Establishing potential for the protection of crops against damage by slugs and aphids using transgenic plants expressing orally toxic fusion proteins.

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Slugs are the most damaging pest of cereal and oil seed rape (OSR) crops in the UK, and are an important pest of potatoes. Current practice for slug control now relies on just two active ingredients, metaldehyde and ferric phosphate, delivered as pelleted baits in the field. Metaldehyde may in the near future be subject to restricted usage as it has been found in concentrations above the permitted EU drinking water standards. Aphids are also a serious pest of cereals, OSR and potatoes, causing direct damage by feeding, and indirect damage via the transmission of plant viruses (eg. Turnip Yellows Virus). Pest control has become increasingly problematic due to the withdrawal of many chemical pesticides from the marketplace and a shortage of new actives concomitant with the widespread development of resistance to the few products currently permitted for use. The applicants have developed and patented an innovative approach that converts naturally derived peptides that have no oral toxicity, such as toxins produced by spiders to capture prey, into orally effective pesticides. Linkage of such toxins to a "carrier" protein able to cross the gut wall enables transport of the toxin to its site of action, typically the central nervous system of the target pest. Fusion proteins produced recombinantly using yeast as an expression host have been shown to be effective against a range of crop pests, including slugs and aphids, but have no deleterious effects on bees [1]. Proof of concept for this approach in transgenic plants has recently been demonstrated by significant mortality of aphids fed on Arabidopsis expressing the insecticidal fusion protein Hv1a/GNA [2]. The aim of the project is to produce transgenic plants expressing candidate pesticidal fusion proteins and to evaluate progeny for protection against damage by the grey field slug (Deroceras reticulatum) and aphids (peach potato Myzus persicae, cereal Sitobion avenae). Exploitation of fusion protein technology through exogenous applications (eg. bait and sprays) for the control of crop pests is currently being developed in collaboration with industry. A more elegant and targeted approach would be to deliver fusion proteins in the field via transgenic plants. If successful this would offer a highly novel method for the future protection of key UK crops against attack by major pests. With potential for the generation of a variety of fusion proteins using different toxins to target different sites of action in crop pests this research may also offer a platform from which to develop a pyramid approach to facilitate effective pest control and combat resistance development. Candidate fusion proteins incorporating animal or plant derived pesticidal toxins and plant derived carrier proteins will be used to generate transgenic plants. Candidate toxins include the atracotoxin Hv1a spider venom peptide that targets insect voltage-gated calcium channels and pea albumin 1b (Pa1b) a peptide from pea seeds that inhibits V-ATPase. Snowdrop lectin Galanthus nivalis agglutinin (GNA) is well established as a carrier component of pesticidal fusion proteins and a recently identified alternative, mannose-binding lectin from ginger Zingiber officinale (ZoA) would also be utilised. Hv1a/GNA has been selected as it has been shown to have equivalent oral toxicity towards slugs to metaldehyde and toxicity to pyrethroid resistant aphids [3]. We have recently demonstrated that recombinant fusion proteins incorporating Pa1b linked to GNA or ZoA have significantly deleterious effects on aphid survival and fecundity in artificial diet bioassays. Proof of concept will be conducted using the well-established floral dipping technique to create transgenic Arabidopsis expressing fusion proteins under the control of constitutive and/or phloem specific promoters. Homozygous lines would be assayed for protection against attack by juvenile D. reticulatum and M. persicae in whole plant and detached leaf assays. A m