Skip to main content
NCBI home page
Plant Physiology logoLink to Plant Physiology
. 2009 Jul;150(3):1576–1586. doi: 10.1104/pp.109.139550

Different Lepidopteran Elicitors Account for Cross-Talk in Herbivory-Induced Phytohormone Signaling1,[W],[OA]

Celia Diezel 1, Caroline C von Dahl 1,2, Emmanuel Gaquerel 1, Ian T Baldwin 1,*
PMCID: PMC2705021  PMID: 19458114

Abstract

Salicylic acid (SA), jasmonic acid (JA), ethylene (ET), and their interactions mediate plant responses to pathogen and herbivore attack. JA-SA and JA-ET cross-signaling are well studied, but little is known about SA-ET cross-signaling in plant-herbivore interactions. When the specialist herbivore tobacco hornworm (Manduca sexta) attacks Nicotiana attenuata, rapid and transient JA and ET bursts are elicited without significantly altering wound-induced SA levels. In contrast, attack from the generalist beet armyworm (Spodoptera exigua) results in comparatively lower JA and ET bursts, but amplified SA bursts. These phytohormone responses are mimicked when the species' larval oral secretions (OSSe and OSMs) are added to puncture wounds. Fatty acid-amino acid conjugates elicit the JA and ET bursts, but not the SA burst. OSSe had enhanced glucose oxidase activity (but not β-glucosidase activity), which was sufficient to elicit the SA burst and attenuate the JA and ET levels. It is known that SA antagonizes JA; glucose oxidase activity and associated hydrogen peroxide also antagonizes the ET burst. We examined the OSMs-elicited SA burst in plants impaired in their ability to elicit JA (antisense [as]-lox3) and ET (inverted repeat [ir]-aco) bursts and perceive ET (35s-etr1b) after fatty acid-amino acid conjugate elicitation, which revealed that both ET and JA bursts antagonize the SA burst. Treating wild-type plants with ethephone and 1-methylcyclopropane confirmed these results and demonstrated the central role of the ET burst in suppressing the OSMs-elicited SA burst. By suppressing the SA burst, the ET burst likely facilitates unfettered JA-mediated defense activation in response to herbivores that otherwise would elicit SA.

Plants are continuously challenged by a variety of biotic agents that attack in different ways, over different spatial scales, and with different consequences for a plant's Darwinian fitness. To survive, plants recognize and respond differently to different attackers deploying chemical or morphological defenses that kill, starve, poison, repel, and trap their attackers or attract the natural enemies of these attackers. Each attacker, depending on its natural history, evolves different counterresponses to these plant defenses, which, in turn, increases the need for a plant to recognize different attackers and tailor specific responses.

How plants cope with these demands is the subject of intensive research, and it is clear that three phytohormones and their interactions play a central role: salicylic acid (SA), jasmonic acid (JA), and ethylene (ET; Reymond and Farmer, 1998; Thomma et al., 2001; Glazebrook, 2005; De Vos et al., 2006). SA is known to play a central role in defense against biotrophic pathogens by containing their spread with a preventive cell suicide, known as the hypersensitive response. Additionally, SA elicits a long-lasting, induced resistance response to a broad range of invading pathogens, known as systemic acquired resistance (Ross, 1961; Ryals et al., 1996; van Loon et al., 1998; Potlakayala et al., 2007). JA, on the other hand, plays a key role as an elicitor of defense responses to necrotrophic pathogens by initiating SA-independent induced systemic resistance (Pieterse et al., 1998; Vijayan et al., 1998) and to herbivores (Halitschke and Baldwin, 2005; De Vos et al., 2006). There is also considerable evidence for dose-dependent antagonism of SA on JA-mediated herbivore defenses (Doherty et al., 1988; Péna-Cortes et al., 1993; Doares et al., 1995; Baldwin et al., 1997), as well as pathogen defenses (Stout et al., 1999; Gupta et al., 2000; De Vos et al., 2006; Mur et al., 2006). This important form of cross-talk has been described in a large number of Arabidopsis (Arabidopsis thaliana) accessions (Koornneef and Pieterse, 2008). Less is known about JA's effects on SA signaling, but in JA- and coronatine-insensitive mutants, SA-mediated gene expression and defenses are typically enhanced (Kloek et al., 2001; Li et al., 2004) and JA treatment suppresses SA-dependent pathogen-related (PR) protein expression (Niki et al., 1998).

