Insect-specific toxins derived from Bacillus thuringiensis (Bt) provide a valuable resource for pest suppression. Here we review the different strategies that have been employed to enhance toxicity against specific target species including those that have evolved resistance to Bt, or to modify the host range of Bt crystal (Cry) and cytolytic (Cyt) toxins. These strategies include toxin truncation, modification of protease cleavage sites, domain swapping, site-directed mutagenesis, peptide addition, and phage display screens for mutated toxins with enhanced activity. Toxin optimization provides a useful approach to extend the utility of these proteins for suppression of pests that exhibit low susceptibility to native Bt toxins, and to overcome field resistance.

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.

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First and foremost, I would like to thank my advisor, Bryony Bonning, for allowing me the opportunity to work in her lab. A special thank you to Nanasaheb Chougule who provided me with my initial training, Teresa Fernandez-Luna for helping troubleshoot, Yuting Chen for assisting with both PCR methodologies and feeding assays, and Mike Rausch for helping with feeding assays. Last but not least, I would like to thank my loving parents, Robert and Terri Deist, for their lifelong encouragement and support.