Euphytica (2006) 147: 273–285 DOI: 10.1007/s10681-006-3608-1

 C

Springer 2006

Resistance to insect pests: What do legumes have to offer? Owain Edwards1 & Karam B. Singh2,∗ 1

CSIRO Entomology, Private Bag 5, Wembley, WA 6913, Australia; 2 CSIRO Plant Industry, Private Bag 5, Wembley, WA 6913, Australia (∗ author for correspondence: e-mail: [email protected])

Received 1 December 2004; accepted 11 March 2005

Key words: chewing insects, herbivores, jasmonic acid, phloem-feeding insects, plant defence, plant-insect interactions, resistance genes

Summary Insect pests are major problems for all crops, worldwide. In this review we will focus on legumes, which are attacked by a range of insect pests including pod/seed feeders, defoliators and sap feeders. We review the history of breeding for resistance to insect pests in legumes, which has had mixed success, and discuss further opportunities in this area. We also review the extraordinary array of direct and indirect mechanisms contributing to insect defence in legumes, the understanding and exploitation of which offer opportunities for both legume and non-legume crops. There is also good potential to improve insect resistance in legume crops through a detailed understanding of the signaling pathways that regulate induced responses to insect feeding, and recent progress in this area, primarily obtained from non-legume systems, is reviewed. The importance legumes play in farming systems, their wide range of novel chemistry and the emergence of model systems suitable for genomic approaches present opportunities for research in this area strongly linked to breeding programs to help develop legume crops with enhanced insect resistance. Introduction Insects are important pests of legumes worldwide because of the damage inflicted by direct feeding, but also because they vector or provide infection sites for plant pathogens. The relative importance of insect pests on legumes compared to other biotic stresses (e.g. pathogens, weeds) depends greatly on the crop species and its location. Tropical crops, such as beans in Central America and pulses in Asia and Africa, will usually suffer more direct feeding damage from a wider variety of insect pests than these same crops in cold season Mediterranean climates (Qaim & Zilberman, 2003). However, crop and pasture legumes grown in temperate areas or in cold seasons are more likely to be attacked by aphids and the viruses they transmit (Heie, 1994). The economic importance of insect herbivores will depend greatly on the life stage and tissues of the plant  CSIRO’s

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they attack, and on their method of feeding. The most economically damaging of legume pests feed on seeds in storage or in the field (Cardona & Kornegay, 1999; Clement et al., 1999). Insects that attack the pods can also cause considerable economic loss, although some legume species are able to compensate effectively for pod and seed damage (Russin et al., 1987; Brier & Rogers, 1991). Foliage- or sap-feeding insects can also inflict serious damage whenever environmental conditions support massive population growth. Foliagefeeding insects can also be economically important at low densities when they attack reproductive tissue (e.g. Helicoverpa spp.) or they destroy seedlings (e.g. bean stem maggot, Ophiomyia spp.), as can sap-feeding insects when they transmit disease or introduce factors into the plant that cause physiological damage symptoms (e.g. Therioaphis trifolii (Monell), Bemisia tabaci (Gennadius)). Like most plants, legumes rely on a suite of defences for protection against insect pests. Legume seeds are often protected by an accumulation of anti-nutritional compounds that remain until

274 germination (Stamopoulos, 1987). Vegetative tissues are most commonly protected by a suite of generic defence mechanisms, both structural and chemical. Chemical defences can be constitutively expressed or induced and may be localized within tissues, translocated throughout the plant, or released as volatile emissions (Kessler & Baldwin, 2002). Both structural and chemical defences can act directly on the herbivorous pests by deterring herbivore feeding (antixenosis), by suppressing herbivore growth and development (antibiosis), or by minimizing damage symptoms (tolerance) (Clement et al., 1994); or can act indirectly by increasing herbivore mortality caused by other environmental factors (Clement et al., 1999). Many mechanisms important to insect defence have been bred out of cultivated legumes, as in other crops, because they also detract from the taste and texture of the crop (Clement, 2002). Breeders attempting to re-introduce these defences often rely on accession libraries with a good representation of wild genotypes, but achieving resistance without also reducing crop quality or yield can be difficult. Signaling or regulatory components of the defence response may also have been bred out of cultivated lines, which may prevent re-introduced defences from functioning effectively. In this paper, we describe the current state of play regarding insect resistance in legumes and the exciting opportunities offered by this diverse family to better understand how plants defend themselves against insects. To begin we present a history of successes and failures in breeding insect resistance into legume crops, then describe using representative examples the wide diversity of mechanisms found in legumes that contribute to insect defence. We then review what is known about the signaling and regulatory components of insect defence in plants, information derived primarily from non-legume systems but with important contributions from legume systems as well. We conclude by discussing the opportunities likely to arise from continuing research in legume defence against insects.

