Abstract
Recombinant fusion protein technology allows specific insecticidal protein and peptide toxins to display activity in orally-delivered biopesticides. The spider venom peptide δ-amaurobitoxin-PI1a, which targets insect voltage-gated sodium channels, was fused to the “carrier” snowdrop lectin (GNA) to confer oral toxicity.The toxin itself (PI1a) and an amaurobitoxin/GNA fusion protein (PI1a/GNA) were produced using the yeast Pichia pastoris as expression host. Although both proteins caused mortality when injected into cabbage moth (Mamestra brassicae) larvae, the PI1a/GNA fusion was approximately 6 times as effective as recombinant PI1a on a molar basis. PI1a alone was not orally active against cabbage moth larvae, but a single 30 μg dose of the PI1a/GNA fusion protein caused 100% larval mortality within 6 days when fed to 3rd instar larvae, and caused significant reductions in survival, growth and feeding in 4th – 6th instar larvae. Transport of fusion protein from gut contents to the haemolymph of cabbage moth larvae, and binding to the nerve chord, was shown by Western blotting. The PI1a/GNA fusion protein also caused mortality when delivered orally to dipteran (Musca domestica; housefly) and hemipteran (Acyrthosiphon pisum; pea aphid) insects, making it a promising candidate for development as a biopesticide.
Highlights
Synthetic pesticides have been widely used for crop protection against herbivorous insects in intensive agricultural production, and are a necessary input to achieve high yields and consequent food security
The expression construct for production of recombinant PI1a/GNA fusion protein contained the same synthetic mature PI1a coding sequence fused to the N-terminus of a coding sequence corresponding to residues 1e105 of mature snowdrop lectin (GNA) via a 3 amino acid linker peptide; again, the fusion protein was arranged in-frame C-terminal to the a-factor prepro-sequence, and N-terminal to a sequence encoding the (His)6 tag, supplied by the vector (Fig. 1B)
The d-amaurobitoxin PI1a was selected as a possible component for biopesticidal fusion proteins for reasons described earlier, and because it is effective against a different target than previous insecticidal neurotoxins used in lectin-based fusion proteins; Sfl1, from the spider Segestria florentina (Fitches et al, 2004) has an unknown target, ButaIT from the scorpion Mesobuthus tamulus (Trung et al, 2006) is assumed to target chloride channels, and Hv1a, from the spider Hadronyche versuta (Fitches et al, 2012) targets calcium channels
Summary
Synthetic pesticides have been widely used for crop protection against herbivorous insects in intensive agricultural production, and are a necessary input to achieve high yields and consequent food security. There are widely held concerns over the indiscriminate use of pesticides in general, and insecticides in particular, including the development of resistance in target pests, detrimental effects in non-pest and beneficial insects, contamination of watercourses and the poisoning of higher animals. Besides naturally occurring protein biopesticides such as Bacillus thuringiensis toxins, biotechnological methods can be used to produce recombinant proteins with insecticidal activity. These include insecticidal fusion proteins containing a toxic peptide or protein fused to a “carrier”, where the carrier confers oral activity on a toxin that must normally be injected into the insect to reach its site of action, by directing transport of the fusion across the insect gut (Fitches et al, 2004)
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