Journal of Integrative Plant Biology 2012, 54 (5): 282299

Invited Expert Review

Metabolic Engineering of Plant-derived (E)--farnesene Synthase Genes for a Novel Type of Aphid-resistant Genetically Modied Crop Plants
F

Xiu-Dao Yu1 , John Pickett2 , You-Zhi Ma1 , Toby Bruce2 , Johnathan Napier2 , Huw D. Jones2 and Lan-Qin Xia1
of Crop Sciences (ICS), Chinese Academy of Agricultural Sciences (CAAS), Beijing 100081, China Research, Harpenden, Hertfordshire AL5 2JQ, UK Corresponding author Tel(Fax): +86 10 8210 5804; E-mail: xialq@mail.caas.net.cn F Articles can be viewed online without a subscription. Available online on 20 February 2012 at www.jipb.net and www.wileyonlinelibrary.com/journal/jipb doi: 10.1111/j.1744-7909.2012.01107.x
2 Rothamsted 1 Institute

Abstract
Aphids are major agricultural pests that cause signicant yield losses of crop plants each year. Excessive dependence on insecticides for long-term aphid control is undesirable because of the development of insecticide resistance, the potential negative effects on non-target organisms and environmental pollution. Transgenic crops engineered for resistance to aphids via a non-toxic mode of action could be an efcient alternative strategy. (E)--Farnesene (EF) synthases catalyze the formation of EF, which for many pest aphids is the main component of the alarm pheromone involved Lan-Qin Xia in the chemical communication within these species. EF can also (Corresponding author) be synthesized by certain plants but is then normally contaminated with inhibitory compounds. Engineering of crop plants capable of synthesizing and emitting EF could cause repulsion of aphids and also the attraction of natural enemies that use EF as a foraging cue, thus minimizing aphid infestation. In this review, the effects of aphids on host plants, plants defenses against aphid herbivory and the recruitment of natural enemies for aphid control in an agricultural setting are briey introduced. Furthermore, the plant-derived EF synthase genes cloned to date along with their potential roles in generating novel aphid resistance via genetically modied approaches are discussed. Keywords:
Aphids; (E)--farnesene sythase; genetic-modied crops; terpenoid.

Yu XD, Pickett J, Ma YZ, Bruce T, Napier J, Jones HD, Xia LQ (2012) Metabolic engineering of plant-derived (E)--farnesene synthase genes for a novel type of aphid-resistant genetically modied crop plants. J. Integr. Plant Biol. 54(5), 282299.

Introduction
Aphids (Aphididae) are major agricultural pests that cause signicant yield losses of crop plants each year by inicting damage both through the direct effects of feeding and by vectoring harmful plant viruses (International Aphid Ge-

nomics Consortium 2010). Annual worldwide crop losses due to aphids are estimated at hundreds of millions of dollars (Blackman and Eastop 1984; Oerke 1994; Morrison and Peairs 1998). Along with the application of nitrogen fertilizer and elevation of atmospheric CO 2 concentration, aphid infestation becomes more serious (Awmack and Harrington 2000; Aqueel

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and Leather 2011). For many crops, insecticides provide a simple and effective strategy for aphid control. However, the application of such chemicals is not desirable in the long term, because of the development of insecticide resistance (Sabater-Munoz et al. 2006) and the potential negative effects on non-target organisms, and the need for more sustainable agricultural practices with fewer external chemical inputs. During a long period of co-evolution, plants and aphids have established a complex interaction. On the one hand, aphids have established complex life cycles involving extensive phenotype plasticity with rapid population growth (Blackman and Eastop 1984), and a very short generation time of as little as 5 d (Morrison and Peairs 1998). They also have different morphs: one genotype can generate winged or unwinged forms depending on environmental stresses. Furthermore, they can alternate between a parthenogenetic (a form of asexual reproduction where growth and development of embryos occur without fertilization of male and female gametes) morph with asexual live-bearing females and a sexual morph with males and egg-laying females during different seasons (International Aphid Genomics Consortium 2010). On the other hand, plants have also evolved delicate and complicated defense systems comprising constitutive defense traits and defense pathways induced upon aphid attack (Chen 2008). Conventional breeding programs have been undertaken and considerable efforts have been expended in the search for aphid resistance in small grain germplasm worldwide (Stoger et al. 1999). However, due to the complexity of plant-aphid interactions and the rapid development of resistant pest biotypes (Stoger et al. 1999), outbreak of aphids causing substantial losses are reported regularly. Breeders and growers are still struggling to nd an efcient strategy for aphid control in major crop plants. Development of aphid-resistant crop plants through genetic engineering would

be a good alternative strategy. To date, efforts on development of aphid-resistant transgenic crops have mainly concentrated on introduction of plant-derived lectin genes (Table 1), which may potentially have harmful effects on aphid natural enemies as well (Birch et al. 1999; Hogervorst et al. 2009). Aphid populations are regulated by natural enemies such as ladybird beetles and parasitoid wasps. Despite the effectiveness of biological control, behavioral responses to the threat of predation may allow aphids to persist as pests (De Vos et al. 2010). For many pest species of aphids, avoidance of predators involves the release of an alarm pheromone comprising (E)--Farnesene (EF), a volatile sesquiterpene, released from the cornicles (aka siphunculi) on the aphids abdomen when attacked by its natural enemies (Bower et al. 1972; Pickett and Grifths 1980; Dixon 1998). In this context, EF could function either as a direct repellent or act as a kairomone (chemical messengers emitted by organisms of one species but benet members of another species) for attraction of natural enemies of aphids (Hatano et al. 2008). Although application of EF to crop plants against pest aphids would be an alternative approach, high oxidation rate of EF has hampered the application of EF either in laboratory tests (Yang and Zettler 1975) or directly on crops (Hille Ris Lambers and Schepers 1978). Essential oils, which protect EF from oxidation, have also been trialed in the laboratory and eld (Bruce et al. 2005), but the aphids are sensitive to other minor components included, which reduce the repellent response. Many plant species produce EF, either constitutively or in response to herbivore damage (Crock et al. 1997; Mumm et al. 2003; Harmel et al. 2007). However, most produce other compounds that interfere with the response of EF (Dawson et al. 1984) although some plants have evolved to avoid this

