Abstract
Aphids are sap-sucking insects (order: Hemiptera) that cause extensive damage to a wide range of agricultural crops. Our goal was to optimize a naturally occurring insecticidal crystalline (Cry) toxins produced by the soil-dwelling bacterium Bacillus thuringiensis for use against the pea aphid, Acyrthosiphon pisum. On the basis that activation of the Cry4Aa toxin is a rate-limiting factor contributing to the relatively low aphicidal activity of this toxin, we introduced cathepsin L and cathepsin B cleavage sites into Cry4Aa for rapid activation in the aphid gut environment. Incubation of modified Cry4Aa and aphid proteases in vitro demonstrated enhanced processing of the toxin into the active form for some of the modified constructs relative to non-modified Cry4Aa. Aphids fed artificial diet with toxin at a final concentration of 125 μg/ml showed enhanced mortality after two days for one of the four modified constructs. Although only modest toxin improvement was achieved by use of this strategy, such specific toxin modifications designed to overcome factors that limit aphid toxicity could be applied toward managing aphid populations via transgenic plant resistance.
Highlights
Aphids can cause extensive economic losses to agricultural crops, with some U.S $1.6 billion in costs attributed to the soybean aphid, Aphis glycines in the United States alone [1]
Purified modified Cry4Aa constructs appeared as two prominent bands at 60 and 65 -kDa in polyacrylamide gels, both of which were detected by western blot (Fig 2B)
By exploiting the major proteases utilized in the aphid gut and modifying a crystal toxins (Cry) toxin that, when activated, is toxic against pea aphids we found enhancement of toxin activation in vitro
Summary
Aphids can cause extensive economic losses to agricultural crops, with some U.S $1.6 billion in costs attributed to the soybean aphid, Aphis glycines in the United States alone [1]. Yield losses occur through direct feeding, transmission of numerous plant viruses [2] and from aphid honeydew which provides a medium for fungal growth [3]. Current aphid management relies primarily on the application of chemical insecticides that may have negative environmental consequences and to which aphids can rapidly develop resistance [4, 5]. Transgenic crops incorporating insecticidal crystal toxins (Cry) isolated from the bacterium Bacillus thuringiensis have been applied for management of other insect pests [6,7,8,9,10,11], resulting in increased yields, and decreased use of chemical insecticides [9,10,11]. Aphids (Hemiptera) use piercingsucking mouthparts to feed on plant phloem resulting in minimal natural exposure to Bt toxins
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