The gaseous hormone ET, in addition to its central role in many physiological processes such as fruit ripening and senescence, modulates defense responses, particularly those mediated by the JA cascade, rather than eliciting defense responses on its own (for review, see von Dahl and Baldwin, 2007). For example, the ET burst elicited by herbivore attack enhances the production of JA-elicited proteinase inhibitors in tomato (Solanum lycopersicum; O'Donnell et al., 1996), but suppresses JA-elicited nicotine production in the native tobacco Nicotiana attenuata (Kahl et al., 2000; Winz and Baldwin, 2001). Whereas both ET-JA and JA-SA interactions are important for various pathogen responses, the ET-SA interaction is not as well studied. Recently, Leon-Reyes et al. (2009) demonstrated that ET modulated the role that NPR1 plays in JA-SA cross-talk in Arabidopsis.

It is well established that a plant's response to herbivore attack frequently differs from that of even careful simulations of the mechanical wounding that herbivore feeding causes (Baldwin, 1988; Baldwin et al., 2001; see also Mithofer and Boland, 2008, for examples in which the timing of mechanical damage can mimic herbivore-specific responses). Insect-specific elicitors, found in insect oral secretions (OS) or oviposition fluids, are frequently responsible for the specificity of the responses. In lepidopteran OS, several types of elicitors have been identified. A Glc oxidase (GOX) was isolated from Helicoverpa zea OS; GOX suppresses plant defense responses (Eichenseer et al., 1999; Musser et al., 2005). β-Glucosidase from the OS of white cabbage butterfly (Pieris brassicae) larvae elicits the production of volatiles from cabbage plants; these volatiles attract parasitic wasps (Cotesia glomerata) to feeding larvae. The volatiles released are comparable to those released by the treatment of leaves with β-glucosidase from almonds, and the parasitic wasps did not discriminate between cabbage leaves treated with almond β-glucosidase and leaves treated with larval OS (Mattiacci et al., 1995). In addition to these proteins, peptides called inceptins from the fall armyworm's OS, proteolytic postingestive products of the host plant's chloroplastic ATP synthase γ-subunit (Schmelz et al., 2006, 2007), as well as fatty acid-amino acid conjugates (FACs) composed of either linolenic acid or linoleic acid and their derivatives and an amino acid moiety, Gln or Glu (Alborn et al., 1997; Pohnert et al., 1999; Spiteller and Boland, 2003; Maffei et al., 2004;), are established elicitors in lepidopteran OS. Volicitin, a hydroxyl FAC (N-17-hydroxylinolenoyl-l-Gln) that was the first FAC identified in beet armyworm (Spodoptera exigua) OS, induces volatile release in maize (Zea mays) seedlings (Alborn et al., 1997). Since then, different forms of FACs have been found in other insect species (Pohnert et al., 1999; Halitschke et al., 2001; Mori et al., 2001; Spiteller and Boland, 2003). The effects of applying the two most abundant FACs in the OS of the specialist herbivore of N. attenuata, tobacco hornworm (Manduca sexta; Lepidoptera, Sphingidae), have been intensively studied. When synthetic FACs are applied to wounded N. attenuata leaves, they simulate most of the responses elicited by attack from tobacco hornworm larvae, including the activation of mitogen-activated protein kinase activity and the elicited changes in transcriptome, proteome, and defensive secondary metabolites (Halitschke et al., 2001; Halitschke and Baldwin, 2003; Giri et al., 2006; Wu et al., 2007).

Less is known about the actual amounts of these elicitors that come in contact with leaf tissues during the feeding process. Peiffer and Felton (2005) showed that H. zea larvae secrete microgram amounts of GOX onto the leaf (e.g. 2.47 μg on tobacco [Nicotiana tabacum] leaves). Peiffer and Felton (2009) recently estimated the amount of total regurgitate that is applied to leaves during larval feeding; frequently <10 nL was applied. Given that tobacco hornworm regurgitate is able to elicit a full JA burst with all of the associated responses when it is diluted 1/1,000 (Schittko et al., 2000), the small quantities of OS that are transferred to leaves during feeding are sufficient to elicit herbivore recognition responses in plants.