Conventional breeding of resistance to insect pests in legumes Though screening of legume genetic resources for insect resistance has showed some promise, successful breeding of insect resistance into commercial cultivars has proven difficult in many legume crops. For example, despite having perhaps the most success-

ful legume screening program for identifying pest resistance, the soybean breeding program in the USA released only four insect-resistant soybean cultivars in their first 26 years and the adoption of these has been limited because of issues of agronomic inferiority (Boethel, 1999). In tropical bean systems, there have been some successes in developing cultivars resistant to single pests, but multiple insect and disease resistant varieties are desperately needed to be commercially viable (Cardona & Kornegay, 1999). In cool season legumes, improvements in insect resistance have rarely been greater than incremental (Muehlbauer, 1996; Clement et al., 1994); perhaps the greatest success has been aphid resistance in cultivated narrow-leafed lupins in Australia (Cowling, 1999). In pasture legumes, there has been more success. Alfalfa/lucerne breeders have had the greatest success in developing insect resistance, with the successful development of resistance against three aphid species, leafhoppers, and the alfalfa weevil (Manglitz & Sorensen, 1999). There have also been some successes with annual pasture legumes, where breeders have exploited wild germplasm to breed resistance to bluegreen aphid, Acyrthosiphon kondoi Shinji, into a number of cultivars (Berlandier et al., 2000; Nair et al., 2003). Importantly, included in these successes is the barrel medic (Medicago truncatula L.), which has been a pasture crop in Australia for several decades but has recently been adopted by the international community as a model system for legume research. This combination makes it an ideal system for studying aphid resistance in legumes, as will be discussed later in this review. Why has there been so little progress in improving insect resistance in many legume crops? While for some species there are limited genetic resources available, the problem in many cases has been that researchers do not access the full range of germplasm resources that are available (Clement, 2002). This was the case for many years in finding resistance in beans to the Mexican bean weevil, Zabrotes subfasciatus (Boheman), until effective resistance controlled by a single, dominant gene was located in a small collection of wild lines by van Schoonhoven et al. (1983). Identifying effective Helicoverpa spp. in chickpea germplasm collections also has met with limited success (Srivastava & Srivastava, 1989), but recent efforts have identified promising levels of resistance in accessions of the wild relatives Cicer bijugum Rech. and C. reticulatum Ladiz. (Sharma et al., 2001).

275 Collections of wild accessions of crop species or their non-domesticated relatives may provide the answer to the problems posed by the limited genetic resources in legume crops (Clement, 2002). Other recent examples include resistance to pod fly and pod wasp in wild relatives of pigeonpea (Sharma et al., 2003), resistance to aphids in wild Vicea spp. (Holt & Birch, 1984), resistance to Apion godmani Wagner in landraces of Phaseolus vulgaris L. (Garza et al., 2001), resistance to Liriomyza cicerina Rond. in wild chickpeas (Singh & Weigand, 1994), and volatiles deterring the brown pod bug, Clavigralla tomentosicollis Stal., in wild Vigna spp. (Koona et al., 2003). For some wild relatives, such as C. bijugum, success will also depend on effective embryo rescue technology. Achieving pest resistance without reducing agronomic quality has been a second problem. This can occur when resistance traits are under polygenic control or have low dominance, resulting in many undesirable qualities being introduced along with the desired trait during the breeding process. Such problems have plagued breeding of resistance for bean weevil (Sitona lineatus (L.)) in beans, and for Mexican bean beetles and leafhoppers in soybeans (Boethel, 1999). A modified recurrent selection procedure was, however, used by CIAT to successfully overcome this problem and improve resistance and tolerance to leafhoppers in common beans (Kornegay & Cardona, 1990). It is particularly difficult to breed for pest resistance when the mechanism of resistance in itself reduces crop quality. This can be true for many physical resistance mechanisms including pod thickness (e.g. pea weevil resistance), and for chemical resistance when the compounds involved are also toxic to mammals (e.g. alkaloids in lupins). Successful breeding for resistance to three aphid species in alfalfa/lucerne is a notable exception to the problems listed above, which is in part due to the simple and dominant inheritance pattern of many aphid resistance genes. Simple, dominant aphid resistance genes has also recently been identified against bluegreen aphid in M. truncatula (AKR) (Klingler et al., in press), and against soybean aphid (Aphis glycines Matsumura) in soybeans (Rag1) (Li et al., 2004a; Roth, 2004). There can, however, still be problems with these aphid-resistant lines. Unfortunately, some of these genes are temperature-dependent, losing efficacy at temperature below approximately 15 ◦ C (Summers & Newton, 1987). In addition, the capacity of aphids to develop resistance-breaking “virulent” biotypes has been demonstrated by A. kondoi on alfalfa in Oklahoma

