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Merging molecular and ecological approaches in plant–insect interactions *Ian T Baldwin, Rayko Halitschke, Andre Kessler and Ursula Schittko The singer–song-writer Paul Simon sang about the ‘50 ways to leave your lover’; plants have at least as many ways of coping with their insect herbivores. Recent research has elucidated the mechanisms of direct and indirect plant defenses, and has provided the first proof of a protective function for indirect defenses in nature. Insect attack elicits a large transcriptional reorganization that differs from that elicited by mechanical wounding. Elicitors in herbivore oral secretions can account for herbivore-specific responses. Patterns of transcriptional changes point to the existence of central herbivore-activated regulators of metabolism. Addresses Department of Molecular Ecology, Max Planck Institute for Chemical Ecology, Carl Zeiss Promenade 10, Jena 07745, Germany *e-mail: [email protected] Current Opinion in Plant Biology 2001, 4:351–358 1369-5266/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations DDRT mRNA differential display reverse transcription FAC fatty-acid–amino-acid conjugate JA jasmonic acid MeJA JA methyl ester ORCA-3 Octadecanoid-Responsive-CatharanthusAPETALA2-domain protein-3 PI protease inhibitor PPO polyphenol oxidase VOC volatile organic compound

Introduction The past year has witnessed unparalleled communication among the previously isolated research communities studying different aspects of plant–insect interactions. Ecologists have reviewed the functional complexity of plant resistance to insects for the benefit of readers with molecular perspectives [1•,2]. These presentations underscore the fact that plant–insect interactions are played out in an ecological arena that is larger than the plant itself. Consequently, the spatial scale of the high throughput functional screens that have been so successfully applied to plant–pathogen interactions may not be appropriate for the study of plant–insect interactions. On the molecular side, sophisticated tools have recently been applied to plant–insect interactions, revealing important mechanisms that mediate direct and indirect defenses. Substantially less is understood about the molecular basis of tolerance. The recent use of DNA microarrays [3•,4] and mRNA differential display reverse transcription (DDRT)-PCR [5••] has provided an assessment of the extensive transcriptional changes that occur after insect attack and allowed tantalizing glimpses of the metabolic coordination underlying induced plant responses. These exchanges suggest that

the gap between experimental tractability and ecological realism will soon be bridged in the study of these prevalent ecological interactions. This review concentrates on advances in the functional understanding of direct and indirect plant defense responses against insects that cause extensive foliar damage. It therefore complements Walling’s excellent review [6••] of a plant’s responses to piercing and sucking insects that cause minimal amounts of damage and elicit pathogenincompatible reactions.

Direct defenses Direct defenses, in contrast to indirect defenses, are plant traits (e.g. primary and secondary metabolites, silica, thorns, trichomes etc.) that by themselves affect herbivore performance and are generally categorized by their mode of action [7]. Two new examples of likely antidigestive or antinutritive proteins, long known to be inducible by herbivory or mechanical damage, have been recently described: a protease inhibitor (PI) that inhibits elastases in the larval midgut [8] and a polyphenol oxidase (PPO) from poplar [9]. PPOs catalyze the oxidation of phenolic secondary metabolites into reactive quinones, which, in turn, polymerize into an insect-trapping glue or reduce the nutritional quality of plants by cross-linking proteins. In addition to PIs that target the major proteolytic digestive enzymes of herbivores, insect attack also elicits the production of a cystein proteinase in maize. When expressed in callus, this proteinase decreases herbivore performance [10]. Its mode of action is unknown, but it may interact with the peritrophic membrane of the lepidopteran midgut. Many secondary metabolites are thought to function as toxins that can poison nonadapted herbivores and force adapted herbivores to invest limited resources in detoxification. Arabidopsis and its relatives produce glucosinolates, and Arabidopsis genomics has facilitated significant advances in understanding the molecular control of the biosynthesis of this diverse class of secondary metabolites [11]. It remains to be determined whether the details of glucosinolate metabolite variation are responsible for shaping the herbivore community of Arabidopsis and its relatives in nature. A field study examining glucosinolate profiles in natural populations of Brassica oleraceae in the UK concluded that differences in current selection pressures were probably not responsible for maintaining the differences in metabolite profiles [12], which contrasts recent results on tropane alkaloid production in Datura [13•]. Three studies with herbivores (i.e. Pieris rapae and Plutella xylostella) that specialize on

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the Brassicaceae found no evidence that resistance is induced by the attack of these herbivores, by ethylene or by jasmonic acid (JA) treatment in Arabidopsis, Raphanus raphanistrum, Lepidium virginicum, R. sativus, and Brassica juncea [14•,15–17]. However, resistance was reported in these species against generalist herbivores, which, in turn, correlated loosely with the concentrations of total glucosinolates in the plants. Hence, glucosinolates are likely functioning to repel opportunistic herbivores and incur detoxification costs in specialized herbivores, the detailed mechanisms of which provide a challenge for future research. The biophysical properties of mechanical defenses that increase the toughness of plant tissues have been recently reviewed [18]. Such traits, which decrease the nutritional value of a plant and therefore slow herbivore growth, may not function by themselves as plant defenses because compromised herbivores are likely to consume more plant material to complete their development. However, if antinutritive and antidigestive compounds are expressed in concert with indirect defenses, which increase the probability of herbivore mortality from predators, parasitoids, or diseases, they can function defensively. Conversely, the expression of toxic compounds may have the opposite effect. Plant toxins that are sequestered by herbivores may protect them from mortality caused by the third trophic level.