Here we compare the ET, SA, and JA responses in N. attenuata plants in response to attack from the two most common Lepidopteran herbivores feeding on N. attenuata plants in the plant's native habitat: the specialist tobacco hornworm and the non-native generalist beet armyworm (Steppuhn et al., 2004). As described previously, feeding by tobacco hornworm larvae elicits prominent ET and JA bursts (Halitschke et al., 2004; von Dahl et al., 2007), and we report here that feeding by beet armyworm larvae results in smaller JA and ET bursts, and a prominent SA burst, which is lacking from the response to tobacco hornworm attack. These contrasting phytohormone responses can be mimicked by applying the larvae's specific OS to mechanical wounds, and we identify GOX activity and its associated hydrogen peroxide (H2O2) production as the elicitor in OSSe responsible for the differences in phytohormone responses. To further understand the role of phytohormone cross-talk, we examined the phytohormone responses in OSMs-elicited N. attenuata plants impaired in their ability to elicit JA (antisense [as]-lox3) and ET (inverted repeat [ir]-aco) bursts and to perceive the ET burst (35s-etr1b) and discovered that both ET and JA bursts antagonize the SA burst. We propose that, by suppressing the SA burst, the ET burst facilitates unfettered JA-mediated defense activation when plants are attacked by herbivores that also elicit SA signaling.

RESULTS

Generalist and Specialist Herbivores Elicit Different Patterns of SA, JA, and ET Accumulation

The specialist herbivore tobacco hornworm and the non-native generalist herbivore S. exigua are the two most common lepidopteran herbivores feeding on N. attenuata plants in their native habitats (Steppuhn et al., 2004). We compared SA, JA, and ET accumulation patterns in N. attenuata plants attacked by these two herbivores. The measures of total SA were consistently 4- to 5-fold higher than the measures of free SA in all experiments, but the kinetics of total SA mirrored those of free SA (Rayapuram and Baldwin, 2007; C. Diezel and I.T. Baldwin, unpublished data), so we report here only the free SA levels. Beet armyworm attack elicited significantly larger SA levels, up to 6-fold compared to unattacked plants, than did attack from tobacco hornworm larvae (ANOVA; F2,14 = 2.602; P < 0.05; Fig. 1A). In contrast, JA values induced by tobacco hornworm attack largely exceeded those measured in response to beet armyworm attack (ANOVA; F2,15 = 13.009; P < 0.05; Fig. 1B). The herbivore-induced ET burst followed the same pattern. Tobacco hornworm attack resulted in 3-fold increases in ET emission compared to unattacked plants, whereas beet armyworm attack did not significantly alter the emission of this phytohormone (ANOVA; F2,7 = 15.183; P < 0.05; Fig. 1C).

Figure 1.

Figure 1.

Open in a new tab

Attack from the generalist lepidopteran herbivore, beet armyworm, elicits higher SA levels but lower JA and ET levels in N. attenuata leaves than does attack from the specialist lepidopteran herbivore, tobacco hornworm. A and B, Mean ± se (n = 8) levels of free SA and JA in untreated wild-type N. attenuata plants (control, white bars) and 3 d after herbivory by tobacco hornworm (gray bars) or beet armyworm (black bars) larvae. C, Mean ± se (n = 8) ET emission of excised leaves during 22 h of herbivory by tobacco hornworm or beet armyworm larvae and of leaves that were left undamaged (control). ET was analyzed using a photoacoustic spectrometer (PAS) equipped with a laser source. Different letters indicate significant differences among treatments (ANOVA; P < 0.05). FW, Fresh weight.

