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

蛞蝓是英国谷物和油菜（OSR）作物最具破坏性的害虫，也是马铃薯的重要害虫。目前的蛞蝓控制实践现在只依赖于两种活性成分，四聚乙醛和磷酸铁，作为颗粒状诱饵在现场交付。在不久的将来，金属醛的使用可能会受到限制，因为它的浓度超过了欧盟饮用水标准。蚜虫也是谷物、OSR和马铃薯的严重害虫，通过取食造成直接损害，并通过传播植物病毒（例如，植物病毒）造成间接损害。芜菁黄化病毒）。由于许多化学杀虫剂从市场上撤出以及新活性物质的短缺，伴随着对目前允许使用的少数产品的抗性的广泛发展，害虫控制已经变得越来越成问题。申请人开发了一种创新方法并获得专利，该方法将没有口服毒性的天然来源的肽（例如蜘蛛捕获猎物时产生的毒素）转化为口服有效的杀虫剂。这种毒素与能够穿过肠壁的“载体”蛋白的连接使得毒素能够运输到其作用部位，通常是目标害虫的中枢神经系统。使用酵母作为表达宿主重组产生的融合蛋白已被证明对一系列作物害虫有效，包括蛞蝓和蚜虫，但对蜜蜂没有有害影响[1]。在转基因植物中，这种方法的概念证明最近已经通过以表达杀虫融合蛋白Hv 1a/GNA的拟南芥为饲料的蚜虫的显著死亡率来证明[2]。该项目的目的是生产表达候选杀虫融合蛋白的转基因植物，并评估后代对灰田蛞蝓（Deroceras reticulatum）和蚜虫（桃马铃薯Myzus persicae，谷物Sitobion avenae）损害的保护。通过外源性应用（例如，目前正在与工业界合作开发用于控制作物害虫的杀虫剂（如毒饵和喷雾剂）。一种更优雅和有针对性的方法是通过转基因植物在田间传递融合蛋白。如果成功，这将为未来保护英国主要作物免受主要害虫的侵袭提供一种非常新颖的方法。利用不同的毒素靶向作物害虫的不同作用位点产生各种融合蛋白的潜力，这项研究也可以提供一个平台，从中开发一种金字塔方法，以促进有效的害虫控制和对抗抗性的发展。掺入动物或植物来源的杀虫毒素和植物来源的载体蛋白的候选融合蛋白将用于产生转基因植物。候选毒素包括Hv 1a蜘蛛毒肽，它靶向昆虫电压门控钙通道和豌豆白蛋白1b（Pa 1b），一种来自豌豆种子的肽，抑制V-ATP酶。雪花莲凝集素雪花莲凝集素（Galanthus nivalis agglutinin，GNA）被公认为杀虫融合蛋白的载体组分，并且最近鉴定的替代物，来自生姜的甘露糖结合凝集素（ZoA）也将被利用。选择Hv 1a/GNA是因为已证明其对蛞蝓的口服毒性与四聚乙醛相当，对拟除虫菊酯抗性蚜虫的毒性相当[3]。我们最近已经证明，重组融合蛋白，将Pa 1b连接到GNA或ZoA有显着的有害影响蚜虫的生存和繁殖力的人工饲料生物测定。概念验证将使用成熟的浸花技术进行，以产生在组成型和/或韧皮部特异性启动子控制下表达融合蛋白的转基因拟南芥。纯合系将被检测以保护免受幼D的攻击。reticulatum和M.在整株植物和离体叶测定中。A m

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