Table 1. Lectin genes have been engineered into the major crop plants for aphid control Gene or gene products Lectins Galanthus nivalis agglutinin Maize Wheat Corn leaf aphid Rhopalosiphum maidis Grain aphid Sitobion avenae Wheat aphid Schizaphis graminum Grain aphid Sitobion avenae and Rhopalosiphum padi Linnaeus Galanthus nivalis agglutinin and wheat -amylase inhibitor Canavalia ensiformis lectin Pinellia ternata agglutinin and cry1Ac Amaranthus caudatus agglutinin Others Trypsin inhibitor mti-2 Potato Peach-potato aphid Myzus persicae Saguez et al. 2010 Cotton Cotton aphid Aphis gossypii Glover Wu et al. 2006a Potato Wheat Peach-potato aphid Myzus persicae Wheat aphid Schizaphis graminum Rondani Gatehouse et al. 1999 Yu and Wei 2008 Potato Peach-potato aphid Myzus persicae Gatehouse et al. 1996 Wang et al. 2005 Stoger et al. 1999 Xu et al. 2004 Liang et al. 2004 Transformed crops Targeted insects Reference

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problem (Gibson et al. 1983). EF synthase genes, which encode enzymes that convert farnesyl diphosphate (FPP) to the acyclic sesquiterpene EF, have been isolated and characterized from Douglas r (Pseudotsuga menziesii) (Huber et al. 2005), Yuzu (Citrus junos) (Maruyama et al. 2001), sweet wormwood (Artemisia annua) (Picaud et al. 2005, Yu et al. 2011) and black peppermint (Mentha piperita) (Crock et al. 1997; Prosser et al. 2006) (Table 2). Studies of tritrophic interactions involving aphids and their natural predators and parasitoids have demonstrated that transgenic Arabidopsis and tobacco plants expressing peppermint and sweetworm wood EF synthase genes, respectively can repel aphids and attract their natural enemies, thus minimizing aphid infestation (Beale et al. 2006; Yu et al. 2011). This demonstrates a potentially valuable strategy of using plant-derived EF synthase genes for aphid control in economically important crops in an environmentally benign way. In this review, we briey introduce the effects of aphids on host plants, plants defenses to aphid herbivory and the recruitment of natural enemies for aphid control in an agricultural setting. Furthermore, the plant-derived EF synthase genes cloned to date are discussed along with the strategies for manipulating the EF synthetic pathway in crop plants and the potential for generating novel aphid resistance via genetically modied (GM) approaches.

Effects of Aphids on Host Plants

Aphids are major arthropod pests of agriculture worldwide; the family Aphididae comprises more than 4 300 species, damaging crops by sucking nutrients from the phloem and/or by transmitting plant viruses. Unlike the majority of insects, aphids can reproduce clonally and an aphids embryonic development begins before its mothers birth (Goggin 2007). These traits allow for rapid population growth of aphids in the eld due to their parthenogenetic lifestyle, short generation times, and the fact that nymphs of certain aphid species can reach maturity in as little as 5 d (Dixon 1988; Goggin 2007; De Vos et al. 2010).

Table 2. Plant-derived EF synthase genes isolated so far Plant species Douglas r Yuzu Sweet wormwood Genbank accession no. AY906867 AF374462 AY835398; GU294840; GU294841 Black peppermint AF024615; AJ86642 Crock et al. 1997 Prosser et al. 2006 References Huber et al. 2005 Maruyama et al. 2001 Picaud et al. 2005 Yu et al. 2011

Aphids damage their host plants by ingesting phloem sap through narrow piercing-sucking mouthparts called stylets. Successful phloem feeding requires overcoming a number of phloem-related plant properties and reactions. The most important hurdle is formed by the phloem wound responses, such as coagulating proteins in the phloem sieve elements of the plant and in the capillary food canal in the insects mouth parts, i.e. the stylets (Tjallingii 2006). In order to actively suppress the plant defense responses, aphids often initiate probing of the leaf epidermis cells immediately upon landing. Each probe takes less than one minute and involves stylet penetration of the epidermis cell wall and membrane, injection of saliva and ingestion of the cell contents, which causes only minor damage to the host plant (Hogenhout and Bos 2011). During probing and feeding, aphids secrete two types of saliva: gelling saliva, which is thought to protect stylets during penetration, and water saliva, which is secreted into various plant host cell types and phloem (Prado and Tjallingii 2007). Once prolonged feeding has been established, aphids repeatedly inject watery saliva into plant cells and phloem during salivation. In addition to having a protective function in the form of polyphenol oxidases and other detoxifying enzymes, aphid saliva likely contains factors that facilitate uptake of phloem sap (Cherqui and Tjallingii 2000; De Vos and Jander 2009) and some salivary proteins share the same features with plant pathogen effectors and therefore may function as aphid effectors by perturbing host cellular processes (Bos et al. 2010). Will et al. (2000) demonstrated that aphid saliva also contains calcium-binding proteins and can inhibit calciumdependent phloem occlusion, which is triggered by a calcium ux in response to wounding. The ability to prevent sieve tube plugging is an important adaptation that allows aphids to remain at a single feeding site for hours at a time (Goggin 2007). Symptoms of aphid attack differ depending on the aphid species and plant species in question. More common symptoms of aphid infestation include chlorosis, necrosis, wilting, stunting, and malformation of new growth (Goggin 2007). As far as cereal aphids are concerned, the Russian wheat aphid (Diuraphis noxia Mordvilko) gives rise to leaf rolling and discoloration, while the greenbug (Schizaphis graminum) gives rise to typical leaf discoloration and eventually necrosis (Delp et al. 2009). In contrast, the bird cherry-oat aphid causes no specic symptoms, except reduced plant growth at high population densities (Delp et al. 2009). Meanwhile, Honek and Martinkova (2004) found that the growth and population density of the cereal aphid Metopolophium dirhodum (W.) was mainly dependent on the quality of the host plant and many other environmental factors are also known to affect the fecundity and longevity of aphids (Aqueel and Leather 2011). Furthermore, aphids also induce physiological changes in their host plants. The pea aphid, for example, diverts nitrogen from