(Zarrabi et al., 1995). Though not yet a problem in alfalfa/lucerne, aphid virulence is a documented problem for potato aphid (Macrosiphum euphorbiae (Thomas)) on tomato and for Russian wheat aphid (Diuraphis noxia (Mordvilko)) on wheat, and as such these resistant lines may not be sustainable (Goggin et al., 2001; Smith et al., 2004). Finally, in many cases the failures arise because proper links have not been established between researchers identifying resistance mechanisms and the breeders that would introduce these mechanisms into commercial lines. The next section gives numerous examples of insect resistance mechanisms that have been identified in legumes, yet it appears that only a small percentage of these have ever been introduced into breeding programs.

Types of insect resistance found in legumes Legumes as a group employ an extraordinary array of direct and indirect defences against insects (Table 1). These include structural defences, secondary metabolites, and antinutritional compounds and in the following sections we will review examples of each of these types of defences. Insect pests of legumes can be separated functionally into three categories, based on the plant tissues being attacked: pod/seed feeders, foliage (leaf and stem) feeders, and sap or cell content feeders. This classification determines to a large part the types of plant defences to which the herbivore will be exposed. Structural defences Structural defences can provide resistance to all categories of insect pests. Simple and glandular hairs (trichomes) have been shown to provide resistance to sapsucking insects like the potato leafhopper in alfalfa/lucerne (Shade et al., 1979). It is likely that these glandular hairs provide some level of resistance to other sapsucking pests, including aphids (Shade & Kitch, 1983). Trichomes also seem to be responsible for preventing heavy aphid colonization of chickpeas, as aphids feed successfully on areas of the plant where trichomes are not found (Edwards, 2001). Young nymphs and larvae of a variety of insect herbivores can often be found immobilized in trichome secretions (Boethel, 1999). In leafhopper-resistant soybeans, resistance has been shown to be related to the orientation and size of trichomes rather than their density (Turnipseed, 1977).

276 Table 1. Genetic control of resistance mechanisms to insect pests of various legumes Legume species

Insect pest

Mechanism

Genetics

References

Common bean (Phaseolus vulgaris)

Leafhoppers, Empoasca spp.

Tolerance, antixenosis

Quantitative

Galwey & Evans (1982); Kornegay & Temple (1986)

Bean pod weevil, Apion godmani

Antibiosis

Agr, Agm

Garza et al. (1996)

Mexican bean weevil, Zabrodes subfasciatus

Antibiosis

Arc

Osborne et al. (1986); Romero-Andreas et al. (1986)

Bean weevil, Acanthoscelides obtectus

Antibiosis

Recessive, quantitative

Kornegay & Cardona (1991)

Mexican bean beetle, Epilachna varivestis

Antibiosis

Recessive, quantitative

Rufener et al. (1989)

Pea aphid, Acyrthosiphon kondoi

Indirect via leaf morphology

st

Kareiva & Sahakian (1990)

af

Soroka & MacKay (1990)

Pea (Pisum sativum)

Pea weevil, Bruchus pisorum

Hypersensitivity

Np

Doss et al. (2000)

Chickpea (Cicer arietinum)

Pod borers, Helicoverpa armigera & H. punctigera

Chemical antibiosis (trichome exudates)

Quantitative

Yoshida et al. (1997)

Soybean (Glycine max)

Various caterpillars: H. zea, H. virescens, Pseudoplusia includens, Spodoptera exigua

Antibiosis, some antixenosis

Recessive or partial dominance, quantitative

Lambert & Kilen (1984); Kilen & Lambert (1986, 1998).