While larval feeding is known to elicit VOCs in many plants [27], herbivore oviposition has only recently been shown to induce VOC emissions. In elm, oviposition by a leaf beetle induces the release of VOCs that attract an egg parasitoid [28•]. The elicitors in the oviduct secretions of the elm leaf beetle have yet to be identified; however, the elicitors of another indirect response to oviposition were recently characterized (see below; [29••]). Plants appear to publicize the presence of future herbivores (i.e. of insect eggs) as well as actively feeding ones. DNA microarrays have allowed researchers to examine the extent to which VOCs can elicit defense-related transcripts in neighboring plants. In one such experiment, transcription of defense-related genes was induced in excised lima bean leaves that were exposed to herbivore-induced VOCs or fumigated with selected terpenoids (i.e. β-ocimene, and the C11 and C16 homoterpenes) [4,26•]. Whether such responses occur within intact plants that are exposed to realistic VOC concentrations and environmental conditions remains to be determined. Indirect defense might be a high-risk strategy; its function depends on the presence and behavior of the herbivore’s natural enemies, and the signals (i.e. VOCs) and rewards (i.e. nectar) that plants release can easily be co-opted by other herbivores [27]. If plants can recognize and tailor their responses to specific herbivores, however, some of these risks can be minimized.

Indirect defenses In addition to direct defenses, plants express traits that facilitate ‘top-down’ control of herbivore populations by attracting predators and parasitoids to the feeding herbivore. Indirect defense mediated by the herbivore-induced release of volatile organic compounds (VOCs) has received the most attention [19], but herbivore-induced nectar production by extrafloral nectaries functions similarly. A recent field study [20••] demonstrated that herbivoryinduced increases in nectar production by Macaranga tanarius reduced subsequent herbivory rates and, like VOC induction [21], is elicited by the octadecanoid cascade. Understanding of the molecular basis of herbivore-induced VOC production in maize has advanced with the identification of STC1, a gene involved in the production of a naphthalene-based sesquiterpenoid [22] and IGL, which codes for indole-3-glycerol phosphate lyase and is involved in the release of indole [23•]. Both IGL and STC1 are induced by volicitin, an elicitor found in caterpillar oral secretions (see below), and are involved in the biosynthesis of components of maize’s herbivore-induced VOC bouquet. In addition, a terpene synthase, (E)-nerolidol synthase, has been characterized in maize [24], cucumber, and lima bean [25]. This enzyme likely catalyzes the first committed step in the biosynthesis of the C11 homoterpene, which is found in many herbivore-induced volatile bouquets. The C11 homoterpene may also play a role in between-plant activation of defense-related genes [26•].

Insect-specific elicitors Volicitin (N-[17-hydroxylinolenoyl]-L-glutamine), which was isolated from the oral secretions of beet armyworm (Spodoptera exigua) larvae and increases the emission of VOCs when applied to maize, was the first reported herbivore-specific elicitor. This year, three years after its discovery, the Tumlinson group published the background information on this compound [30,31]. The absolute configuration of the hydroxylinolenoyl moiety of volicitin has been determined and the enantiomers separated [32]. Unfortunately, neither enantiomer of volicitin was active in the induction of VOCs in lima bean [32]. Volicitin is a member of a family of fatty-acid–amino-acid conjugates (FACs) whose conjugation appears, in part, to be mediated microbially [33••]. It may function as a digestion-aiding surfactant in the insect. Other oral secretions have been shown to contain glucose oxidase [34], which may function to increase H2O2 production at the site of herbivory and potentiate elicitation by the formation of reactive oxygen singlets. Lastly, a channel-forming peptide mixture from Trichoderma viride was reported to elicit a VOC release in lima bean that is comparable to that occurring when the JA cascade is antagonized by the salicylic acid cascade [35]. Whether such channel-forming peptides are found in herbivore saliva remains to be determined. A novel class of herbivore-specific elicitors has been reported to elicit a novel type of defense response in peas.

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Bruchins (i.e. long chain diols that are mono- or diesterified with 3-hydroxypropanoic acid), which are found in pea and cowpea weevils, elicit neoplastic growth at the oviposition site in certain genotypes of peas [29••]. The neoplastic growth lifts the recently hatched larva out of the oviposition site and forces it to re-burrow into the pea pod. Although this is not lethal in itself, it exposes the young larvae to predators, parasites, and desiccation, thereby functioning as an indirect defense.