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the apical growth zones of alfalfa to nitrogen source tissues and increases deposition rate of nitrogen in the middle part of the infested tissue (Girousse et al. 2005). Infestations of grasses by two species of aphids, greenbug and Russian wheat aphid, increase the concentrations of amino acids and the proportions of essential amino acids in the phloem sap of the host plants (Sandstrom 2000). Greenbugs also greatly increase the concentrations of free amino acids in the wheat leaves at the infestation sites (Dorschner et al. 1987). Many of the symptoms or the physiological changes of the host plants induced by aphid infestation are clearly benecial to aphids and deleterious to their host plants. For example, during the compatible interactions with Russian wheat aphid, susceptible wheat plants react to the injection of aphid saliva by discoloration and rolling of the leaves longitudinally around the main leaf vein to form a tubular refuge that protects aphids from predators (Smith et al. 2010); the discoloration can signicantly reduce the plant photosynthetic efciency and results in weakened plants with substantially lower grain yields (Smith et al. 1991). Each year, worldwide crop losses due to aphids are estimated at hundreds of millions of dollars (Blackman and Eastop 1984; Oerke 1994; Morrison and Peairs 1998). For example, in 20102011 crop seasons, 62.5% of the 26 million hectares of the Chinese wheat growing area suffered severe aphid infestations (Xia et al. 2011). The major aphid species infesting wheat in China are grain aphids (Sitobion avenae F.), greenbug (S. graminum), Rhopalosiphum padi Linnaeus and Metopolophium dirhodum Walker. Among them, grain aphids are the more dominant and destructive species, affecting wheat production areas in Yellow Huai and the Northern China Plain, the Southwest, Northwest and the Middle Yangtze River regions (Zhang et al. 2009) and cause as much as 15 to 60% reduction in wheat yield (Wang et al. 2011). Aphids also infest other agriculturally important crops such as maize, cotton, oilseed and soybean with the affected area estimated as 4.3, 4.7, 2.3 and 2.05 million hectares, respectively, resulting in yield reductions and economic losses to farmers in spite of wide-spread use of chemicals. The area of aphid infestation represents approximately 14%, 90%, 32% and 23% of the total growth area of maize, cotton, oilseed and soybean, respectively, in China in the 2010/11 crop seasons (www.natesc.moa.gov.cn; the statistical data of the affected acreages of soybean came from personal communication with Professor Kunjun Zhao, China Northeast Agricultural University).

physical and chemical barriers that exist before aphid attack. For example, trichomes are modied epidermal cells found on the surface of the aerial organs of most plants, which constitute a physical barrier to aphid movement and settlement (Tingey and Laubengayer 1981; Vargas et al. 2005); trichomes are also involved in the secretion of large amounts of sucrose esters, which play a major role in deterring the settling and probing of aphids (Neal et al. 1990). In addition, some plant secondary metabolites, including phenylalanine ammonia-lyase (PAL), polyphenol oxidase (PPO), peroxidase (POD) and 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA) are considered important biochemical markers in cereal plants resistance against aphids (Escobar et al. 1999; Han et al. 2009). DIMBOA is the main hydroxamic acid aglucone in wheat and maize extracts, and when added to articial diets, DIMBOA is deleterious to aphids (Argandona et al. 1983; Escobar et al. 1999). Negative relationships were also found between the DIMBOA content of wheat plants and the number of aphids feeding on them (Niemeyer et al. 1989). Relative to the constitutive defenses, inducible defenses become activated upon aphid attack. Two types of inducible defenses, direct defenses and indirect defenses, have been observed in plants. Direct defenses indicate any plant traits that by themselves affect the susceptibility of host plants to insect attacks (Kessler and Baldwin 2001), including host cell wall fortication, R gene-mediated hypersensitive responses and anti-manipulation of hosts to limit food supply of aphids; removing essential nutrients to reduce nutrient value supplied for aphids and producing antibiotic chemicals (Goggin 2007; Chen 2008). Indirect defenses, on the other hand, include plant traits that by themselves do not affect the susceptibility of host plants, but can serve as attractants for natural enemies of the attacking insect (Chen 2008). For example, certain plants such as the aphid-infested potato specially emit EF and the compound attracts egg-laying aphidophagous predators Episyrphus balteatus (Harmel et al. 2007). Other more common volatiles that are induced by aphid herbivory and attract natural enemies include (Z)-3-hexenyl acetate (Yu et al. 2008), methyl salicylate (Zhu and Park 2005) and cis-jasmone (Birkett et al. 2000; Diezel et al. 2011).

Interactions between Aphids and their Natural Enemies

When attacked by predators or parasitoids, aphids secrete sticky defensive droplets from the siphunculi, a pair of tube-like structures on their dorsal surface (Kunert et al. 2005). These droplets contain an alarm pheromone comprising a mixture of compounds with EF as the predominant and, in some aphid species, the only component (Bowers et al. 1972; Pickett and Grifths 1980; Kunert et al. 2005). EF can also function as a kairomone in attracting aphid predators, including ladybirds

Plant Defense Against Aphid Infestation

To defend against aphid attacks, plants have evolved a broad range of defense mechanisms. These mechanisms can be generalized into two categories: constitutive defenses and inducible defenses (Chen 2008). Constitutive defenses include

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(Nakamuta 1991; Zhu et al. 1999; Al Abassi et al. 2000; Mondor and Roitberg 2000; Acar et al. 2001; Francis et al. 2004), syrphids (Francis 2005a; Harmel et al. 2007; Verheggen et al. 2008b), lacewings (Zhu et al. 1999) and parasitoids (Dawson et al. 1983; Micha and Wyss 1996; Du et al. 1998; Foster et al. 2005). A large guild of parasitoids and predators in agro-ecosystems are increasingly recognized as important sources of biocontrol for invasive agricultural aphids. The species of aphid natural enemies in certain regions are correlated with the type of agroecosystem. For example, coccinellids, chrysopids, hemerobiids, syrphids, braconids, aphelinids, and to a lesser extent, nabids and anthocorids comprise the specialist natural enemy fauna using cereal aphids in North America (Brewer and Elliott 2004). Generalist predators are also predominant in the natural enemy complex of soybean, among which, coccinellids including the Harmonia axyridis Pallas and the anthocorid Orius insidiosus (Say), have been shown to successfully suppress pockets of Aphis glycines in soybean even when the overall eld densities were low (Fox et al. 2005; Desneux et al. 2006). The efcacy of biological control is highest during aphid early colonization stages (Edwards et al. 1979; Chiverton 1986). The impact of generalist predators is thought to be critically important in agro-ecosystems under organic farming (Zehnder et al. 2007), where reduced pesticide application may enhance densities of generalist predators, thereby potentially strengthening their contribution to pest control (Hole et al. 2005; Birkhofer et al. 2008).

