Abstract
A pyramid strategy combining the Cry1A and Cry2A toxins in Bt crops has been widely used throughout the world to delay pest adaption to transgenic crops and broaden the insecticidal spectrum. Midgut membrane-bound cadherin (CAD), aminopeptidase-N (APN) and alkaline phosphatase (ALP) are important for Cry1A toxicity in some lepidopteran larvae, but the proteins that bind Cry2A in the midgut of target insects and their role in the Cry2A mechanism of action are still unclear. In this study, we found that heterologously expressed CAD, APN4 and ALP2 peptides from the midgut of Helicoverpa armigera could bind to the Cry2Aa toxin with a high affinity. Additionally, the efficiency of Cry2Aa insecticidal activity against H. armigera larvae was obviously reduced after the genes encoding these proteins were silenced with specific siRNAs: CAD- and ALP2-silenced larvae showed significantly similar reductions in mortality due to the Cry2Aa toxin (41.67% and 43.06%, respectively), whereas a larger reduction in mortality was observed in APN4-silenced larvae (61.11%) than in controls. These results suggest that CAD, APN4 and ALP2 are involved in the mechanism of action of Cry2Aa in H. armigera and may play important functional roles in the toxicity of the Cry2Aa toxin.
Highlights
Has been widely used in many countries to replace first-generation Bacillus thuringiensis (Bt) cotton, producing only the Cry1A toxin
The expressed peptides were evaluated through SDS-PAGE, and the results demonstrated that the expressed CAD, APN4 and ALP2 proteins exhibited the expected sizes of 47 kDa, 40 kDa and 51 kDa, respectively, and that there was only one main visible band for these three proteins after purification (Fig. 1, lane 1, lane 2 and lane 3)
The purified proteins were detected with the corresponding antibodies against HaCAD, HaAPN4 and HaALP2, and the results indicated that all three purified recombinant proteins could strongly hybridize with the corresponding polyclonal antibodies (Fig. 1, lane 4, lane 5 and lane 6)
Summary
Has been widely used in many countries to replace first-generation Bt cotton, producing only the Cry1A toxin. In contrast with Cry1A proteins, Cry2A toxins can effectively control lepidopteran pests other than H. armigera, such as Spodoptera exigua and Agrotis ypsilon[24]. Introduction of Cry1A/Cry2A Bt cotton in China is expected to provide more effective control activity against many target lepidopteran pests and assist in delaying the development of insect resistance to Bt toxic proteins[25,26]. To determine the roles of CAD, APN and ALP from the midgut of H. armigera in the Cry2Aa toxin mechanism of action, we studied the binding characteristics of the Cry2Aa toxin with recombinant H. armigera CAD, APN4 (a cluster of lepidopteran APNs) and ALP2 (a cluster of lepidopteran ALPs) peptides[32,33]. We hypothesize that CAD, APN4 and ALP2 may function as receptors for the Cry2Aa toxin in H. armigera and play important functional roles in the mechanism of action of the Cry2Aa toxin
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