Stink bugs Nezara viridula

Antibiosis and antixenosis

Quantitative

Lopes et al. (1997)

Mexican bean beetle, E. varivestis

Antibiosis, some antixenosis

2–3 major genes,

Kogan (1972); Sisson et al. (1976)

Soybean aphid, Aphis glycines

Antibiosis

Rag1

Li et al. (2004a,b); Roth (2004)

Potato leafhopper, E. fabae Pea aphid, Acyrthosiphon pisum

Antixenosis (trichomes) Antibiosis

Quantitative Polygenic

Elden & Elgin (1992) Julier et al. (2004)

Potato leafhopper, E. fabae

Tolerance

Quantitative

Sorensen & Horber (1974)

Bluegreen aphid (A. kondoi)

Antibiosis, antixenosis, tolerance

AKR

Klingler et al. (2005)

Alfalfa (Medicago sativa)

Barrel medic (Medicago truncatula)

Soybean trichome length and density has also been implicated in resistance to beanflies, whiteflies, pod borers, and bean leaf beetles (Chiang & Norris, 1983; Lambert et al., 1995; Lam & Pedigo, 2001; Talekar & Lin, 1994). Pod trichomes have been implicated in resistance to coreid bugs (Koona et al., 2002). Pod and seed morphology can also contribute to insect resistance. In cowpea, resistance to weevils correlates strongly with seed coat thickness (Kitch et al., 1991) while resistance to hemipteran and lepidopteran

pod pests in this species correlated with a variety of morphological characters including pod toughness, hull thickness, peduncle length, and seed location in the pod (Tayo, 1989; Koona et al., 2002). There is also evidence for structural, indirect defences in legumes. In peas, aphid damage is reduced on leafless (af) and semi-leafless (st) phenotypes because of a combination of increased predation (Kareiva & Sahakian, 1990), increased vulnerability to adverse weather (Soroka & MacKay, 1990), and the reduction

277 in preferred feeding spaces (Soroka & MacKay, 1990). In lima bean, there are induced, indirect defences in the form of secreted extrafloral nectar that attracts ants which then predate upon the feeding herbivores, a response that can also be elicited by jasmonic acid treatment (Heil, 2004). Secondary metabolites Though it is generally assumed that secondary metabolites play a role in legume defence against insect herbivores, there is surprisingly little information on what specific phytochemicals contribute to insect resistance. The role of phytoalexins in contributing to pathogen defence in legumes is well-documented (see review by Dakora & Phillips, 1996), but their role in insect herbivore defence is not as clear. Coumestrol has been shown to contribute to Mexican been beetle (Epilachna varivestis Mulsant) resistance in some soybean lines, but cannot alone account for the full antifeeding effects (Burden & Norris, 1992). A suite of leaf surface volatiles, including isoflavonoids, contribute significantly to feeding deterrence of the red-legged earth mite, Halotydeus destructor (Tucker), in subterranean clover, Trifolium subterraneum L. (Wang et al., 1998, 1999) and of Helicoverpa armigera (Hubner) in pigeonpea, Cajanus cajan (L.) (Green et al., 2003). Other classes of compounds are also likely to be important in insect defence in legumes. There is some evidence that seed tannins and saponins contribute to seed weevil resistance (Stamopoulos, 1987). Total saponins also correlate with Spodoptera littoralis (Boisduval) resistance in alfalfa (Agrell et al., 2003). In chickpeas, oxalic and malic acid exudates from leaf trichomes have been shown to contribute to resistance to the foliage and pod-feeding caterpillars H. armigera and H. punctigera (Yoshida et al., 1997). In lupins, feeding deterrence of vertebrate and invertebrate pests correlates strongly to leaf and seed tissue alkaloid levels (Wink, 1984). Density of a leaf galler correlates to total alkaloid concentration in Lupinus arboreus Sims (Adler & Kittelson, 2004). Damage by red-legged earth mite has been shown to be negatively correlated with leaf alkaloid concentrations in narrow-leafed lupins, L. angustifolius L. (Wang et al., 2000). Phloem alkaloids appear to confer aphid resistance in this species through antibiosis, as aphid feeding is not deterred on resistant lines (Edwards et al., 2003). Breeding for aphid resistance in this species appears to have selected for increased phloem concentrations