Current Status of Development of GM Crops for Insect Resistance

Compared with conventional crop breeding programs, genetic engineering of crop plants could not only widen the potential pool of useful genes but also permit the simultaneous introduction of several different desirable genes in a single event. Ever since the rst report of transgenic plants appeared in 1984 (Horsch et al. 1984), insect-resistant transgenic crops have developed very fast and more than 20 different genes conferring insect resistance have been genetically engineered into the major crops. This progress has had a signicant benecial effect on global agriculture, at least in terms of pest reduction and improved quality (Gatehouse et al. 2011). On 22 October 2009, the Chinese Ministry of Agriculture issued its rst two biosafety certicates for commercial production of two Bacillus thuringiensis endotoxin (Bt) rice lines (Chen et al. 2011), providing a broad and stable foundation for future growth of GM wheat and other edible crop plants both in China and the rest of the world (Xia et al. 2011). Although insect-resistant GM crops such as Bt cotton, maize and soybean have had great success in commercialization and planting in a global context,

less progress has been made in development of aphid resistant GM crops. So far, efforts on development of aphid-resistant GM crops have mainly concentrated on introduction of plant-derived lectin genes (Table 1). Many plant lectins show a carbohydrate specicity for glycoconjugates present in organisms (such as viruses, micro-organisms, fungi, nematodes or phytophagous insects) outside the plant kingdom, whereas these glycoconjugates (e.g., galactose, sialic acid) have low abundance or are absent in plants (Vandenborre et al. 2011). The effect of a broad spectrum of plant lectins has been tested on several insect species, some of which are toxic to insects, especially for aphids (Michiels et al. 2010). Up to now, more than four plant lectin genes have been engineered for aphid-resistance in crops. Among them, Galanthus nivalis agglutinin (GNA) has been introduced into wheat (Stoger et al. 1999), maize (Wang et al. 2005) and potato (Gatehouse et al. 1996), respectively. Bioassay results showed that transgenic wheat plants from lines expressing GNA at levels greater than 0.04% of total soluble protein decrease the fecundity, but not the survival of grain aphids (Stoger et al. 1999). Other lectin genes, Canavalia ensiformis lectin (Gatehouse et al. 1999), Amaranthus caudatus agglutinin (Wu et al. 2006a) and Pinellia ternata agglutinin (PTA) (Yu and Wei 2008), have also been proved to be toxic to aphids in transgenic plants. However, GNA could cause adverse effects on predatory ladybirds and parasitoids Aphidius ervi via aphids in the food chain (Birch et al. 1999; Hogervorst et al. 2009), resulting in major concerns over the biosafety of application of these lectin genes in agriculturally important crops for use in aphid control. Therefore, other safe and more effective genes/genetic strategies for aphid control need to be exploited. Recently, Pitino et al. (2011) developed the plantmediated RNAi technology for silencing a peach aphid salivary gland-specic gene MpC002, which encodes a salivary protein that may function as an aphid effector by perturbing host cellular processes, and the gut-specic gene Rack-1; MpC002 and Rack-1 expression were knocked down by up to 60% through feeding on transgenic tobacco and Arabidopsis plants, and silenced aphids produced less progeny, exemplifying the feasibility of plant-mediated RNAi approach for aphid control. However, while MpC002 is an aphid gland-specic gene, Rack-1 is an intracellular receptor that binds activated protein kinase G (PKG), an enzyme primarily involved in signal transduction cascades (Rigas et al. 2003). Rack-1 protein is conserved among plants and animals (Chen et al. 2006), suggesting the application of this gene in genetic engineering of agriculturally important crop plants is also not feasible because biosafety issues are of major concern. Therefore, although the plant-mediated RNAi approach for pest control is currently a hotspot, efforts are still needed to screen for the aphidspecic genes necessary for effective aphid control using this technology.

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Plant Biosynthesis of Terpenoids and their Roles in Pest Resistance

Terpenoids are major components of plant volatile blends and play a predominant role in the attraction of enemies or predators of herbivores (Kopke et al. 2008). All terpenoids are either derived from the mevalonate pathway (MVA) or the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway, which lead to the formation of the C5 unit isopentenyl diphosphate (IPP) and its allylic isomer, dimethylallyl diphosphate DMAPP, the basic terpenoid biosynthesis building blocks (Mahmoud and Croteau 2002). The sequential head-to-tail addition of IPP units to DMAPP in condensation reactions initially yields geranyl diphosphate (GPP, C10), farnesyl diphosphate (FPP, C15) and geranylgeranyl diphosphate (GGDP), which are the precursors of monoterpene, sesquiterpene and diterpene, respectively; in most cases, biosynthesis of sesquiterpenes is assumed to take place at the cytosol/endoplasmic reticulum boundary, whereas monoterpene and diterpene biosynthesis are compartmentalized in plastids (Bohlmann et al. 1998) (Figure 1). Some plant-derived terpenoids have been shown to act as aphid feeding deterrents and are often toxic at higher levels. The sesquiterpene p-benzoquinone perezone isolated from Perezia adnata and even its non-natural derivatives exhibited strong antifeedant activity against peach aphid (Bur-

gueno-Tapia et al. 2008). Other sesquiterpenes farnesol and bisabolene, and monoterpenes geraniol could signicantly inhibit aphid settling in assays using apterous aphids Myzus persicae and leaf discs embedded in agar (Gutierrez et al. 1997). -Citronellol, linalool and oils distilled from several species of Artemisia or Achillea millefolia (yarrow) also showed aphid repellence (Halbert et al. 2009). Apart from acting as feeding deterrents to insects, volatile terpenoids released from different plant parts also act as host location cues for insect natural enemies (Kappers et al. 2005) as well as play crucial roles in pollinator attraction and interaction with the surrounding environment (Weir et al. 2004; Nieuwenhuizen et al. 2009). In a natural environment, caterpillar-damaged maize-releasing EF repelled the corn leaf aphid Rhopalosiphum maidis (Bernasconi et al. 1998), and the trichomes of wild potato Solanum berthaultii release EF, which acts as a kairomone and repels aphids at short distances (Gibson and Pickett 1983). Plant breeders have successfully obtained hybrid potatoes bearing glandular trichomes from the wild potato, which are well defended against infestation and colonization by aphids (Tingey et al. 1982). However, some agronomically important crop plants such as wheat, rice and others do not produce this compound upon aphid infestation, and although EF can also be synthesized by certain plants, in most cases, it is often contaminated with inhibitory compounds.