Because differences in feeding behavior (timing and biomechanics of leaf damage) could be responsible for the different SA, JA, and ET responses, we used a standardized mechanical wounding treatment to determine whether OS elicitation could account for the differences in plant responses. Treatment of standardized puncture wounds produced by a fabric pattern wheel with OS from the two herbivore species recapitulated the differences in phytohormone signaling elicited by herbivore attack (Fig. 2). Elicitation with OS from tobacco hornworm (OSMs) or beet armyworm (OSSe) larvae qualitatively reproduced the phytohormone patterns observed during active feeding, but the absolute values of the JA and ET bursts were different from those observed during insect feeding, which is likely due to the differences in the time of leaf harvesting and the differences in the wounding event between the OS elicitation and real herbivore feeding. SA levels increased up to 3-fold in plants elicited with OSSe compared to in plants wounded and treated with water or OSMs (ANOVA; F2,6 = 9.663; P < 0.05 Fig. 2A). JA levels, on the other hand, increased up to 4-fold in plants treated with OSMs compared to in water-treated control plants, whereas OSSe-treated plants accumulated JA to levels that did not differ from levels in wound- and water-treated plants (ANOVA; F2,6 = 56.608; P < 0.05; Fig. 2B). ET emissions from plants treated with OSMs were significantly higher than those in water- and OSSe-elicited plants (ANOVA; F2,6 = 15.523; P < 0.05; Fig. 2C).

Figure 2.

Figure 2.

Open in a new tab

OS of beet armyworm larvae elicit higher levels of SA, but lower levels of JA and ET compared to tobacco hornworm OS when applied to puncture wounds in N. attenuata leaves. A and B, Mean ± se (n = 3) levels of free SA and JA in wild-type N. attenuata plants 90 min after elicitation by wounding leaves with a fabric pattern wheel and applying 20 μL of either water (w, white bars), tobacco hornworm (OSMs, gray bars), or beet armyworm OS (OSSe, black bars). C, Values are the mean ± se (n = 3) ET levels released from wild-type N. attenuata plants after elicitation by water (w, open bar), OSMs (gray bar), or OSSe (black bar). ET in the headspace of the elicited leaves was collected for 5 h. Different letters indicate significant differences among treatments (ANOVA; P < 0.05). FW, Fresh weight.

OS Components and Their Role in Eliciting SA via H2O2 Production

Different classes of OS-derived elicitors are known to regulate different phytohormone responses. In N. attenuata, FACs found in tobacco hornworm OS are able to mimic most of the known OSMs-elicited changes in transcripts, proteins, and metabolites (Halitschke et al., 2003; Voelckel and Baldwin, 2004; Giri et al., 2006; von Dahl et al., 2007; Pandey et al., 2008; Gaquerel et al., 2009). Notably, the two most abundant FACs—N-linolenoyl-l-Gln (C18:3-Gln) and N-linolenoyl-l-Glu (C18:3-Glu)—found in tobacco hornworm OS are necessary and sufficient to elicit the OS-specific JA (Halitschke et al., 2003) and ET (von Dahl et al., 2007) bursts. Levels of these FACs were significantly lower in plants treated with OSSe compared to those treated with OSMs (Fig. 3), which is consistent with the reduced JA and ET bursts after OSSe elicitation. In contrast, adding C18:3-Gln and C18:3-Glu to wounds in wild-type leaves did not significantly elicit SA accumulation compared to adding these compounds to the leaves of the detergent-treated control (ANOVA; F2,14 = 2.717; P = 0.87; Supplemental Fig. S1).

Figure 3.

Figure 3.

Open in a new tab

FAC profiles in tobacco hornworm OS (OSMs) and beet armyworm OS (OSSe). Values are relative concentrations of the most abundant FACs in OSMs (black bars) and OSSe (white bars) of three pooled samples from 30 third- to fifth-instar larvae of each species that were actively feeding on wild-type N. attenuata plants. N-linolenoyl-l-Glu (C18:3-Glu), N-linoleoyl-l-Glu (C18:2 Glu), N-palmitoyl-l-Glu (C16:0-Glu), N-linolenoyl-l-Gln (C18:3-Gln), N-linoleoyl-l-Gln (C18:2- Gln), N-17-hydroxylinolenoyl-l-Gln (volicitin), and N-palmitoyl-l-Gln (C16:0-Gln). Note the changes in y-axis scale (4× and 40×) to better visualize less abundant FACs.

β-Glucosidase is another insect-derived elicitor of plant defense responses (Mattiacci et al., 1995). This hydrolytic enzyme has been postulated to target SA-glucoside conjugates to release free SA; however, treating puncture wounds with commercial almond β-glucosidase at concentrations similar to those found in OSSe did not significantly increase SA levels compared to treating puncture wounds with water (ANOVA; F2,11 = 0.204; P = 0.82; Supplemental Fig. S2).

