Expression of recombinant and mosaic Cry1Ac receptors from Helicoverpa armigera and their influences on the cytotoxicity of activated Cry1Ac to Spodoptera litura Sl-HP cells

Abstract

Bacillus thuringiensis (Bt) toxin receptors play important roles in the killing of pests, and investigation on characterization of the receptors is essential for utilization of Bt and management of insect resistance. Here, recombinant and mosaic receptors of Bt Cry1Ac toxin from Helicoverpa armigera were expressed in Spodoptera litura Sl-HP cells and their influences on cytotoxicity of activated Cry1Ac toxin were investigated. When H. armigera aminopeptidase N1 (APN1), alkaline phosphatase 2 (ALP2) and cadherin fused with or without GFP tag were, respectively, expressed in Sl-HP cells, live cell-immunofluorescence staining detection revealed that the quantity of the toxin binding to cadherin or cadherin-GFP was much more than that binding to ALP2 and APN1 or their fusion proteins with GFP, and only the cadherin- or cadherin-GFP-expressing cells showed aberrant cell morphology after the treatment of the toxin at low concentrations. ALP2 and APN1 fused with or without GFP tag did not significantly enhance the cadherin-mediated cytotoxicity of the toxin. The mosaic ALP-TBR-GFP-GPI was located on cell membrane, but did not bind to the toxin. The mosaic truncated cadherin-GFP-GPI was not located on cell membrane even if the signal peptide was sustained. The concentrations of the toxin resulting in swelling of 50 % cells for noncadherin-expressing Sl-HP cells and cadherin-expressing Hi5 cells were 5.08 and 9.50 µg/ml within 1 h, respectively. Taken together, our data have indicated that the binding affinity of ALP2 and APN1 to activated Cry1Ac toxin is much weaker than that of cadherin and both ALP2 and APN1 do not enhance the cytotoxicity of the toxin even though cadherin is co-expressed, and the mosaic receptor of ALP2 inserted with cadherin toxin binding domain does not mediate cytotoxicity of the toxin. In addition, the noncadherin-expressing Sl-HP cells are more susceptible to activated Cry1Ac than the cadherin-expressing Hi5 cells.

Keywords: Alkaline phosphatase; Aminopeptidase N; Bacillus thuringiensis; Cadherin; Trichoplusia ni.

Fig. 1

Constructs of plasmids. S signal peptide, GPI glycosyl phosphatidyl inositol modification signal of GPI-anchored protein, GFP green fluorescence protein, B region of Hacadher (H. armigera cadherin) in binding to activated Cry1Ac (TBR), T trans-membrance region of Hacadherin, C cytosolic part of Hacadherin. Sig presents the signal peptide of ALP2 or APN1. A9 indicates that there were 9 amino acid residues between GFP and GPI-anchoring site. Tcadherin represents the C-terminal truncated cadherin without the transmembrane domain and cytosolic domain

Fig. 2

Expression and location of various ALP2 proteins fused with GFP in Sl-HP cells. a Expression of the fusion proteins detected using anti-GFP antibody as primary antibody. Lane 1 ALP-GFP-GPI; lane 2 ALP-GFP-A9-GPI; lane 3 Sig-GFP-ALP-GPI; lane 4 ALP-GPI-GFP, showing GPI-GFP cleaved from ALP-GPI-GFP; lane 5 Control (cells without transfection). b Expression of various fusion proteins detected using anti-ALP2 antibody as a primary antibody. *Non-specific band. A9. There were 9 amino acid residues between GFP and the cleavage site of GPI modification signal. ALP2 with GPI modification signal can not be distinguished from ALP2 without GPI in panel a because the molecular weight of GPI modification signal is very low. c, d Location of various ALP2 fusion proteins on cell membrane of Sl-HP cells assayed via live cell-immunofluoscence staining

Fig. 3

Expression and location of APN1 and cadherin fused with GFP in Sl-HP cells. The cells expressing the fluorescence proteins were fixed and stained with Hoechst 33258 (Sigma-Aldrich Co.) at 24 h of post-transfection. Then they were subjected to observation using fluorescence microscope. The fused proteins were abundant on cell membrane and partial fused proteins were located in cytoplasm in the form of small dots

Fig. 4

Binding of the activated Cry1Ac to ALP-GFP-GPI, APN-GFP-GPI and cadherin-GFP on cell membrane. The cells were treated with activated Cry1Ac at 0.04 μg/ml for 0.5 h at 24 h of post transfection. Then the cells were fixed after washing twice, and immunofluorescence assay was performed using anti-Cry1Ac serum as a primary antibody. Arrows point to the positive cells binding to activated Cry1Ac. Red fluorescence signal was very weak for the cells expressing ALP-GFP-GPI or APN-GFP-GPI, compared to the cells expressing cadherin-GFP

Fig. 5

Effects of the three recombinant receptors on the cytotoxicity of activated Cry1Ac to Sl-HP cells. a The receptors fused with GFP. b The receptors without GFP fusion. Arrows point to the aberrant cells treated with the activated Cry1Ac at 0.04 μg/ml for 1 h at 24 h of post transfection

Fig. 6

Influence of the co-expression of ALP-GFP-GPI or APN-GFP-GPI with cadherin-mCherry on the cytotoxicity of activated Cry1Ac to Sl-HP cells. Arrows point to the aberrant cells treated with activated Cry1Ac at various concentrations for 1 h at 24 h post transfection

Fig. 7

Localization of mosaic receptors and their influences on cytotoxicity of activated Cry1Ac to Sl-HP cells. a, b The cells expressing ALP2-TBR-GFP-GPI. a1 The cells under fluorescence microscope; a2 live-cell immunofluorescence staining of the cell using anti-GFP antibody as a primary body; a3 merged; b1 ALP2-TBR-GFP-GPI expressing cells treated with activated Cry1Ac at 0.08 μg/ml for 1 h; b2 live-cell immunofluorescence staining of the cells treated with activated Cry1Ac at 0.08 μg/ml for 1 h, using anti-Cry1Ac antibody as primary body; b3 merged; c1 ALP2-TBR-GFP-GPI expressing cells treated with activated Cry1Ac at 0.08 μg/ml for 1 h, showing no swollen cells; d1 tcadherin-GFP-GPI expressing cells under fluorescence microscope; d2 live-cell immunofluorescence staining of the tcadherin-GFP-GPI expressing cells treated with activated Cry1Ac at 0.04 μg/ml for 0.5 h at 24 h of post transfection, using anti-GFP antibody as a primary antibody