Figure 1. Terpene biosynthesis pathway in plants (Cited from Bohlmann et al. 1998) DMADP, dimethylallyl diphosphate; FPP, farnesyl diphosphate; G3P, glyceraldehyde-3-phosphate; IPP, isopentenyl diphosphate; GGDP, geranylgeranyl diphosphate; GPP, geranyl diphosphate; MEP, 2-C-methyl-D-erythritol 4-phosphate pathway; MVA, the mevalonate pathway.

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EF and its Potential Role in Aphid Control

Aphids release alarm pheromone from the cornicles on their abdomen when attacked by their natural enemies (Bower et al. 1972; Pickett and Grifths 1980; Dixon 1998). EF as the main, and generally the only component of the aphid alarm pheromone, that can interrupt aphid feeding and cause other aphids in the vicinity to become agitated or disperse from their host plant (Bowers et al. 1972; Pickett and Grifths 1980; Wohlers 1981; Francis et al. 2005b). The quantity and composition of aphid alarm pheromones varies with the aphids morphological type and age (Gut and Oosten 1985). The amount of EF released by aphid increases positively with its body weight but the concentration declines exponentially (Byers 2005). Aphids possess the OBP3 protein in their olfactory system, capable of detecting low concentrations of the alarm pheromone EF (Qiao et al. 2009) and using EF levels to assess their environment with regard to other aphids (Verheggen et al. 2009). However, the release of EF is not contagious, i.e. a non-stressed aphid receiving the alarm signal does not release additional EF (Verheggen et al. 2008a; Hatano et al. 2008). Furthermore, EF also plays a role in the control of aphid morphological types, causing lower weights for adult aphids and lower fecundity and prolonged development when aphids are stimulated with EF during early instars (Su et al. 2006). Therefore, aphid development and total offspring numbers can be negatively affected by EF perception (Mondor and Roitberg, 2003). Generally speaking, EF can increase the proportion of alate offspring (Kunert et al. 2005; Podjasek et al. 2005), while alate aphids are usually more sensitive to EF than the apterae ones and often leave host plants emitting EF (Crock et al. 1997), thereby resulting in lower aphid numbers and reduced virus transmission in host plants. EF can also substantially enhance the effectiveness of pesticides and mycoinsecticide by increasing mobility of aphids (Grifths and Pickett 1980; Elagamy and Haynes 1992; Roditakis et al. 2000). Most importantly, EF can function as a kairomone for natural enemies of aphids such as ladybirds (Nakamuta 1991; Zhu et al. 1999; Al Abassi et al. 2000; Mondor and Roitberg 2000; Acar et al. 2001; Francis et al. 2004), syrphids (Francis 2005a; Harmel et al. 2007; Verheggen et al. 2008b), lacewings (Zhu et al. 1999) and parasitoids (Dawson et al. 1983; Micha and Wyss 1996; Du et al. 1998; Foster et al. 2005), and recruit these parasitoids or predators for aphid control in an agricultural setting. Based on this, continuous efforts have been devoted to the application of synthetic EF in the eld for aphid control in recent years. Application of synthetic EF to crop plants against pest aphids is not feasible because it is a volatile hydrocarbon and is not persistent in the environment. Furthermore, it is susceptible to oxidation due to the presence of several double bonds in the

molecule (Qiao et al. 2009), has a short atmospheric life time, only 11 min by the inuence of OH radicals and 16 min by the inuence of ozone (Bouvier-Brown et al. 2009), and this has hampered the application of EF either in laboratory tests (Yang and Zettler 1975) or directly on crops (Hille Ris Lambers and Schepers 1978). Attempts have been made to overcome this problem by the synthesis of analogs, derivatives, or exploitation of natural essential oils. Some EF derivatives give products that are more stable and less volatile, which inhibit aphid settling and virus transmission, including the pesticideresistant aphids (Dawson et al. 1982, 1988). However, the chemical synthesis of EF and its derivatives is complicated and expensive, and application of these chemicals on a large scale in the eld is not cost-effective. EF is a component of the essential oil of many plants (Flamini et al. 2003; Miyazawa and Tamura 2007) and using essential oils with high EF content is another potential option to counter this problem. In eld plot experiments, numbers of pea aphid (Acyrthosiphon pisum) were signicantly reduced when sprayed with the essential oil from Hemizygia petiolata rich in EF (more than 70% of the component was EF) (Bruce et al. 2005). However, this strategy encountered problems with insufcient purity of EF. Genetic engineering of crop plants to biosynthesize and emit EF for aphid control could be a good alternative.

Characterization of EF Synthase Genes Isolated from Plants

EF can also be synthesized by certain plants. The genes encoding EF synthases, which convert farnesyl diphosphate (FPP) to the acyclic sesquiterpene EF, have been isolated and characterized from Douglas r (Huber et al. 2005), Yuzu (Maruyama et al. 2001), sweet wormwood (Picaud et al. 2005; Yu et al. 2011) and black peppermint (Crock et al. 1997; Prosser et al. 2006) (Table 2). For example, Crock et al. (1997) cloned an EF synthase by random sequencing of a peppermint oil gland library; the recombinant enzyme expressed in Escherichia coli could convert FPP to EF (85%), (Z)--farnesene (8%), -cadinene (5%) and other minor unidentied sesquiterpene products, whereas only EF (98%) and (Z)--farnesene (2%) were synthesized when Mn2+ was used as the co-factor. Cluster analysis (Figure 2A) and phylogenetic reconstruction analysis (Figure 2B) revealed that plant-derived EF or sesquiterpene synthase genes from the same or relative plant species were more closely related to each other than that of their counterparts from distant species. Despite the remarkable differences among sesquiterpene synthases, crystal structure analyses revealed their three-dimensional similarities contain highly-conserved amino acids. Among these there are three important domains (Lesburg et al. 1997; Starks et al. 1997), i.e., an Asp-rich DDxxD motif in the middle part of sweet wormwood EF synthase genes AaFS1 and AaFS2 isolated by Yu et al. (2011) (corresponding to amino acids 327331 in AaFS1 with

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Figure 2. Sequence analyses of plant-derived EF synthase genes.