Labial salivary gland extracts treated with active GOX from OSSe have been shown to increase SA-mediated PR-1a protein levels in cultivated tobacco plants (Musser et al., 2005). We measured GOX activity in the two OS and found that GOX activity was 2.5-fold lower in OSMs compared to in OSSe (Student's t test; P # 0.001; Supplemental Fig. S3). Moreover, treating leaf puncture wounds with a solution containing GOX and Glc at concentrations similar to those measured in OSSe significantly increased free SA levels (ANOVA; F4,15 = 2.269; P < 0.05; Fig. 4A). No significant increases in SA levels were observed when leaves were treated with GOX or Glc separately, demonstrating that it was GOX activity, and likely elevated H2O2 levels, that were responsible for the SA burst. Boiling OSSe to denature its proteins inactivated its ability to elicit SA accumulation (Fig. 4B). Interestingly, boiled OSSe elicited higher JA levels than did unboiled OSSe or even OSMs (ANOVA; F4,11 = 27.435; P < 0.05; Fig. 4C), suggesting that it is the GOX activity of OSSe that suppresses the JA burst when wounds encounter OSSe during beet armyworm attack. In contrast, boiling OSMs, which contains low GOX activity levels (Supplemental Fig. S2), did not alter the JA-eliciting activity of OSMs.

Figure 4.

Figure 4.

Open in a new tab

GOX elicits SA production. A, Mean ± se (n = 5) levels of free SA in wild-type N. attenuata leaves of untreated plants (c) or plants that were wounded and subsequently treated with water (w), Glc, and GOX (g + GOX), Glc (g), or GOX alone. B, Mean ± se (n = 5) levels of SA are shown of plants that were wounded and subsequently treated with water (w), tobacco hornworm OS (OSMs), beet armyworm OS (OSSe), or boiled beet armyworm OS (OSSeb). Control plants (c) remained untreated. C, Values are the mean ± se (n = 5) JA levels in N. attenuata leaves that were treated the same way as in B. Different letters indicate significant differences among treatments (ANOVA; P < 0.05). FW, Fresh weight.

GOX catalyzes the oxidation of d-Glc, resulting in the concomitant production of d-gluconic acid and H2O2. Consistent with this mechanism, we found that H2O2 concentrations were higher in OSSe than in OSMs. Moreover, we measured H2O2 concentrations in boiled OSMs and OSSe and found that boiling reduced H2O2 levels in OSSe to levels found in OSMs. When GOX or Glc alone was added to OSMs, there was also no increase in H2O2 levels, suggesting that both Glc and GOX are at levels too low in OSMs to significantly increase H2O2 levels (ANOVA; F5,12 = 30.406; P < 0.05; Fig. 5). Supplementing the OS of both species with Glc and GOX resulted in similarly high levels of H2O2 in the OS (ANOVA; F2,6 = 2.645; P > 0.05; Supplemental Fig. S4), demonstrating that H2O2 scavenging is not the explanation for the low H2O2 levels in OSMs.

Figure 5.

Figure 5.

Open in a new tab

H2O2 levels in fresh or boiled OS of tobacco hornworm and beet armyworm larvae: Glc (g) or GOX supplementation to OSMs does not increase levels of H2O2. Values are concentrations of H2O2 in OSMs and OSSe. OS samples include three pooled samples each of the OS from 30 different third- to fifth-instar caterpillars of each species actively feeding on wild-type N. attenuata plants. Supplementing the OS of both species with Glc plus GOX produces similar high levels of H2O2 (Supplemental Fig. S4), demonstrating that H2O2 scavenging is not the explanation for the low levels in OSMs.

JA Suppresses the FAC-Specific SA Burst

JA and SA have long been thought of as antagonists even though more subtlety in their interplay has been recently described (Mur et al., 2006). To determine whether the OSMs- and FAC-elicited JA bursts antagonize SA accumulation during tobacco hornworm feeding, we used plants genetically silenced for lipoxygenase3 (as-lox3), a key JA biosynthetic gene (Fig. 6). As previously reported (Halitschke and Baldwin, 2003), FAC-elicited as-lox3 plants accumulated 4-fold less JA than did similarly treated wild-type plants (ANOVA; F4,15 = 6.162; P < 0.05; Fig. 6B). Concomitantly, SA levels in FAC-elicited as-lox3 plants were significantly elevated, which was not observed in wild-type plants (ANOVA; F5,24 = 3.118; P < 0.05; Fig. 6A). These results demonstrate that while FACs do not elicit an SA burst in wild-type plants, they do in plants with silenced JA signaling.

