Bacillus thuringiensis Vip1 Functions as a Receptor of Vip2 Toxin for Binary Insecticidal Activity against Holotrichia parallela

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.

Keywords: Bacillus thuringiensis; Holotrichia parallela; Vip1Ad and Vip2Ag binary toxin; binding.

Figure 1

Analysis of vegetative insecticidal proteins (Vip)1Ad and Vip2Ag purified by ion-exchange chromatography. (A) Step gradient elution profile of Vip1Ad purified using a HiTraq Q HP column. (B) Sodium dodecyl sulfate polyacrylamide gel electrophores (SDS-PAGE) analysis of purification of Vip1Ad using an anionic column. (C) Step gradient elution profile of Vip2Ag purified using a HiTraq SP HP column. (D) SDS-PAGE analysis of the purification of Vip2Ag using a cationic column. In Figure 1A, C, the black arrows indicate the HDVIP1 and HDVIP2 supernatants, blue line: UV280 nm, red line: conductivity, green line: percentage of eluant.

Figure 2

Histopathological effects of Vip1Ad and Vip2Ag binary toxin on the midgut of H. parallela larvae observed by transmission electron microscope (TEM). Sections of the midgut epithelium from larvae fed (A) phosphate-buffered saline (PBS, negative control), (B) Vip1Ad (30 μg/g soil), (C) Vip2Ag (30 μg/g soil), (D) Bt strain HBF-18 (positive control), or (E) a mixture of Vip1Ad and Vip2Ag (molar ratio 1:1; 30 μg/g soil). L, lumen; BBM, brush border membrane.

Figure 3

Binding assays for Vip1Ad, Vip2Ag and BBM vesicles (BBMVs) of H. parallela. (A) SDS-PAGE analysis of Vip1Ad activation by trypsin. (B) Western blot analysis of binding between Vip1Ad, Vip1Ad-t and BBMVs. The Vip1Ad on gel as a positive control and BBMV on gel as a negative control. (C) ELISA analysis of binding between Vip1Ad and BBMVs. (D) Western blot analysis of binding between Vip2Ag and BBMVs. Vip2Ag on gel as positive control and BBMV as negative control.

Figure 4

Analysis of the interaction between Vip1Ad and Vip2Ag. (A) Ligand blot analysis of the binding of Vip1Ad, Vip1Ad-t and Vip1Ad-t-monomer with Vip2Ag. The Vip1Ad on gel as a positive control. (B) Panel above: Dot blot analysis of the binding of Vip2Ag with Vip1Ad, Vip1Ad-t and Vip1Ad-t-monomer. The Vip2Ag on membrane as a positive control and Vip1Ad as a negative control. Panel lower: Dot blot analysis of the binding of Vip1Ad, Vip1Ad-t and Vip1Ad-t-monomer with Vip2Ag. The Vip1Ad on membrane as a positive control and Vip2Ag as a negative control. (C) Ligand blot analysis of the Vip2Ag with Vip1Ad, Vip1Ad-t-monomer or Vip1Ad-t. Vip2Ag on gel as a positive control. Vip1Ad and Vip2Ag are protoxins, Vip1Ad-t is the products of activated Vip1Ad by trypsin, Vip1Ad-t-monomer is monomer fragment (70 kDa) purified from Vip1Ad-t.

Figure 5

Ultracentrifugation analysis of the oligomerization of Vip1Ad-t and Vip2Ag. (A) Ultracentrifugation analysis of the oligomerization of Vip1Ad. (B) Ultracentrifugation analysis of the binding between Vip2Ag and Vip1Ad-t. Different oligomers were shown by various colors. Grey arrows indicate deployer, red arrows indicate trimers, yellow arrows indicate tetramers, green arrows indicate pentamers, blue arrows indicate hexamers and purple arrows indicate heptamers.