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(A) Comparison of the deduced amino acid sequences of plant-derived representative sesquiterpene synthases. 1, sweet wormwood EF synthase AaFS1; 2, sweet wormwood EF synthase AaFS2; 3-5, EF synthase from Yuzu, black peppermint and Douglas r, respectively; 6, 5-epi-aristolochene synthase from tobacco with known crystal structure. Amino acids identical in all six genes are marked in blue. Amino acids identical in ve genes are marked in green. The highly conserved DDxxD region is marked with a bar, solid circles indicate highly conserved His, triangle indicates highly conserved Arg, and pentagram indicates highly conserved amino acid residues in J-K loop. (B) Phylogenetic tree of the so far isolated plant-derived sesquiterpene synthase genes. The joint enrooted tree was generated using MEGA4 by the neighbor-joining method. Bootstrap values were indicated at each branch. Sesquiterpene synthase 1 was under accession no. AJ271793, Sesquiterpene synthase 2 was under accession no. AF304444, the GenBank accession no. of other genes were showed in the Results section. (C) The Gene Structure Display Server (GSDS) (http://gsds.cbi.pku.edu.cn/) output of the sweet wormwood EF synthase AaFS1 gene structure. The exons, and introns are indicated by blue rectangles and green lines, respectively. 0, 1, 2 represent the intron phase. (D) The GSDS output of the Asian peppermint EF synthase MaFS1 gene structure. (E) The GSDS output of the Douglas r EF synthase PmFS2 gene structure.

the GenBank accession number of GU294840), two arginine residue positions corresponding to AaFS1 at Arg290 and Arg292 , an aspartic acid residue Asp551 and a lysine residue Lys561 , respectively (Figure 2A). The DDxxD motif was involved in the binding of the required divalent metal cofactor (Starks et al. 1997). Based on the results of X-ray crystallographic analysis of 5-epi-aristolochene synthase from tobacco, it was suggested that Arg290 and Arg292 were located in A-C loop, while Asp551 and Lys561 were located in J-K loop; the two loops played an important role in binding with substrate (Starks et al. 1997). At the same time, His, Cys and Arg residues have been conrmed to be essential for sesquiterpene synthase activity through inactivation experiments (Rajaonarivony et al. 1992; Savage et al. 1995). Furthermore, ve arginine residues (Arg137,140,290,292,468 ) and one histidine (His103 ) were also highly conserved among the compared sesquiterpene synthases (Figure 2A). The terpene synthase genes were divided into three classes by comparison of intron/exon patterns (Trapp and Croteau 2001). Class I contained 1214 introns, while class II had nine introns and class III six introns. Intron phases are dened as relative positions of an intron within or between codons (Intron phase 1 means that the intron occurs between the rst and second bases of a codon; phase 2 means that the intron occurs between the second and third letters of a codon; phase 0 means that the intron occurs between codons), which is a conserved character of eukaryotic gene structures, because any phase change requires either compensatory double mutations or more complex molecular mechanisms (Long and Deutsch 1999). So far, among the isolated EF synthase genes, the AaFS1 gene with six introns fell into class III (Figure 2C), with its intron number and phase identical to the Asian mint (Mentha asiatica) (E)--farnesene synthase genes MaFS1 (Figure 2D). However, the Douglas r EF synthase PmFS2 gene had 11 introns (Figure 2E), and the black peppermint EF synthase gene isolated by Prosser et al. (2006) had seven introns,

and neither fall into any of the categories mentioned above. It remains unclear how the variations in intron number in these plants affect the production of EF or other terpenes.

Metabolic Engineering of EF Synthesis in Crop Plants as a Potential Important Alternative Strategy for Aphid Control
Genetic manipulation of mono-, sesqui- and diterpenes in crop plants could provide an alternative tool for improving pest aphid control. For example, P450-suppressed transgenic tobacco plants producing higher levels of diterpene cembratriene-ol had higher aphidicidal activity; in vivo assays with these plants could greatly diminish aphid colonization (Wang et al. 2001). Recombinant linalool/nerolidol synthase (FaNES1) catalyzes the biosynthesis of the monoterpene alcohol linalool and its sesquiterpene counterpart nerolidol; in dual-choice assays with Myzus persicae, the FaNES1 transgenic Arabidopsis plants signicantly repelled the aphids (Aharoni et al. 2003). Genetic engineering of crop plants to constitutively emit EF for aphid control would be a good strategy (Beale et al. 2006; Yu et al. 2011). Compared with the maize terpene synthase 1 which could produce three sesquiterpenes: (E)-farnesene (26%), (E)--nerolidol (29%) and (E, E)-farnesol (45%) (Schnee et al. 2002), the EF synthase genes from Douglas r, Yuzu, sweet wormwood and black peppermint produced only one major sesquiterpene, EF, indicating these genes were good candidates for manipulating crop plants for aphid control (Yu et al. 2011). Rothamsted scientists have previously demonstrated that Arabidopsis can be genetically engineered to emit extremely pure EF. These plants, expressing an EF synthase cloned from peppermint (Mentha piperita), elicited potent effects on the behavior of the peach aphid Myzus persicae (alarm and repellent responses) and its natural enemy, the parasitoid Diaeretiella rapae (an arrestant

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response). This was the rst example of plant genetic engineering to produce an insect pheromone and demonstrated that the resulting emission affects behavioral responses at two trophic levels such that the plants become more resistant to aphids (Beale et al. 2006). Recently, the ICS, CAAS lab found that overexpression of EF synthase genes AaFS1 or AaFS2 from sweet wormwood in tobacco plants also resulted in the emission of pure EF (Figure 3). Behavioral studies involving the peach aphids, and predatory lacewings (Chrysopa septempunctata) demonstrated that the transgenic tobacco expressing AaFS1 and AaFS2 could repel peach aphids, but not as strongly as expected, probably due to other deterrent plant volatiles emitted by tobacco plants. However, AaFS1 and AaFS2 lines exhibited strong, statistically-signicant attraction to lacewings. Further experiments combining aphids and lacewing larvae in an octagonal arrangement showed transgenic tobacco plants could repel aphids and attract lacewings, thus minimizing aphid infestation (Yu et al. 2011). Therefore, the EF-emitting transgenic crop plants may have practical applications in agriculture as a result of not only repellence

to aphids and reducing aphids population growth, but also recruiting the natural enemies of aphids by increasing predation on habituated aphids for aphid control (De Vos et al. 2010). These ndings demonstrate that genetic engineering of crop plants to constitutively emit EF for aphid control is a feasible and promising strategy. However, the low sesquiterpene (including EF) production of transgenic plants overexpressing sesquiterpene synthase genes were observed in some studies, indicating that engineering sesquiterpene production in plants is a more challenging task. For example, transgenic Arabidopsis plants overexpressing the FaNES1 gene targeted to chloroplast emitted the sesquiterpene nerolidol, but the expression level was 100 to 300-fold lower than that of linalool (Aharoni et al. 2003). Attempts to engineer sesquiterpene synthesis in tobacco have also been performed with a fungal trichodiene synthase and only small amounts of the expected sesquiterpenes could be detected (Hohn and Ohlrogge 1991). When another sesquiterpene synthase, amorpha-4,11-diene synthase gene from Artemisia annua, was transformed into tobacco, this only