Figure 6.

Figure 6.

Open in a new tab

JA signaling suppresses FAC-elicited SA elicitation. A, Mean ± se (n = 5) levels of SA (ng g fresh weight [FW]−1) in wild-type (WT, black) and transformed N. attenuata plants silenced for JA production (as-lox, gray) of untreated plants (time point zero) or plants that were wounded and subsequently treated with the two most abundant FACs (linolenic acid-Gln and linolenic acid-Glu) found in OSMs (FAC, black symbols, solid lines) or with the triton detergent containing solution (tri, white symbols, dotted lines) used in the FAC treatment. Leaves were harvested 10, 30, 45, 60, 90, and 120 min after the treatments. B, Mean ± se (n = 5) JA levels (ng g FW−1) of wounded N. attenuata wild-type (black) and as-lox (gray) plants 10, 30, 45, 60, 90, and 120 min after immediately treating wounds with FACs (FAC, black symbols, solid lines) or the triton detergent (tri, white symbols, dotted lines). JA levels of untreated plants are shown as zero time points. Asterisks indicate significant differences among the treatments at the individual time points (* = P < 0.05; ** = P < 0.01; *** = P < 0.001).

GOX Suppresses the FAC-Specific ET Burst

We next examined the effect of GOX activity on ET emission. Boiling OSMs did not alter the well-described FAC-elicited ET burst, which is not surprising given that boiling does not denature the FACs that elicit the ET burst (von Dahl et al., 2007). However, supplementing OSMs with GOX decreased ET emissions by 25% compared to supplementing with OSMs alone. In addition, plants treated with OSSe elicited 33% more ET after the OS were boiled (ANOVA; F5,17 = 15.391; P < 0.05; Fig. 7A), which suggested that GOX activity antagonizes ET production. Wound-induced levels of ET were not statistically altered by treating leaves with GOX and Glc. Rather, the FAC-elicited ET burst was reduced by more than one-third when GOX and Glc were supplemented with the FAC solution, which is consistent with the hypothesis that GOX activity is a negative regulator of ET production in N. attenuata (ANOVA; F4,14 = 18.616; P < 0.05; Fig. 7B).

Figure 7.

Figure 7.

Open in a new tab

GOX suppresses OSMS and FAC-elicited ET emission. A, Values are the mean ± se (n = 5) ET levels (nL h−1 g fresh weight [FW]−1) in N. attenuata leaves of untreated plants (c) or plants that were wounded and subsequently treated with tobacco hornworm OS (OSMs), boiled tobacco hornworm OS (OSMsb), tobacco hornworm OS supplemented with GOX, beet armyworm OS (OSSe), or boiled beet armyworm OS (OSSeb). B, Mean ± se (n = 5) ET values are shown of plants that were wounded and subsequently treated with the triton detergent-containing solution used in the FAC treatment (tri), FACs (FAC), FACs plus Glc plus GOX (FAC +g + GOX), or Glc plus GOX (g + GOX) alone. Different letters indicate significant differences between treatments (ANOVA; P < 0.05).

ET Suppresses the OSMs-Elicited SA Burst

To further explore the role of ET signaling in modulating the SA burst, we measured the OSMs-elicited SA bursts in transgenic N. attenuata plants rendered ET insensitive by the ectopic expression of the mutant etr1-1 receptor of Arabidopsis (35s-etr1b), as well as in plants impaired in their ability to produce ET (ir-aco). OSMs elicitation resulted in a 3-fold higher SA burst in these two genetic backgrounds compared to untransformed plants (Fig. 8A). Treating wounded wild-type leaves of N. attenuata with ethephone, an ET releaser, reduced the SA burst to 30% of that elicited by wounding and water treatments (ANOVA; F2,10 = 4.891; P < 0.05; Fig. 8B). Moreover, the OSMs-elicited SA burst increased by 25% when wild-type plants were rendered ET insensitive by a prior overnight exposure to 1-methylcyclopropane (1-MCP), an ET receptor antagonist (ANOVA; F4,19 = 8.388; P < 0.05). Nonwounded plants and plants that were wounded following a 1-MCP treatment did not differ in their SA levels from 1-MCP-free plants (Supplemental Fig. S5).