Insect attack results in a large transcriptional reorganization The use of cDNA microarrays [3•,4] and DDRT-PCR [5••,14•] has extended studies on herbivore-induced gene expression to large-scale investigations of the insectresponsive transcriptome, assessing the extent of different genes coordinately affected by herbivory in wild-type and mutant plants. In the first microarray study of plant–insect interactions, 150 Arabidopsis genes were probed for their response to Pieris larval feeding. Many of these genes were induced by both wounding and larval feeding. Moreover, analysis of the JA-insensitive coi1-1 mutant showed that 50% of the wound-inducible genes require intact octadecanoid signaling for their response [3•]. A central role for the JA cascade is also supported by the similarity of transcript accumulation in Nicotiana attenuata plants that were either treated with JA methyl ester (MeJA) or exposed to herbivory [5••]. Extensive overlap with wound and octadecanoid signaling has similarly been reported for plant–pathogen interactions [36,37] and is not surprising; herbivores have an array of feeding apparatuses with which they cause substantial wounding. Differential transcript accumulation in response to herbivory and mechanical tissue damage has also been found, complementing earlier reports on herbivore-specific hormone and secondary metabolite accumulation [38]. The Arabidopsis microarray study [3•] demonstrated that many putatively drought-implicated transcripts were not induced as dramatically by P. rapae feeding damage as by mechanical wounding, suggesting that herbivores might be selected to minimize drought-induced responses. Moreover, herbivore-specific transcript accumulation in N. attenuata plants suggests that insects may be able to suppress wound-associated defense responses [39••]. Minute amounts of Manduca sexta oral secretions were sufficient to antagonize the wound-induced transcript accumulation of some genes (type I) whereas transcript accumulation of others (type II) was synergistically effected (Figure 1). While the use of ‘boutique’ chips allows the analysis of a pre-selected suite of stress-induced genes, future work with larger fractions of the Arabidopsis genome will reveal the full extent of the transcriptional changes that occur after herbivory. DDRT-PCR has been used in N. attenuata to gain an unbiased view of the transcriptional changes induced by its specialist herbivore, M. sexta [5••]. From this study, it was estimated that more than 500 genes

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respond to herbivore attack, of which 27 were analyzed in detail. These herbivore-regulated genes could be crudely classified as being involved in photosynthesis, electron transport, the cytoskeleton, carbon and nitrogen metabolism, signaling and responses to stress, wounding or invasion by pathogens. In general, transcripts that are involved in photosynthesis were strongly downregulated, whereas those that respond to stress, wounding and pathogens, or are involved in shifting carbon and nitrogen to defense, were strongly upregulated. These coordinated changes parallel the metabolic reconfiguration following pathogen attack [40] and point to the existence of central herbivore-activated regulators of metabolism. An exciting example of such a transcriptional regulator has recently been discovered: ORCA-3 (Octadecanoid-ResponsiveCatharanthus-APETALA2 [AP2]-domain protein-3) directs metabolic fluxes into the biosynthesis of terpenoid indole alkaloid by regulating four genes in the alkaloids’ metabolic pathway [41••]. ORCA-3 is an AP2/ethyleneresponsive binding-factor (ERF)-domain protein similar to the transcription factors that respond to ethylene (e.g. ERF1), drought (dehydration-responsive-element-binding protein 2A [DREB2A] and DREB2B) and cold (C-repeat-Binding Factor [CBF]), and to pto-receptor kinases, which mediate tomato’s recognition of the pathogen Pseudomonas syringae. Ecologists have long understood that plants have many different ways of coping with their herbivores (including direct defenses, indirect defenses, and tolerance), and that the mechanisms responsible for these defenses are likely to generate metabolic tradeoffs that could result in fitness costs [42]. The molecular mechanisms underlying this complicated metabolic coordination have, however, remained elusive. These new studies that ‘ask-the-plant’ by investigating the plant’s herbivore-induced transcriptome provide tantalizing hints as to how these complicated responses are coordinated within metabolism. These studies have also highlighted how much we still have to learn.

An integrative system: Nicotiana attenuata Plant–insect interactions are played out in an ecological arena that is larger than the plant itself and incorporates many community-level components, as indirect defenses so clearly illustrate. These higher-order interactions can reverse the fitness outcome of a trait, as occurs when plant chemical defenses are sequestered by herbivores and used against their own predators. N. attenuata is a plant that is native of the Great Basin Desert of North America. It interacts with various herbivore species with a set of inducible defensive traits and provides a good system for the functional analysis of the full complement of insect–plant interactions. This rapid cycling (i.e. short generation time) diploid ‘chases’ fires by timing its germination from long-lived seedbanks with smoke-related germination cues. Synchronized germination in such ephemeral high-resource habitats has selected for rapid growth. The plant’s responses to insect attack can

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therefore be examined in the context of intense intraspecific competition. When attacked by the nicotine-tolerant Solanaceae specialist M. sexta, N. attenuata plants ‘recognize’ the attack, as evidenced by an endogenous JA burst that is propagated throughout the damaged leaf ahead of the rapidly foraging

herbivore [43]. JA treatment elicits dramatic increases in direct defenses, including the production of toxins (i.e. nicotine [44], phenolics, flavonoids, phenolic putrescine conjugates and diterpene sugar esters [45]), antidigestive proteins (i.e. proteinase inhibitors [46]), and antinutritive enzymes (i.e. PPOs [47]). In addition, the emission of various VOCs is elevated as an indirect