Figure 3. Gas chromatography-mass spectra (GC-MS) prole of volatiles emitted by transgenic tobacco plants with EF synthase gene from sweet wormwood (Adapted from our data published previously by Yu et al. 2011). (A) The volatiles from non-transgenic control plants. (B) The volatiles from transgenic plants. The compounds were as follows: 1, Tridecane; 2 and 6, siloxane contaminant; 3 and 5, unkown; 4, Tetradecane; Peak A, EF. (C) Mass spectrum of peak A. (D) Mass spectrum of authentic EF.

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resulted in the production of amorpha-4,11-diene at level of 0.2 to 1.7 ng/d per g fresh weight (Wallaart et al. 2001). Our previous study indicated that in tobacco transgenic lines engineered with two sweet wormwood EF synthase genes, the EF emission level ranged from approximately 1.55 to 4.65 ng/d per g fresh weight. This was lower than expected but these transgenic lines still exhibited repellence to aphid and attraction to lacewing (Yu et al. 2011). In summary, although genetic engineering of crop plants to consecutively emit EF for aphid control is a feasible and promising strategy, engineering crop plants synthesizing signicant amounts of EF remains a challenge.

Challenges and Perspectives

The use of genetic engineering to produce novel crop plants emitting alarm pheromone for aphid control has two important challenges. One is that the GM crop plants should release high purity and sufcient EF to mimic the natural aphid alarm response as well as increasing foraging by aphid predators or parasitoids (Beale et al. 2006). A second is the sub-cellular localization of both the precursor pool and the introduced EF synthases. The third is to generate an appropriate concentration of EF at a time when aphids present a threat to the crop by using wound-inducible promoters, which are underway at Rothamsted laboratory. An important consideration for the use of EF to elicit an alarm response in aphids is that it needs to be of very high purity. EF is a main component of certain plant volatiles and it has been shown that aphids are very sensitive to even small amounts of EF but do not respond to EF in the same way when it is released together with other plant volatiles (Dawson et al 1984, Bruce et al. 2005) as mentioned briey in the Introduction. For example, wild potato Solanum berthaultii, releasing high quantities of EF, from foliar trichomes, is more repellent to the green peach aphid than the commercial varieties of Solanum, which produce lower levels of EF along with some inhibitory compounds such as (E)-caryophyllene (Dawson et al. 1984; Ave et al. 1987). In this context, the EF synthase genes from Douglas r, Yuzu, sweet wormwood and black peppermint produced only one major sesquiterpene, EF, indicating that these genes were good candidates for manipulating crop plants for aphid control (Yu et al. 2011). In order to increase the amount of EF synthesized, the following strategies may be considered. First, multipoint metabolic engineering may be needed to increase the ux through the pathways leading EF synthesis. The availability of precursors plays an important role in plant sesquiterpene engineering (Aharoni et al. 2006), so this must be studied in the context of the whole pathway level,

including the precursor synthesis and supply, rather than single-gene engineering strategies. HMGR (3-hydroxy-3methylglutaryl CoA reductase) catalyzes the irreversible conversion of HMG-CoA to mevalonate, and FPP synthase catalyzes the synthesis of FPP from IPP and DMAPP; these two enzymes have been proposed to be key regulatory steps controlling isoprenoid metabolism (Figure 1) (Bach 1995; Cunillera et al. 1997). Transgenic wheat plants constitutively expressing both codon-optimized FPP synthase gene from cow (Bos taurus) and EF synthase gene from black peppermint emitted more EF than plants constitutively expressing EF synthase alone (Pickett et al. unpublished data by Rothamsted laboratory). Furthermore, coupled overexpression of a patchoulol synthase (PTS) with an avian FPP synthase gene increased sesquiterpene patchoulol accumulation two- to ve-fold higher than that of plants harboring only PTS, and this could be further augmented by another two- to six-fold by the concomitant overexpression of a truncated HMGR (Wu et al. 2006b). Second, foreign sesquiterpene synthases should be better targeted to a suitable subcellular compartment. An FPP synthase isoform existed in the mitochondria of Arabidopsis (Cunillera et al. 1997), so Kappers et al. (2005) presumed that FPP should be available in this cell compartment and generated higher levels of nerolidol by targeting the strawberry FaNES1 gene to mitochondria. Besides, a chloroplast form of FPP synthase exists in rice, wheat and tobacco (Sanmiya et al. 1999), so the chloroplast could also be an ideal compartment for sesquiterpene engineering in these crop plants. Third, the sesquiterpene biosynthetic pathway could also be redirected from its natural cytosolic location to the plastids (indicated in Figure 1). Wu et al. (2006b) generated transgenic tobacco plants producing high levels of sesquiterpene by overexpressing both an avian FPP synthase and an appropriate sesquiterpene synthase in the chloroplast; the transgenic plants could increase the synthesis of the sesquiterpenes patchoulol and amorpha-4, 11-diene more than 1 000-fold. Farnesyl diphosphate is a ubiquitous isoprenoid intermediate involved in sesquiterpene biosynthesis in the cytoplasm (Figure 1) (Crock et al. 1997), suggesting that cytoplasm pools of FPP might be accessible to foreign EF synthases. We formerly transferred AaFS1 and AaFS2 into tobacco under the control of a constitutive cauliower mosaic virus 35S promoter and observed relatively low EF production, as well as non-linear correlation between the levels of EF emission and AaFS1/AaFS2 expression levels (Yu et al. 2011), indicating that the availability of the precursor (FPP) supply might be a major limiting factor in the biosynthesis of sesquiterpene in tobacco or other crop plants.