of one particular alkaloid, which is highly bioactive against aphids (Edwards, unpublished data). Volatile secondary metabolites have also been shown to contribute to indirect defence in legumes. In lima beans, volatiles including terpenoids released from plants in response to insect feeding attract predators that help to control the herbivores (Petitt & Wietlisbach, 1992; Shimoda et al., 1997; Takabayashi et al., 1994). These same volatile cues attract predators to neighbouring, conspecific plants due to passive and active accumulation of these volatiles cues (Dicke & Dijkman, 2001). In lima beans, methyl salicylate is critical to the attraction of predatory mites and is not released simply by jasmonic acid treatment (De Boer & Dicke, 2004). Though the above studies have contributed somewhat to our understanding of how legumes defend themselves against insect herbivory, there is little evidence that this information on bioactive secondary metabolites has ever been used effectively to improve subsequent breeding for insect resistance. Anti-nutritional compounds Anti-nutritional compounds are an important component of the insect defence arsenal in legumes, and are particularly important in protecting seeds from herbivory (Elden, 2000; Stamopoulos, 1987). In fact, human consumption of legume sprouts rather than seeds is due to an increase in palatability associated with biochemical changes in anti-nutritional compounds during germination (Nakamura et al., 2001). Unlike the lowmolecular weight compounds described in the previous section, selection for anti-nutritional compounds has been conducted successfully as part of legume breeding programs. A good example is the major seed storage protein in common beans, arcelin, which appears to be poorly digested by insects. This protein, found only in wild Mexican P. vulgaris accessions, was found to confer Mexican bean weevil resistance and was inherited as a single dominant gene with seven known allelic variants (Osborne et al., 1986; Romero-Andreas et al., 1986). Successful backcrossing to cultivated lines was achieved using biochemical testing for the presence of arcelin in addition to feeding bioassays. Arcelin, along with phytohemagglutins (PHA) and α-amylase inhibitors (αAI), are in beans the representatives of a family of proteins collectively called lectins. Defence lectins are anti-nutritional, sometimes lethal proteins often found accumulated in legume seeds. Lectins are often resistant to proteolytic activity

278 and function by binding to chitin or to carbohydrate targets in the insect midgut, thereby blocking nutrient assimilation (Zhu-Salzman et al., 2002). Most provide effective resistance against insects and only a few, such as concanavalin A in Jack beans, are also sufficiently toxic to vertebrates to prevent human consumption (Oliveira et al., 1999). Most have variable, and often sub-lethal, effects depending on the herbivore. For example, African yam bean lectin provides effective resistance against the cowpea weevil, Callosobruchus maculates (Fabricius), but not the legume pod borer, Maruca vitrata Fabricius (Machuka et al., 2000). Several studies have shown that lectins affect the growth and development of non-seed feeding insects in in vitro assays and in transgenic plants (Fitches et al., 1997; Gatehouse et al., 1997; Stoger et al., 1999; Foissac et al., 2000; Down et al., 2003). In particular, the snowdrop lectin gene, GNA, has been evaluated in many crops (Fitches et al., 1997; Gatehouse et al., 1997; Stoger et al., 1999; Foissac et al., 2000; Down et al., 2003), and an α-amylase transgene from beans has been shown to provide resistance to pea weevil, Bruchus pisorum (L.), in Pisum sativum L. (Morton et al., 2000), and to the cowpea weevil and Azuki bean weevil (C. chinensis (L.)) in chickpea (Sarmah et al., 2004). Other seed proteins have been shown to have bioactivity against insects. Pea albumin I b, or the homologue leginsulin in soybeans, has insecticidal activities against weevils (Louis et al., 2004; Gressent et al., 2003). Also common in some legume species, and again concentrated in the seeds, are digestive enzyme inhibitors that interfere with the function of insect digestive proteases or alpha-amylases. Proteinase inhibitors (PIs) are one of many plant defence mechanisms known to be induced by insect feeding (see below), and specialized insect herbivores can overcome this defence by expressing proteases that are insensitive to the action of the PIs (Bown et al., 2004; Zhu-Salzman et al., 2003; Moon et al., 2004). Legume seed-feeding insect herbivores will often have proteases that are insensitive to PIs from plants within their host range, but not from plants outside their host range (Patankar et al., 2001). For this reason, PI genes from distantly related plants are often evaluated as potential transgenes to achieve insect resistance (Jouanin et al., 1998). For example, the soybean cysteine protease inhibitor soyacystatin N gene (scN) has been evaluated against non-legume pests using diet bioassays (Pompermayer et al., 2001) and transgenic plants (McManus et al., 1999).