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Figure 1 legend Insect oral secretions contain FACs that are necessary and sufficient for the elicitation of herbivore-specific alterations to Nicotiana attenuata’s wound response. (a) (i) High-performance-liquidchromatography–mass-spectrometry (HPLC–MS) analysis of oral secretions and regurgitant (Reg) of M. sexta larvae identified (ii) several FACs. (b) The identified FACs were synthesized and their functional significance in regurgitant was examined by removing them from regurgitant by ion-exchange chromatography (FAC –) and then adding back the synthetic FACs to ion-exchanged regurgitant (FAC +) (see [56••] for experimental details). The right panel depicts herbivore-specific responses of N. attenuata to regurgitant. (c) Addition of regurgitant to leaf wounds, even in minute quantities, results in a rapid burst of endogenous JA and ethylene that clearly differs from the normal wound response (W). The ethylene burst is responsible for the suppression of wound-induced nicotine production [48•] by downregulating the accumulation of putrescine methyl transferase transcripts in the roots (not shown; [44]).

(d) Addition of regurgitant to leaf wounds also alters the expression patterns of other genes in N. attenuata [39••]. Threonine deaminase (TD) is encoded by a gene with a type-I expression pattern: the addition of regurgitant to wounds antagonizes the wound-induced increase in its transcripts. In contrast, regurgitant application amplifies the wound-induced increase in the transcription of genes with type IIa expression patterns (e.g. the gene encoding pathogen-induced peroxidase [PIOX]) and the wound-induced suppression of transcripts of genes with type IIb expression (e.g. that encoding the lightharvesting complex subunit, LHB C1). (e) The addition of regurgitant to wounds also elicits the systemic release of terpenoid compounds, which are readily detected in the headspace of N. attenuata plants by gas chromatography-mass spectrometry (GC-MS) analysis [49•] and by generalist predators and ovipositing adult moths (Figure 2). (f) The effects of the removal and re-addition of FACs to M. sexta regurgitant on the three plant responses (i.e. JA burst, mRNA responses, and VOC release) are summarized.

defense [48•,49•]. The employment of such jasmonateinduced resistance traits [50] comes at a substantial fitness cost to the plant, in part because of the high nitrogen demands of nicotine biosynthesis. This cost is accentuated when plants compete with other uninduced conspecifics [51,52].

the adult moths (Figure 2; see also Update). The release of VOCs, which mediates both top-down (i.e. attracting predators) and ‘bottom-up’ effects (i.e. oviposition avoidance), is estimated to reduce potential herbivore loads by more than 90% [56••], suggesting a strong fitness benefit for plants that use appropriate indirect defense traits.

The large resource demands of nicotine biosynthesis may explain why plants downregulate nicotine biosynthesis when attacked by the nicotine-tolerant larvae of M. sexta. Attack by Manduca larvae, or simply the addition of larval oral secretions to mechanical wounds, suppresses the wound-induced transcription of the nicotine biosynthetic genes, NaPMT1 (N. attenuata Putrescine N-Methyltransferase1) and NaPMT2 [44], and the accumulation of nicotine, but sustains the VOC release [48•]. This alteration in defense responses is mediated by yet another herbivore-specific hormonal response, an ethylene burst [48•]. When induced plants are grown in competition with uninduced plants, the ethylene burst reduces the fitness costs of MeJA-induced resistance [53] and may therefore represent an optimization of resource allocation. Moreover, because parasitoids of M. sexta are known to be negatively affected by the nicotine in their larval hosts, the downregulation of nicotine induction while maintaining VOC emissions to attract parasitoids may also allow N. attenuata to optimize the function of its indirect defense. Until recently, herbivore-induced indirect defenses have largely been a laboratory phenomenon. Two reviews have been very critical of the supporting evidence for the role of such defenses [54•] and significance in non-agricultural systems [55]. A recent study of N. attenuata plants growing in natural populations demonstrated, by manipulating the release of single compounds in the herbivore-induced VOC bouquet, that VOC emission resulted in increased predation rate of Manduca eggs by a generalist predator and decreased oviposition rate by

The tailoring of direct and indirect defense is part of a larger transcriptional reconfiguration of N. attenuata that is, in part, orchestrated by oral secretions of Manduca larvae (Figure 1; [5••,39••]). The factors in the oral secretions that are necessary and sufficient for not only the transcriptional changes but also the JA burst and the VOC release are a suite of FACs (Figure 1; [57••]). If these FACs are removed from the oral secretions, the eliciting activity is lost but is regained when synthetic FACs are added back. Remarkably, functionalized FACs (e.g. volicitin), such as those often found in the oral secretions of other lepidopteran larvae [30,31], could not be detected in the oral secretions of M. sexta or the closely related M. quinquemaculata. As the oral secretions of other tobaccofeeding insects (e.g. Heliothis virescens) also elicited similar changes in wound-induced mRNA [39••], maybe all tobacco-feeding Lepidoptera are ‘recognized’ by a similar suite of FACs. Diet does not seem to influence the biological activity of the Manduca’s oral secretion [39••,43], but the presence of similar microbial fauna throughout the lepidopteran herbivores may account for the similar responses in the plants [33••]. If microbes are indeed involved in the production of the elicitors that plants use to recognize their herbivores and call for help from the third trophic level, then this plant–insect system involves a fourth interaction level. This complexity is likely to increase when the influence of mycorrhizae and endophytes are considered. The N. attenuata system illustrates that an intimate understanding of the ecology involved in defense against insects is necessary to decipher the transcriptional ‘Rosetta Stone’.