Figure 8.

Figure 8.

Open in a new tab

ET signaling suppresses OS-elicited SA accumulation. A, Values are mean ± se (n = 5) free SA levels in wild-type (WT) and transgenic N. attenuata plants impaired in their ability to perceive (35s-etr1b) or to produce (ir-aco) ET 120 min after wounding and treating the wounds either with water (w) or with tobacco hornworm OS (OSMs). Control plants (c) remained untreated. B, Mean ± se (n = 5) free SA levels in wild-type N. attenuata plants 120 min after wounding and treating the wounds either with buffer, oral secretions of tobacco hornworm (OSMs), or ethephone (etp). Also shown are wild-type plants that were previously exposed to 1-MCP (MCP + OSMs) to block their ET receptors 120 min after OSMs elicitation. Control plants remained untreated. 1-MCP treatment did not influence SA levels of control or wounded plants (see Supplemental Fig. S3). Different letters indicate significant differences among treatments (ANOVA; P < 0.05). Asterisks indicate significant differences among the genotypes within a treatment (** = P < 0.01; *** = P < 0.001). FW, Fresh weight.

DISCUSSION

In response to herbivore and pathogen attack, plants activate not one, but many, signal cascades; these in turn recruit a suite of defenses. The specificity of the defense response elicited against a particular attacker is in part tailored by the cross-communication among these signal transduction pathways (Glazebrook, 2005; Koornneef and Pieterse, 2008; Leon-Reyes et al., 2009). Biotic attackers, to borrow from Shakespeare, “come not [as] single spies, but in battalions,” and many herbivores function as Trojan horses, vectoring and inoculating plants with pathogens during feeding. Given the high probability that many homopteran insects, such as aphids and whiteflies, transmit disease-causing viruses and pathogens, it is not surprising that plants activate SA signaling in response to their attack (Zarate et al., 2007). Moreover, there is the distinct possibility that certain herbivores deliberately elicit SA responses in plants to suppress effective JA-dependent defenses (Moran et al., 2002; Kaloshian and Walling, 2005; Pegadaraju et al., 2005; De Vos et al., 2006).

Here, we show that when beet armyworm larvae attack wild-type plants, they elicit a 5-fold larger SA burst than when tobacco hornworm larvae attack (Fig. 1A). The response to beet armyworm attack also differs from the response to tobacco hornworm attack in that no ET and lower JA bursts are elicited (Fig. 1, B and C). Remarkably, these differences in phytohormone responses are faithfully mimicked when puncture wounds in leaves are treated with either OSMs or OSSe (Fig. 2).

GOX has been implicated as a potential key mechanism used by caterpillars to counteract induced plant defenses by interfering with JA-dependent signaling (Musser et al., 2002). GOX activity and its reaction product, H2O2, were 2.5-fold higher in OSSe in comparison to OSMs (Supplemental Figs. S3 and S5). When Glc and GOX were applied to puncture wounds at concentrations found in OSSe, SA levels increased significantly in comparison to those in wound- and water-treated plants (Fig. 4A). In addition, when OSSe was boiled to inactivate GOX and its associated H2O2 production (Fig. 5), SA levels decreased to levels found in plants treated with water and OSMs (Fig. 4B). Because FACs do not elicit SA in wild-type plants (Supplemental Fig. S1) and are not degraded by boiling (Roda et al., 2004), and because the addition of GOX and Glc (but neither alone) to boiled OSSe or OSMs produces similarly elevated H2O2 levels (Supplemental Fig. S4), we conclude that the amount of active GOX found in OSSe is sufficient to account for the SA burst either directly or via the production of H2O2. Interestingly, because GOX and Glc-supplemented FAC solutions elicited attenuated ET and JA bursts compared to pure FAC solutions (Fig. 7B), the GOX activity of OSSe can also account for the lower JA bursts and the lack of ET bursts elicited by OSSe. In summary, we propose that the elevated GOX activity of OSSe is sufficient to account for observed changes in the classical FAC-elicited phytohormone response, but additional work needs to be done to completely exclude a potential role of the different FAC profiles of the OS.