Non-protein amino acids are anti-nutritional compounds often found in leaf and stem tissues of legumes as well as seeds, and are thought to function primarily in herbivore defence. For example, a suite of non-protein amino acids from the tropical legume Calliandra spp. has been shown to have activity against the black bean aphid, Aphis fabae Scopoli (Simmonds et al., 1988). Perhaps the best-characterized of the legumes in terms of non-protein amino acids is Lathyrus latifolius L., which contains five major non-protein amino acids with variable effects against insects and vertebrates (Bell et al., 1996). This variation may indicate that manipulation of these non-protein amino acids could be a feasible strategy to improve insect defence. While conventional breeding will continue to have a valuable role in providing insect resistant cultivars in many legume crop species, it appears that in some systems there will continue to be barriers to achieving insect resistance in this way. Fortunately, there are exciting opportunities arising from our growing understanding of signaling mechanisms that coordinate inducible responses in plants following insect attack. We believe that this approach promises to provide novel strategies to achieve insect resistance in previously intractable systems.

Signaling pathways and downstream changes implicated in insect resistance As in other plant systems, there is growing evidence that insect resistance in legumes can be elicited or enhanced by herbivory. For example, resistance to bluegreen aphid in M. truncatula has been shown to be enhanced by prior feeding (Klingler et al., 2005). Also, S. littoralis larvae showed reduced preference for foliage that had been damaged 5 or 7 days previously (Agrell et al., 2003). Resistance to Mexican bean weevil in a number of soybean varieties is elevated in previously damaged leaves (Underwood et al., 2000). It is unlikely that resistance induction will occur in seeds, but induced resistance in pods can affect seed-feeding insects in the field (Doss et al., 1995). Any chance of utilizing induced defences to improve insect resistance requires a much better understanding of the mechanisms involved, and in particular the complex network of signaling pathways that control the induction response. While our knowledge of the signaling pathways and downstream changes involved in insect resistance has lagged behind what we know about plant-pathogen

279 interactions, this situation is changing and the advent of genomic approaches involving transcriptomics, proteomics and metabolomics are likely to see major advances over the next few years. It is not our intention to cover this area in detail, since there have been a number of excellent recent reviews on plant responses to insects (see for example, Walling, 2000; Bostock et al., 2001; Kessler & Baldwin, 2002; Ferry et al., 2004). Rather we will try to focus on recent progress that highlights the potential for these types of studies to provide new insight and opportunities for generating insect resistance in legumes. While legume systems have not played a major role in contributing to our understanding of signaling pathways involved in insect resistance, there have been some exceptions including the discovery of a novel form of insect resistance in pea in response to cowpea or pea weevils (Doss et al., 2000). This example shows that induced defences can be important against pests of seeds and pods. The oviposition fluid from these weevils contains a novel class of elicitors called bruchins, which stimulate cell division at the sites of egg attachment, resulting in neoplastic growth beneath the egg that impedes larval entry into the pod and potentially also exposes the larvae to harmful situations such as desiccation and predation. A similar type of response appears to occur in beans to the bean pod weevil (Garza et al., 2001). Our understanding of plant responses and signaling are most advanced for chewing insects where potential elicitors of plant defence responses have been identified, including those isolated from oral secretions, such as lytic enzymes like B-glucosidase and glucose oxidase, as well as fatty-acid-amino-acid conjugates (reviewed in Kessler & Baldwin, 2002). What remains to be determined is how these elicitors are recognized in the plant. Unlike the situation with a range of pathogens and some other types of insect pests, R-gene type recognition has not been observed for chewing insects. What is becoming clear is that there is a lot of overlap between plant signaling to chewing insects and those resulting from mechanical damage or wounding (see Walling, 2000; Kessler & Baldwin, 2002; for recent reviews). A major wound response is activation of the octadecanoid pathway which has been well studied in Solanaceous plants like tomato and tobacco. In tomato, a number of inducers of the octadecanoid pathway have been found, including a local induction by oligogalacturonides (OGA) and local and systemic induction by an 18 amino acid peptide called systemin. Systemin is transported via the phloem and interacts with a cell-surface receptor called SR160, belonging to the