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Herbivore-induced VOC emissions influence both (a) predation and (b) oviposition rates and thereby function as potent plant defenses in nature. (a) Elevated emission of cis-3-hexene-1-ol (1), linalool (2) and cis-α-bergamotene (3), as well as the VOC emission induced by MeJA, decrease the survival rate of Manduca eggs and young larvae compared to those on untreated control plants (C). This decrease is caused by increased predation pressure by the generalist predator Geocoris pallens. (b) In a similar experiment, MeJA and linalool (2) applications, as well as previous caterpillar damage (Cat), decreased oviposition rates of adult M. quinquemaculata moths compared to those on untreated and undamaged control plants [56••]. VOCs from herbivore-attacked plants are used by both the herbivore and its generalist predator. Hence, they mediate bottom-up as well as top-down control over herbivore populations.

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Conclusions

References and recommended reading

Plants must discriminate among different environmental challenges in order to optimize the allocation of their resources to growth, defense, and reproduction. Phytophagous insects display a great diversity of feeding modes and life histories, and it is important for plants to distinguish among insects that have different fitness consequences for a plant. N. attenuata clearly uses both chemical and physical attributes of herbivory to ‘recognize’ attack from its specialized herbivores. Understanding the complex transcriptional changes that occur after insect attack in functional terms, however, remains a difficult challenge. Clearly, the activation of induced defense responses entails a complex reorganization of metabolism and the transcriptional changes involved will likely reveal fundamental control mechanisms. Studying the multitude of induced responses that are elicited after herbivore attack may provide a unique opportunity to address Stahl’s century old question: “How much of what we call a ‘plant’ is generated by its interactions with other organisms?”[58].

Papers of particular interest, published within the annual period of review, have been highlighted as:

Update A recent laboratory study [59] of Nicotiana tabacum demonstrated that specific night-released VOCs from attacked plants repel ovipositing Heliothis virescens moths. This study confirms the results of a field study in another plantherbivore system [56••], that demonstrated the importance of VOCs in altering oviposition preferences.

• of special interest •• of outstanding interest 1. •

Agrawal AA: Overcompensation of plants in response to herbivory and the by-product benefits of mutualism. Trends Plant Sci 2000, 5:309-313. This review provides an extensive taxonomy of mutualistic interactions. 2.

Paul ND, Hatcher PE, Taylor JE: Coping with multiple enemies: an integration of molecular and ecological perspectives. Trends Plant Sci 2000, 5:220-225.

3. •

Reymond P, Weber H, Damond M, Farmer EE: Differential gene expression in response to mechanical wounding and insect feeding in Arabidopsis. Plant Cell 2000, 12:707-719. The first use of a cDNA microarray enriched in stress-induced genes to study plant responses to insect attack and mechanical wounding. This study revealed that many transcripts were coordinately expressed not only after wounding and herbivore attack but also in response to transient changes in key oxylipin metabolites. 4.

5. ••

Arimura G, Tashiro K, Kuhara S, Nishioka T, Ozawa R, Takabayashi J: Gene responses in bean leaves induced by herbivory and by herbivore-induced volatiles. Biochem Biophy Res Commun 2000, 277:305-310.

Hermsmeier D, Schittko U, Baldwin IT: Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata. I. Large scale changes in the accumulation of growth and defense-related plant mRNAs. Plant Physiol 2001, 125:683-700. This differential display reverse transcription (DDRT)-PCR study is the first in a series of papers [5••,39••,57••] examining a particular plant–herbivore interaction. The authors of this paper demonstrate that feeding by the specialist herbivore Manduca sexta on its host plant, Nicotiana attenuata, results in a dramatic transcriptional reorganization in the plant, which, in turn, provides unique insights into the molecular basis of plant tolerance and resistance mechanisms.

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6. Walling LL: The myriad plant responses to herbivores. J Plant •• Growth Regul 2000, 19:195-216. This review of responses to phloem-feeding insects is a ‘must-read’. It not only covers the author’s own outstanding contributions to whitefly-induced responses in plants (i.e. the discovery of SLW1 and SLW3) but also provides a comprehensive review of herbivore-induced signaling, signaling cross-talk between herbivore- and pathogen-induced responses, comparisons of signaling in Arabidopsis with that in Lycopersicon, and a summary of the elicited genes.

