Abstract
Bacillus thuringiensis is a well-known entomopathogenic bacterium that produces vegetative insecticidal proteins (Vips, including Vip1, Vip2, Vip3, and Vip4) during the vegetative phase. Here, we purified Vip1 and Vip2 from B. thuringiensis and characterized the insecticidal effects of these protoxins. Bioassay results showed that a 1:1 mixture of Vip1Ad and Vip2Ag, purified by ion-affinity chromatography independently, exhibited insecticidal activity against Holotrichia parallela larvae, with a 50% lethal concentration value of 2.33 μg/g soil. The brush border membrane (BBM) in the midgut of H. parallela larvae was destroyed after feeding the Vip1Ad and Vip2Ag mixture. Vacuolization of the cytoplasm and slight destruction of BBM were detected with Vip2Ag alone, but not with Vip1Ad alone. Notably, Vip1Ad bound to BBM vesicles (BBMVs) strongly, whereas Vip2Ag showed weak binding; however, binding of Vip2Ag to BBMV was increased when Vip1Ad was added. Ligand blotting showed that Vip2Ag did not bind to Vip1Ad but bound to Vip1Ad-t (Vip1Ad was activated by trypsin), suggesting the activation of Vip1Ad was important for their binary toxicity. Thus, our findings suggested that Vip1Ad may facilitate the binding of Vip2Ag to BBMVs, providing a basis for studies of the insecticidal mechanisms of Vip1Ad and Vip2Ag.
Highlights
White grubs are important insect pests that severely damage the roots of many crops, including soybeans, corn, peanuts, turf, and some vegetables [1]
Different insecticidal proteins were produced by B. thuringiensis, such as the vegetative insecticidal proteins (Vip) during the vegetative phase and the crystal proteins (Cry) during the sporulation phase of growth [8]
The recombinant strains HDVIP1 and HDVIP2 were grown in LB medium and cultural supernatants were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
Summary
White grubs are important insect pests that severely damage the roots of many crops, including soybeans, corn, peanuts, turf, and some vegetables [1]. The management of H. parallela larvae is highly dependent on the use of chemical pesticides [2], and many efforts are being made to develop environment friendly means of controlling pests. As an alternative to chemical pesticides, Bacillus thuringiensis (Bt) biopesticides have been employed in pest control for decades [4], and many H. parallela specific Bt strains are currently being evaluated [5,6,7]. The 3d-Cry family represents the biggest family, which are globular molecules containing three distinct domains connected by single linkers [8]. Cry proteins receive their mnemonic names and four hierarchical ranks depending on their primary sequence identity. The third rank gives a lowercase letter to distinguish proteins that share more than 78% identity and less than 95% with other Cry proteins [8]
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