LRR receptor-like kinase family (reviewed in Ryan & Pearce, 2003). Remarkably, SR160 also functions as a receptor for brassinosteroid signaling in tomato leaving open the intriguing possibility that wound responses need to be coupled with brassinosterol signaling (Wang & He, 2004). Activation of the octadecanoid pathway by OGA and systemin eventually leads to the production of jasmonic acid (JA) and its methyl ester (MeJA), which in turn activate a range of defence responses including many that have been described in previous sections of this review (e.g. PI proteins, volatiles). Recently other systemin-like peptides have been isolated in tomato and tobacco, which are also involved in defence signaling (Ryan & Pearce, 2003). The importance of the octadecanoid pathway and JA for defence against chewing insects has been clearly shown by genetic/reverse genetic approaches involving mutants in JA perception or synthesis (see for example, McConn et al., 1997; Royo et al., 1999; Halitschke & Baldwin, 2003; Li et al., 2004b; Kessler et al., 2004). For example, in a recent study it was shown that silencing three key enzymes in this pathway in native tobacco, resulted in plants that were not only more vulnerable to adapted insect pests but also attracted new pests (Kessler et al., 2004). The octadecanoid pathway is also regulated by other plant signals such as abscisic acid (ABA), ethylene and salicylic acid (SA) (Walling, 2000). The pathways activated by these signals do not function as independent linear pathways but form networks where both synergistic and antagonistic interactions occur. The dynamics of such a signaling network can be manipulated by an attacking insect as illustrated by work with Heliothis zea (Boddie), whose saliva contains glucose oxidase, which can suppress the production of nicotine in tobacco plants thereby preventing an effective defence (Musser et al., 2002). It has yet to be determined whether Helicoverpa spp. use a similar strategy of defence suppression when feeding on legumes. These interesting results demonstrate the potential importance of turning off genes for successful insect infestation and interfering with this process may be another useful avenue to explore, in the quest for enhanced insect resistance. Genomic approaches are being used to study plant responses to chewing insects (reviewed in Ferry et al., 2004). These studies, which have primarily involved transcriptomics, have shown clear overlap with both pathogen induced and wound induced responses as well as specific effects caused by chewing insects. These and similar studies on other classes of insect pests are

280 throwing up many new genes implicated in insect resistance and which are likely to have counterparts in legumes. The combination of reverse genetics and genomics also offers much promise as illustrated by studies on an insect-inducible lipoxygenase gene in tobacco (Halitschke & Baldwin, 2003). Silencing this gene resulted in a weakened defence response, as measured in part through microarray studies, and an increase in insect damage. Plant responses to sap-sucking insects are less well understood, although in contrast to the situation with chewing insects, the involvement of R genes has been clearly established with the cloning of the Mi gene in tomato (Rossi et al., 1998). Interestingly, this gene, which belongs to the NBS-LRR class of R genes, confers resistance both to nematodes and two types of sap sucking insect pests, aphids and whiteflies (Milligan et al., 1998; Rossi et al., 1998; Nombela et al., 2003). Other aphid resistance genes are also likely to be members of the NBS-LRR class including the Vat gene in melon, which confers resistance to cotton-melon aphid A. gossypii Glover (Dogimont et al., 2003) and AKR, which confers resistance to bluegreen aphid in M. truncatula (Klingler et al., 2005). Elicitors in sapsucking insects that are recognised, either directly or indirectly, by R gene-encoded proteins remain unknown although potential candidates include components of aphid saliva. Signaling downstream of these aphid resistance genes is likely to have significant overlap with pathogen signaling. Analysis of gene expression changes following aphid infestation, using a variety of approaches including suppressive subtractive hybridization (SSH) and microarrays, has shown up-regulation of both JA and SA responsive genes as well as induction of other types of aphid-responsive genes (see Moran & Thompson, 2001; Moran et al., 2002; de Ilarduya et al., 2003; Zhu-Salzman et al., 2004; Voelckel et al., 2004). For example, infestation of Arabidopsis with green peach aphid Myzus persicae Sulzer, a susceptible interaction, resulted in the up-regulation of both JA and SA responsive genes (Moran & Thompson, 2001; Moran et al., 2002) and similar results were seen in tomato during both a susceptible (green peach aphid) and a resistant (potato aphid) interaction (de Ilarduya et al., 2003). However, in sorghum, infestation with greenbug, Schizaphis graminum Rondani, led to a strong induction of SA-responsive genes, but only a weak induction of JA-regulated genes, even though treatment with MeJA deterred greenbug infestation (Zhu-Salzman et al., 2004). A study look-

