 protoxin by midgut juice(MJ)of S.exigua was identified at Lys~198 using N terminal sequencing.The results of proteases inhibitor assays showed that trypsin was the main protease in the activation of Vip3Aa60.Compared with the Vip3Aa60 protein,the mutants,keeping any one of three Lys sites(Lys~195,Lys~197 or Lys~198),had no distinct differences on the protoxin activation and toxicity against S.exigua.Only when all of three sites were replaced,the protoxin could not be processed into activated toxin by trypsin and the activation rate of protoxin by MJ became more slowly and its insecticidal activity decreased.The characteristic of existing three trypsin cleavage sites made Vip3Aa60 protoxin could be more efficiently activated in the midgut of insects and further exerted its insecticidal toxicity.When Vip3Aa60 protoxin was not activated by trypsin,the insects could still activate Vip3Aa60 by other ways.But both the activation efficiency and insecticidal toxicity of Vip3Aa60 were decreasing.(2)The similarity of amino acid sequences between Vip3Ad5 and Vip3Aa60 was 85%,and the differences were mainly in the C terminal which was the activated toxin sequence.The activated toxin played an important role in the receptor binding and toxicity against insects.The activation partern of Vip3Ad5 was similar to that of Vip3Aa60.Howere,the results of stability of activated toxin assays showed that Vip3Aa60 activated toxin was stable,while Vip3Ad5 activated toxin was unstable and easily further degraded by MJ with the prolongation of the activation time.The stability of activated toxin might be related to the toxicity of Vip3A.The receptor binding assays showed that both Vip3Aa60 and Vip3Ad5 could bind to the BBMV of S.exigua,and shared the same binding site.The results of protein and protein interacts test showed that the dissociation constant of Vip3Aa60 with the receptor was only 6%of Vip3Ad5,indicating that Vip3Aa60 had greater affinity with BBMV than Vip3Ad5.It was suggested that the affinity between activated toxin and receptor might be associated with Vip3A toxicity.In this work,we studied the insecticidal mechanism of Vip3A from protoxin activation and receptor binding.We identified the trypsin cleavage sites of Vip3A protoxin and explored the reasons of Vip3Ad5 without insecticidal toxicity,laying the foundation for the analysis of the mechanism of Vip3A toxins.