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The supply of FPP for the biosynthesis of sesquiterpenes probably depends on the crop plant species in question and their physiological status. Compared with the higher sesquiterpene levels in sweet wormwood and grand r (Steele et al. 1998; Wallaart et al. 1999), the production of endogenous sesquiterpene in tobacco was very low, even undetectable (Chappell and Nable 1987; De Moraes et al. 1998). This suggested that only a low amount of free FPP could be used for the biosynthesis of sesquiterpene in tobacco, and the foreign sesquiterpene synthase had to compete with other endogenous ones for the limited supply of FPP. However, Schnee et al. (2006) overexpressed the maize tps10 gene in Arabidopsis plants to generate high quantities of TPS10 sesquiterpene products, which were identical to those released by maize, suggesting that FPP in Arabidopsis plants was not limiting. On the other hand, the amount of FPP for sesquiterpene biosynthesis can also be related to the plant physiological status. Sterols play a structural role in membrane uidity and are important for plant vitality; FPP could be directed to the pathway of sterol biosynthesis under normal conditions (Chappell and Nable 1987). Upon exposure to biotic stresses and suitable elicitors (e.g. fungus elicitor, herbivore elicitor, etc.), the production of sesquiterpenes in tobacco can increase dramatically at the expense of sterol production (Vogeli and Chappell 1988; De Moraes et al. 1998), indicating that the proportion of FPP, which is partitioned for sesquiterpene biosynthesis can be altered by elicitors. Indeed, the expression of FPP synthase genes in wheat was greatly upregulated upon aphid infestation, although different expression patterns were observed in a tissue-specic manner (unpublished data by ICS, CAAS lab). At last, there can be phenotypic variation during the manipulation of terpene and/or sesquiterpene synthesis pathways and this might pose another challenge in manipulation of EF synthase genes in economically important crop plants in which people desire GM plants with improved aphid resistance without affecting other agronomic traits. For example, although relatively low levels of EF were emitted by AaFS1/AaFS2 transgenic tobacco plants, we observed some phenotype differences compared with the control, such as long leaf petiole, slim stem, small leaves and more axillary buds (Yu et al. 2011). Interestingly, this is not a single case because not only are the transgenic tobacco plants expressing AaFS1 and AaFS2, but also the plants expressing their counterparts from peppermint exhibited the similar phenotype variation phenomena (unpublished data by ICS, CAAS laboratory). The reason might lie in the fact that targeting EF synthase genes into the cytoplasm of cells in tobacco plants might partly deplete the

limited FPP pool, which in turn affects the biosynthesis of abscisic acid or other important metabolites, the same effects as observed by other authors in engineering the terpene synthesis pathway (Fray et al. 1995; Aharoni et al. 2003; Aharoni et al. 2006). For example, transgenic tomato plants constitutively expressing the carotenogenic enzyme phytoene synthase showed a dwarf phenotype resulting from the over-production of phytoene synthase, which converted GGDP to phytoene and thereby diverted this intermediate away from the gibberellin and phytol biosynthetic pathways (Fray et al. 1995). Furthermore, overexpression of the FPP synthase, a key enzyme in biosynthesis of sesquiterpenes (Figure 1), in transgenic Arabidopsis plants led to a reduction of IPP and DMAPP available for the biosynthesis of the growth regulator cytokinin, which might have caused at least in part the phenotype alteration observed (Masferrer et al. 2002). Moreover, Arabidopsis plants expressing a dual linalool/nerolidol synthase (FaNES1) also showed retarded growth compared with wild-type plants under the same greenhouse conditions, and the phenotype was correlated with levels of linalool production (Aharoni et al. 2003), and transgenic potato plants that expressed FaNES1 produced high levels of linalool, but also showed yellow and damaged regions on their leaf blades (Aharoni et al. 2006). The major explanation of this phenotype change might be due to the reduction in the availability of substrates for other metabolites produced in the plastids, which play an important role in plant growth and development, including the growth regulators gibberellin, abscisic acid and other vital components such as carotenoids, chlorophyll, and quinones (Aharoni et al. 2003). However, these effects on phenotype variations seemed to be species-dependent as transgenic Arabidopsis plants transformed with peppermint EF synthase gene could produce high levels of EF without any observed phenotypic variations (Beale et al. 2006). Thus the cost of increasing sesquiterpenes level should be evaluated when deciding the strategies for genetic engineering of EF synthase gene in economically important crop plants. It is worth mentioning that, by using the rice Rubisco small subunit promoter (rbcS), which is reported to direct foreign gene expression specically to leaves and other green tissues (Huang and Lin, 2007), we successfully generated EF synthase gene transgenic wheat plants by Agrobacterium-mediated transformation method; so far, no phenotype variations were observed in the progeny of transgenic lines except for improved aphid resistance, implying there is sufcient substrate FPP in wheat green tissues for synthesizing EF (unpublished data by ICS, CAAS laboratory).

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Acknowledgments
Some work mentioned in this review is partly funded by the Research Initiative on Development of Disease and Insect Resistance Transgenic Wheat Plants supported by the Chinese Ministry of Agriculture (2008ZX08002001), Natural Science Foundation of China (31171618) and the EU FP7 OPTICHINA Project (266045). Rothamsted Research receives grant-aided support from the Biotechnology and Biological Sciences Research Council (BBSRC) of the UK.

affects aphid and parasitoid behaviour. Proc. Natl. Acad. Sci. USA 103, 1050910513. Bernasconi ML, Turlings TCJ, Ambrosetti L, Bassetti P, Dorn S (1998) Herbivore-induced emissions of maize volatiles repel the corn leaf aphid, Rhopalosiphum maidis. Entomol. Exp. App. 87, 133142. Birch ANE, Geoghegan IE, Majerus MEN, McNicol JW, Hackett CA, Gatehouse AMR, Gatehouse JA (1999) Tritrophic interactions involving pest aphids, predatory 2-spot ladybirds and transgenic potatoes expressing snowdrop lectin for aphid resistance. Mol. Breed. 5, 7583. Birkett MA, Campbell CAM, Chamberlain K, Guerrieri E, Hick AJ, Martin JL, Matthes M, Napier JA, Pettersson J, Pickett JA, Poppy GM, Pow EM, Pye BJ, Smart LE, Wadhams GH,

Received 28 Nov. 2011

Accepted 9 Feb. 2012

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