23. Frey M, Stettner C, Pare PW, Schmelz EA, Tumlinson JH, Gierl A: An • herbivore elicitor activates the gene for indole emission in maize. Proc Natl Acad Sci USA 2000, 97:14801-14806. The authors of this paper describe a new enzyme (and its gene) that is responsible for volicitin-induced indole release in maize. The enzyme diverts indole-3 glycerol phosphate from the synthesis of direct defense metabolites (DIMBOA [2,4-dihydroxy-7-methoxy-2H-1,4-benzoxanzin-3(4H)-one]) into the production of an indirect defense (indole).

7.

Duffey SS, Stout MJ: Antinutritive and toxic components of plant defense against insects. Arch Insect Biochem Physiol 1996, 32:3-37.

8.

Tamayo MC, Rufat M, Bravo JM, San Segundo B: Accumulation of a maize proteinase inhibitor in response to wounding and insect feeding, and characterization of its activity toward digestive proteinases of Spodoptera littoralis larvae. Planta 2000, 211:62-71.

24. Degenhardt J, Gershenzon J: Demonstration and characterization of (E)-nerolidol synthase from maize: a herbivore-inducible terpene synthase participating in (3E)-4,8,-dimethyl-1,3,7nonatriene biosynthesis. Planta 2000, 210:815-822.

9.

Constabel CP, Yip L, Patton JJ, Christopher ME: Polyphenol oxidase from hybrid poplar. Cloning and expression in response to wounding and herbivory. Plant Physiol 2000, 124:285-295.

10. Pechan T, Ye L, Chang Y-m, Mitra A, Lin L, Davis FM, Williams WP, Luthe DS: A unique 22-kD cysteine proteinase accumulates in response to larval feeding in maize genotypes resistant to fall armyworm and other lepidoptera. Plant Cell 2000, 12:1031-1040. 11. Quiros HCd, Magrath R, McCallum D, Kroymann J, Schnabelrauch D, Mitchell-Olds T, Mithen R: alpha-Keto acid elongation and glucosinolate biosynthesis in Arabidopsis thaliana. Theor Appl Genet 2000, 101:429-437. 12. Moyes CL, Collin HA, Britton G, Raybould AE: Glucosinolates and differential herbivory in wild populations of Brassica oleracea. J Chem Ecol 2000, 26:2625-2641. 13. Shonle I, Bergelson J: Evolutionary ecology of the tropane • alkaloids of Datura stramonium L. (Solanaceae). Evolution 2000, 54:778-788. This study demonstrates that two toxic tropane alkaloids found in Datura stramonium are under active natural selection, and that insect herbivores are likely responsible for the negative directional selection on scopolamine and the stabilizing selection on hyoscyamine. 14. Stotz HU, Pittendrigh BR, Kroymann J, Weniger K, Fritsche J, • Bauke A, Mitchell-Olds T: Induced plant defense responses against chewing insects. Ethylene signaling reduces resistance of Arabidopsis against Egyptian cotton worm but not diamondback moth. Plant Physiol 2000, 124:1007-1017. The first use of DDRT-PCR to discover insect-induced genes in Arabidopsis. 15. Agrawal AA: Induced responses to herbivory in wild radish: effects on several herbivores and plant fitness. Ecology 1999, 80:1713-1723. 16. Agrawal AA: Benefits and costs of induced plant defense for Lepidium virginicum (Brassicaceae). Ecology 2000, 81:1804-1813. 17.

Li Q, Eigenbrode SD, Stringam GR, Thiagarajah MR: Feeding and growth of Plutella xylostella and Spodoptera eridania on Brassica juncea with varying glucosinolate concentrations and myrosinase activities. J Chem Ecol 2000, 26:2401-2419.

18. Lucas PW, Turner IM, Dominy NJ, Yamashita N: Mechanical defences to herbivory. Ann Botany 2000, 86:913-920. 19. Agrawal AA: Mechanisms, ecological consequences and agricultural implications of tri-trophic interactions. Curr Opin Plant Biol 2000, 3:329-335. 20. Heil M, Koch T, Hilpert A, Fiala B, Boland W, Linsenmair KE: •• Extrafloral nectar production of the ant-associated plant, Macaranga tanarius, is an induced, indirect, defensive response elicited by jasmonic acid. Proc Natl Acad Sci USA 2001, 98:1083-1088. This tour-de-force study demonstrates that herbivore attack and JA treatments elicit an increase in extrafloral nectar production, which in nature increases the visitation rates of certain insects and decreases herbivory. 21. Ozawa R, Arimura G, Takabayashi J, Shimoda T, Nishioka T: Involvement of jasmonate- and salicylate-related signaling pathways for the production of specific herbivore-induced volatiles in plants. Plant Cell Physiol 2000, 41:391-398. 22. Shen BZ, Zheng ZW, Dooner HK: A maize sesquiterpene cyclase gene induced by insect herbivory and volicitin: characterization of wild-type and mutant alleles. Proc Natl Acad Sci USA 2000, 97:14807-14812.

