Bt toxin engineering to promote protoxin activation in the aphid gut

Aphids cause extensive damage to agricultural crops by feeding on phloem and by spreading numerous plant viruses. Use of crops expressing insecticidal crystalline (Cry) toxins derived from the bacterium Bacillus thuringiensis (Bt) for control of other insect pests suggests that this technology could also be used to control aphid populations. However aphids experience only low levels of mortality when exposed to Cry toxins. A crucial step in Cry toxicity is cleavage of the protoxin into its active form by serine proteases active at alkaline pH in the midgut of some insects. The major proteases in the pea aphid (Acyrthosiphon pisum) are cathepsin L/B which are active at slightly acidic or neutral pH. We introduced cathepsin L and B cleavage sites into Cry4A-S1 for rapid activation and generation of the active toxin in the aphid gut environment. Using overlap extension polymerase chain reaction (OE-PCR) we generated four constructs with specific cleavage sites for Cry4Aa-S1 activation. In vivo and in vitro assays will be conducted to assess if the modified Cry constructs are activated and stable in the aphid gut environment. Toxicity against the pea aphid will be evaluated using membrane feeding assays.