Suppression of Calcineurin Enhances the Toxicity of Cry1Ac to Helicoverpa armigera

Abstract

Insect resistance to Bacillus thuringiensis (Bt) insecticidal proteins has rapidly evolved with the expansion of the planting area of transgenic Bt crops. Pyramiding RNA interference (RNAi) and Bt in crops is urgently needed to counter the rapid increase in pest resistance. The ideal "pyramid" strategy simultaneously targets different action pathways that exert synergetic effects on each other. Here, we identified a dephosphatase, namely, Helicoverpa armigera calcineurin (HaCAN), which might enhance the insecticidal activity of Cry1Ac against Helicoverpa armigera by regulating immune gene expression via dephosphatase activity, but not by acting as a receptor. Notably, blocking enzyme activity or knocking down endogenous HaCAN significantly promoted the enhancement in Cry1Ac toxicity to insect larvae and cells. Correspondingly, the increase in HaCAN activity reduced the cytotoxicity of Cry1Ac as shown by the heterologous expression of HaCAN. Our results provide a probable that HaCAN is an important candidate gene for pyramiding RNAi and Cry1Ac crops to control cotton bollworm.

Keywords: Cry1Ac; Helicoverpa armigera; calcineurin; cell toxicity; membrane yeast two-hybrid.

FIGURE 1

The effect of feeding with CAN inhibitor FK506 on the expression level of HaCAN. Each error bar represents the standard error of the mean from three biological replicates. Significant differences between CK (buffer control) and treatments (feeding Cry1Ac) at different time points are indicated with asterisks (*P < 0.05, based on Student’s t-tests, DPS7.05).

FIGURE 2

The effect of feeding with FK506, Cry1Ac, and Cry1Ac + FK506 on HaCAN activity in the midgut of larvae. Each error bar represents the standard error of the mean from three biological replicates. Significant differences among the different treatments are indicated with different lowercase letters (P < 0.05, based on Tukey’s test, DPS7.05).

FIGURE 3

Expected versus observed mortalities caused by the combinations of Cry1Ac and FK506 against H. armigera. Each error bar represents the standard error of the mean from three biological replicates. 10, 20, and 50 indicate the concentrations of FK506 in μM. Asterisk shows significant differences between observed and expected mortality at P < 0.05 level (based on Student’s t-tests, DPS7.05).

FIGURE 4

The interactions between Cry1Ac and HaCAN based on yeast two-hybrid system. Diagrammatic representation of the bait (A) and prey (B) constructs. SD/-LT/X (SD-leucine-tryptophan + 40 mg/L X-Gal media) (X-Gal soluted in N,N-dimethylformamide), SD/-LTH/X (SD-leucine-tryptophan-histidine + 40 mg/L X-Gal media), SD/-LTHA/X (SD-leucine-tryptophan-histidine-adenine + 40 mg/L X-Gal media), and SD/-LTHA/X/10mM3-AT (SD-leucine-tryptophan-histidine-adenine + 40 mg/L X-Gal media + 10 mM 3-AT) plates, respectively. (C) Representative growth of NMY51 yeast cells co-transformed with one of the seven pairs of prey and bait constructs on four SD medium plates with increasing selection stringency. The treatment of pTSU2-APP and pNubG-Fe65 was positive control. The treatment of pBT3-N-Cry1Ac and pPR3-N was negative control.

FIGURE 5

Impact of the heterologously expressed HaCAN on the cytotoxicity of Cry1Ac to midgut cells. (A) The Sf9 cells that transfected GFP-pIEx (empty vector). (B) The Sf9 cells that transfected GFP-pIEx-CAN. (C) Western blot analysis of HaCAN expression in the transfected cells. (D) The determination of HaCAN activity. (E) Cell mortalities caused by 100 μg/mL activated Cry1Ac. (F,G) Photographs are representative of 400x views of the two treatments under an inverted microscope (OPTIKA IM-5). The blue cells represent dead cells, which were stained blue by trypan blue. Each error bar represents the standard error of the mean from three transfection replicates. Asterisks show significant differences in expression levels, enzyme activities, and mortalities between each treatment (P < 0.05, Student’s t-test, DPSSOFT: DPS7.05).

FIGURE 6

Impact of silencing HaCAN on the cytotoxicity of Cry1Ac to midgut cells. (A) Expression levels of HaCAN in the midgut cells. (B) The determination of HaCAN activity. (C) Cell mortalities caused by 50 μg/mL activated Cry1Ac. (D,E) Photographs are representative of 400x views of the two treatments of the cell lines under an inverted microscope (OPTIKA IM-5). The dead cells were stained blue by trypan blue. Each error bar represents the standard error of the mean from three transfection replicates. Asterisks show significant differences in expression levels, enzyme activities, and mortalities between each treatment (P < 0.05, Student’s t-test, DPSSOFT: DPS7.05).