25. Bouwmeester HJ, Verstappen FWA, Posthumus MA, Dicke M: Spider mite-induced (3S)-(E)-nerolidol synthase activity in cucumber and lima bean. The first dedicated step in acyclic C11-homoterpene biosynthesis. Plant Physiol 1999, 121:173-180. 26. Arimura G, Ozawa R, Shimoda T, Nishioka T, Boland W, Takabyashi J: • Herbivory-induced volatiles elicit defence genes in lima bean leaves. Nature 2000, 406:512-515. VOCs released from attacked excised leaves are shown to alter the expression of defense-related transcripts in unattacked excised leaves in the same chamber. Hence, VOCs provide a potential mechanism for between-plant chemical signaling about herbivore attack (e.g. ‘talking trees’ [46]). 27.

Dicke M, van Loon JJA: Multi-trophic effects of herbivore-induced plant volatiles in an evolutionary context. Entom Exp Applicata 2000, 97:237-249.

28. Meiners T, Hilker M: Induction of plant synomones by oviposition of • a phytophagous insect. J Chem Ecol 2000, 26:221-232. The first study to show that egg laying by a beetle that causes extensive wounding produces a VOC release from the host plant. 29. Doss RP, Oliver JE, Proebsting WM, Potter SW, Kuy SR, Clement SL, •• Williamson RT, Carney JR, DeVilbiss ED: Bruchins: insect-derived plant regulators that stimulate neoplasm formation. Proc Natl Acad Sci USA 2000, 97:6218-6223. New chemical elicitors from the oviposition fluid of bruchid beetles that cause certain genotypes of peas to undergo neoplastic growth and expel the oviposited eggs are reported. The chemical composition of these newly discovered elicitors, dubbed ‘bruchins’, suggests that lipid-based signals will be prevalent in plant–insect interactions. 30. Turlings TCJ, Alborn HT, Loughrin JH, Tumlinson JH: Volicitin, an elicitor of maize volatiles in oral secretion of Spodoptera exigua: isolation and bioactivity. J Chem Ecol 2000, 26:189-202. 31. Alborn HT, Jones TH, Stenhagen GS, Tumlinson JH: Identification and synthesis of volicitin and related components from beet armyworm oral secretions. J Chem Ecol 2000, 26:203-220. 32. Spiteller D, Pohnert G, Boland W: Absolute configuration of volicitin, an elicitor of plant volatile biosynthesis from lepidopteran larvae. Tetrahedron Lett 2001, 42:1483-1485. 33. Spiteller D, Dettner K, Boland W: Gut bacteria may be involved in •• interactions between plants, herbivores and their predators: microbial biosynthesis of N-acylglutamine surfactants as elicitors of plant volatiles. Biol Chem 2000, 381:755-762. This study provides the first evidence to show that gut microbes are involved in the biosynthesis of FAC elicitors in plant–insect interactions. 34. Felton GW, Eichenseer H: Herbivore saliva and its effects on plant defense against herbivores and pathogens. Induced Plant Defenses Against Pathogens and Herbivores: Ecology, and Agriculture. St Paul, Minnesota: American Phytopathological Society Press; 1999:19-36. 35. Engelberth J, Koch T, Schueler G, Bachmann N, Rechtenbach J, Boland W: Ion channel-forming alamethicin is a potent elicitor of volatile biosynthesis and tendril coiling. Cross talk between jasmonate and salicylate signaling in lima bean. Plant Physiol 2000, 125:369-377. 36. Durrant WE, Rowland O, Piedras P, Hammond-Kosack KE, Jones JDG: cDNA-AFLP reveals a striking overlap in race-specific resistance and wound response gene expression profiles. Plant Cell 2000, 12:963-977. 37.

Schenk PM, Kazan K, Wilson I, Anderson JP, Richmond T, Somerville SC, Manners JM: Coordinated plant defense responses in Arabidopsis revealed by microarray analysis. Proc Natl Acad Sci USA 2000, 97:11655-11660.