ing at changes in gene expression to whiteflies in squash has also found induction of JA-responsive genes as well as some whitefly specific genes (van de Ven et al., 2000). Interestingly, our preliminary findings with M. truncatula and bluegreen aphid, suggest that JA-regulated responses are most pronounced in the resistant interaction, while SA responsive genes are induced comparably in both the susceptible and resistant interactions (Gao, Edwards & Singh, unpublished observation). The importance of JA signaling for aphid resistance has also been observed with the Arabidopsis cev1 mutant, which has constitutive JA signaling and as a consequence, enhanced resistance to green peach aphid (Ellis et al., 2002). Moreover, depletion of a specific hydroperoxidase lyase, key enzymes in oxylipin production, resulted in an increase in green peach aphid performance in potato (Vancanneyt et al., 2001). Jasmonic acid has also been shown to regulate resistance to cell content-feeding insects, such as mites and thrips in tomato (Li et al., 2002a). In these studies, a mutant deficient in JA biosynthesis resulted in decrease resistance while constitutive activation of the octadecanoid pathway, through overexpression of the prosystemin gene, led to transgenic plants that were highly resistant. Our understanding of induced defences against insect herbivores will be further enhanced as the focus of these studies broadens to include induced changes in the proteome and metabolome. Regulation at the posttranscriptional level can be just as important as transcriptional regulation. Metabolomic approaches can help to identify the chemical or biochemical basis for resistance regulated by the signaling pathways described in this section. Only with such a holistic approach can we fully understand the complex interactions involved in insect defence. The potential value of coupling these approaches is evident in some recent studies by Ian Baldwin and colleagues. In an interesting study, they looked at transcriptional changes resulting from two different types of insect, sap feeding mirids and chewing hornworms (Voelckel & Baldwin, 2004). They compared the changes brought about by single, sequential or simultaneous attack. They found quite different transcriptional profiles resulting from individual attack by each pest and these also differed significantly from the profiles obtained following sequential or simultaneous attack. In a complementary study, they examined changes in volatiles and secondary metabolites in response to mirids and hornworms and found both species resulted in the production of similar volatiles and secondary metabolites

281 although some metabolites, like cryptochlorogenic acid and caffeoylputrescine, were increased only after mirid attack (Kessler & Baldwin, 2004).

How do insects respond to plant defences? While we have concentrated in this review on the plant side of the interaction with insect herbivores, additional opportunities should arise from a better understanding of what insects are doing to overcome resistance. While we cannot cover this area in any depth, legume systems have contributed here as well. For example, legumes systems have provided valuable information on how insects can overcome plant proteinase inhibitors (Patankar et al., 2001; Zhu-Salzman et al., 2003), and in particular how H. armigera and C. maculata regulate gene expression in response to PIs, including the upregulation of presumably insensitive proteases (Bown et al., 2004; Moon et al., 2004). In addition, insects contain many detoxification enzymes, such as glutathione S-transferases, and cytochrome P450s, which detoxify secondary metabolites from the plant. It has been known for some time that insects increase their production of some detoxification enzymes as a consequence of plant defence responses (see Schuler, 1996). In an exciting development, it has been shown that H. zea actually uses the same signals plants use to coordinate their defence response, SA and JA, as signals to activate specific cytochrome P450 genes that are associated with detoxification of plant derived toxic compounds (Li et al., 2002b,c). Research on the insect side of the interaction should lead to novel strategies to achieve insect resistance through disruption of the mechanisms utilized by insects for successful feeding.

communication between researchers and plant breeders is essential to ensure that promising research outputs are properly evaluated for delivery to farmers. These outputs are likely to arise from many research avenues. Screening of genetic resources coupled with conventional breeding still offers high discovery potential, especially if researchers in this area take full advantage of the landraces and wild species available in worldwide collections (Clement, 2002). Research on defence signaling is likely to generate novel R genes to incorporate into conventional breeding programs, or to use as transgenes. This research is also likely to identify insect-responsive promoters and other regulatory elements, which might be used to express seed-localised defences in other tissues. Novel metabolic engineering approaches to achieve insect resistance are likely to be identified from insect-induced changes in the proteome and metabolome (Dixon et al., 1996). Continued research in all these areas with strong links to breeding programs will be essential to deal with the many problems insect pests pose for legume crops, and will lead to new approaches to improve insect resistance across a range of cropping systems.

Acknowledgments We thank Steve Clement, John Klingler, and Lingling Gao for helpful comments on this manuscript. We apologize for not being able to cite several important references because of length limitations. The work in the authors groups on plant/insect interactions is supported in part by the Grains Research and Development Corporation (GRDC) and the Department of Education, Science and Training (DEST) in Australia.

References Conclusions Legumes have much to offer to the study of insect resistance in plants by virtue of their wide range of novel chemistry, importance to agricultural systems and the emergence of model systems suitable for molecular genetic and genomic approaches. What is therefore clear is the need for an integrated approach that involves research on both sides of the plant/insect interaction into both constitutive and induced defences, as well as the establishment of links between those researchers using conventional approaches and those focusing at the molecular level. Perhaps most importantly,

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