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.

A Single Point Mutation Resulting in Cadherin Mislocalization Underpins Resistance against Bacillus thuringiensis Toxin in Cotton Bollworm

“…Construction of Fusion Proteins-The HaCad genes from 96S and 96CAD strains were amplified by RT-PCR using the total RNA extracted from the midgut tissue of the larvae and cloned into the plasmid pie2-EGFP-N1 as described previously (50) for expression of GFP fusion proteins (HaCad-GFP and mHaCad-GFP). Primers used are listed in supplemental Table S1.…”

“…Plasmid pie2-EGFP expressing EGFP was used as a control. The percentage of swollen cells was determined as an indication of cytotoxicity as described previously (50).…”

Cadherin repeat 5 mutation associated with Bt resistance in a field-derived strain of pink bollworm

“…We detected the transformed cells using a fluorescence microscope after treatment with activated Cry1Ac (six groups ranging from 0 to 40 μg per mL PBS) for one hour. We evaluated cell toxicity using the ratio of swollen cells as described previously 47 . We performed three repeats for each group and applied the ratio of swollen cells for six observed visual fields per repeat per group to determine cells toxicity.…”

Establishment and characterization of a novel cell line from midgut tissue of Helicoverpa armigera (Lepidoptera: Noctuidae)

“…Since columnar cells of lepidopteran midgut are susceptible to B. thuringiensis crystal toxin, and the HNU-Ha-MG1 cell line was derived from midgut tissue of the Cry1Ac-susceptible H. armigera larvae, we investigated whether the cell line was susceptible to B. thuringiensis toxins. Although the activated Cry1Ac was toxic to S. litura Sl-HP cells (Xu et al 2014) and H. armigera larvae (data not shown), it was non-toxic to the HNU-Ha-MG1 cells. The mortality of the activated Cry1Ac-treated HNU-Ha-MG1 cells was 12.0± 4.6% when the final concentration of the toxin was 60 μg/ml at 1 h of post-treatment.…”

“…Although H. armigera larvae are not susceptible to Cry1C, a few Cry1C receptors may exist in the larvae. It is well known that the expression of cadherin in insect cell lines or mammalian cell lines enhanced the susceptibility of the tested cells to activated Cry1A toxin (Xu et al 2014). If HNU-Ha-MG1 cell line was susceptible to the activated Cry1C, a new receptor of Cry1C might be found in the cell line in the future because not much is known about these receptors of Cry1C.…”