38. Stout MJ, Bostock RM: Specificity of induced responses to arthropods and pathogens. Induced Plant Defenses Against

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Pathogens and Herbivores: Ecology, and Agriculture. St Paul, Minnesota: American Phytopathological Society Press; 1999:183-210. 39. Schittko U, Hermsmeier D, Baldwin IT: Molecular interactions •• between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata. II. Accumulation of plant mRNA in response to insect-derived cues. Plant Physiol 2001, 125:701-710. This second part of a series of papers [5••,39••,57••] on the Manduca sexta– Nicotiana attenuata interaction disentangles the wound-response from the response to herbivore oral secretions. This study identifies discrete patterns in the transcriptional changes described in [5••], which point to the existence of herbivore-specific transactivating factors that orchestrate these patterns. 40. Batz O, Logemann E, Reinold S, Hahlbrock K: Extensive reprogramming of primary and secondary metabolism by fungal elicitor or infection in parsley cells. Biol Chem 1998, 379:1127-1135. 41. van der Fits L, Memelink J: ORCA3, a jasmonate-responsive •• transcriptional regulator of plant primary and secondary metabolism. Science 2000, 289:295-297. An ingenious T-DNA activation tagging selection approach allowed the authors to identify the third in a series of ORCAs, which transactivates a suite of genes in the alkaloid biosynthetic pathway. 42. Stowe KA, Marquis RJ, Hochwender CG, Simms EL: The evolutionary ecology of tolerance to consumer damage. Annu Rev Ecol Syst 2000, 31:565-595. 43. Schittko U, Preston CA, Baldwin IT: Eating the evidence? Manduca sexta can not disrupt specific jasmonate induction in Nicotiana attenuata by rapid consumption. Planta 2000, 210:343-346. 44. Winz R, Baldwin IT: Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata. IV. Insect-induced ethylene reduces jasmonate-induced nicotine accumulation by regulating putrescine N-methyltransferase transcripts. Plant Physiol 2001, 125:2189-2202. 45. Keinanen M, Oldham NJ, Baldwin IT: Rapid HPLC screening for jasmonate-induced increases in tobacco alkaloids, phenolics and diterpene glycosides in Nicotiana attenuata. J Agric Food Chem 2001, in press. 46. van Dam NM, Horn M, Mares M, Baldwin IT: Ontogeny constrains the systemic proteinase inhibitor response in Nicotiana attenuata. J Chem Ecol 2001, 27:547-568. 47.

Karban R, Baldwin IT, Baxter KJ, Laue G, Felton GW: Communication between plants: induced resistance in wild tobacco plants following clipping of neighboring sagebrush. Oecologia 2000, 125:66-71.

48. Kahl J, Siemens DH, Aerts RJ, Gäbler R, Kühnemann F, Preston CA, • Baldwin IT: Herbivore-induced ethylene suppresses a direct defense but not a putative indirect defense against an adapted herbivore. Planta 2000, 210:336-342. The authors describe how a herbivore-induced ethylene burst suppresses nicotine accumulation (a direct defense) but not VOC release (an indirect defense).

49. Halitschke R, Kessler A, Kahl J, Lorenz A, Baldwin IT: • Ecophysiological comparison of direct and indirect defenses in Nicotiana attenuata. Oecologia 2000, 124:408-417. This first physiological comparison of a direct defense (i.e. nicotine induction) with an indirect defense (i.e. VOC release) reveals that the resource demands of the direct defense dwarfs that of the indirect defense. 50. van Dam NM, Hadwich K, Baldwin IT: Induced responses in Nicotiana attenuata affect behavior and growth of the specialist herbivore Manduca sexta. Oecologia 2000, 122:371-379. 51. van Dam N, Baldwin IT: Competition mediates costs of jasmonateinduced defences, N acquisition and transgenerational plasticity in Nicotiana attenuata. Functional Ecol 2001, in press. 52. Baldwin IT, Hamilton W: Jasmonate-induced responses of Nicotiana sylvestris result in fitness costs due to impaired competitive ability for nitrogen. J Chem Ecol 2000, 26:915-952. 53. Voelckel C, Schittko U, Baldwin IT: Herbivore-induced ethylene burst reduces fitness costs of jasmonate- and oral secretioninduced defenses in Nicotiana attenuata. Oecologia 2001, 127:274-280. 54. van der Meijden E, Klinkhamer PGL: Conflicting interests of plants • and the natural enemies of herbivores. Oikos 2000, 89:202-208. This review summarizes the controversy surrounding the evolutionary value of indirect defense, stressing the lack of studies on natural systems. 55. Hawkins BA, Mills NJ, Jervis MA, Price PW: Is the biological control of insects a natural phenomenon? Oikos 1999, 86:493-506. 56. Kessler A, Baldwin IT: Defensive function of herbivore-induced •• plant volatile emissions in nature. Science 2001, 291:2141-2144. The first field study of the protective effect of VOC emission in nature demonstrates that VOCs can function as a double-edged sword that protects plants by attracting predators and by repelling ovipositing herbivores. 57. ••

Halitschke R, Schittko U, Pohnert G, Boland W, Baldwin IT: Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata. III. Fatty acid-amino acid conjugates in herbivore oral secretions are necessary and sufficient for herbivore-specific plant responses. Plant Physiol 2001, 125:711-717. This third part of a series of papers [5••,39••,57••] on the Manduca sexta–Nicotiana attenuata interaction demonstrates that fatty–acid–aminoacid conjugates in herbivore oral secretions are necessary and sufficient to cause the herbivore-specific transcriptional changes described in [39••], as well as other phenotypic responses (i.e. JA burst and VOC release) that are specific to herbivore attack. In short, the series of papers [5••,39••,57••] demonstrates that the elicitor-based approach, which has proved invaluable in unraveling plant–pathogen interactions, also works for the study of plant–herbivore interactions. 58. Stahl E: Pflanzen und Schnecken. Eine biologische Studie über die Schutzmittel der Pflanzen gegen Schneckenfrass. Jenaische Z Naturwiss Med 1888, 22:1-126. [Title translation: .]

59. De Moraes CM, Mescher MC, Tumlinson JH: Caterpillar-induced nocturnal plant volatiles repel non-specific females. Nature 2001, 410:577-580.