Abstract
Recombinant fusion proteins containing arthropod toxins have been developed as a new class of biopesticides. The recombinant fusion protein Hv1a/GNA, containing the spider venom toxin ω-ACTX-Hv1a linked to snowdrop lectin (GNA) was shown to reduce survival of the peach-potato aphid Myzus persicae when delivered in artificial diet, with survival <10% after 8 days exposure to fusion protein at 1 mg/ml. Although the fusion protein was rapidly degraded by proteases in the insect, Hv1a/GNA oral toxicity to M. persicae was significantly greater than GNA alone. A construct encoding the fusion protein, including the GNA leader sequence, under control of the constitutive CaMV 35S promoter was transformed into Arabidopsis; the resulting plants contained intact fusion protein in leaf tissues at an estimated level of 25.6 ± 4.1 ng/mg FW. Transgenic Arabidopsis expressing Hv1a/GNA induced up to 40% mortality of M. persicae after 7 days exposure in detached leaf bioassays, demonstrating that transgenic plants can deliver fusion proteins to aphids. Grain aphids (Sitobion avenae) were more susceptible than M. persicae to the Hv1a/GNA fusion protein in artificial diet bioassays (LC50 = 0.73 mg/ml after 2 days against LC50 = 1.81 mg/ml for M. persicae), as they were not able to hydrolyze the fusion protein as readily as M. persicae. Expression of this fusion protein in suitable host plants for the grain aphid is likely to confer higher levels of resistance than that shown with the M. persicae/Arabidopsis model system.
Highlights
Aphids significantly impact agricultural and horticultural crops, either by causing direct damage to plants through feeding on the phloem, or indirectly by acting as vectors for plant pathogenic viruses
The present study demonstrates that the fusion protein Hv1a/Galanthus nivalis agglutinin (GNA) is toxic toward both the peach-potato aphid and the grain aphid
Survival curves differed from each other (p < 0.001), and pairwise multiple comparisons showed significant differences between all BIOASSAYS WITH TRANSGENIC PLANTS Transgenic F3 Arabidopsis plants homozygous for the gene expressing Hv1a/GNA were used in bioassays with M. persicae only, as S. avenae does not feed on crucifers
Summary
Aphids significantly impact agricultural and horticultural crops, either by causing direct damage to plants through feeding on the phloem, or indirectly by acting as vectors for plant pathogenic viruses. There are major drawbacks to the use of these peptides, as topical sprays, as they are unlikely to be rapidly absorbed through the insect cuticle to reach their site of action and are prone to degradation in the environment (Fitches et al, 2004a) Should they survive the application process and be taken up by the insect, they are unlikely to survive the conditions of the insect gut (Fitches et al, 2004a) or be delivered across the midgut epithelium to the correct targets within the insect (Tedford et al, 2004). The discovery that snowdrop lectin Galanthus nivalis agglutinin (GNA) remains stable and active within the insect gut after ingestion, and that it is able to cross the midgut epithelium (Powell et al, 1998), provided an opportunity for its use as a ‘carrier molecule’ to deliver other peptides to the circulatory system of target insect species (Fitches et al, 2002)
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