Whereas the elevated GOX activity of OSSe can account for all of the observed differences in elicited phytohormone responses, the different FAC profiles found in the two OSs (Fig. 3) may also play a role in tailoring the phytohormone responses. The two most abundant FACs in OSMs (linolenic acid- and linoleic acid-Gln conjugates) occurred at low concentrations in OSSe and the two other highly abundant OSMs FACs (linolenic acid- and linoleic acid-Glu conjugates) were not detected. In addition, one FAC (palmitoyl acid-Gln conjugate) was more abundant in OSSe than in OSMs. These differences in the FAC profiles may contribute to the different ET bursts observed in OSSe- and OSMs-treated plants. OSSe contained only trace quantities of volicitin (8% of total conjugates), which is consistent with the results of Pohnert et al. (1999); they found that beet armyworm larvae reared on lima bean (Phaseolus lunatus) produced only minor amounts of volicitin in their OS and large quantities of N-palmitoyl-l-Gln, N-linoleoyl-l-Gln, and N-linolenoyl-l-Gln. These results suggest that the FAC composition of OSSe is highly diet dependent. Our results also differ from those of Halitschke et al. (2001), which did not detect volicitin in OSMs, but, given that volicitin is a minor constituent compared to N-linolenoyl-l-Glu, N-linoleoyl-l-Glu, N-palmitoyl-l-Glu, and N-linolenoyl-l-Gln, we propose that it plays a minor role. It is important to emphasize that, whereas the two most abundant FACs of OSMs are not elicitors of the SA burst in wild-type plants, they are effective elicitors in JA-deficient plants (Fig. 6A). This difference underscores that it is not the elicitor alone that determines the phytohormone response, but the interactions of the elicitor with the plants' endogenous signal interactions that determine which phytohormone accumulates.

Salivary GOX is commonly found among different caterpillar species, but its activity is highly variable. For example, high activity is reported in salivary homogenates of beet armyworm caterpillars and the Mamestra configurata (Bertha armyworm), but not in the true armyworm (Pseudaletia unipuncta), or the specialist alfalfa butterfly (Colias eurytheme; Merkx-Jaques and Bede, 2004). Salivary GOX activity originates predominantly from the labial glands, with <11% of total activity detected in mandibular glands and only minor amounts from the hemolymph (Eichenseer et al., 1999). H. zea larvae reared on different host plants produce varying amounts of GOX in their labial glands (Peiffer and Felton, 2005). GOX has been shown to play important antimicrobial roles in H. zea (Musser et al., 2005) and honeybees are known to secrete GOX into honey, which suppresses bacterial growth (White and Subers, 1963). The antimicrobial role of GOX in insects may have facilitated their amplification and use in eliciting plant pathogen defenses, perhaps as a means of counteracting JA-mediated defenses.

Recently, Leon-Reyes et al. (2009) showed that in Arabidopsis, ET modulates NPR1's mediation of SA-JA cross-signaling. Whereas the NPR1 homolog of N. attenuata plays the opposite role in mediating SA signaling than it plays in Arabidopsis (Rayapuram and Baldwin, 2007), we show here that ET signaling plays a similar role to the role it plays in Arabidopsis: that of suppressing the SA response. Rayapuram and Baldwin (2007) showed that silencing Na-NPR1 did not affect the amount of ET released after Bertha armyworm attack or after OSSe elicitation in the leaves of ir-npr1 glasshouse-grown plants and that silencing Na-NPR1 reduced JA levels, but increased SA in field-grown OSMs-elicited plants, which reveals JA-SA, but not ET-SA antagonism. We show that in OSMs-elicited transgenic 35s-etr1b and ir-aco plants, which are impaired in ET perception or production, as well as in OSMs-elicited wild-type plants pretreated with a competitive inhibitor of ET receptors, 1-MCP, SA accumulates up to 3-fold more than in OSMs-elicited wild-type plants (Fig. 8; Supplemental Fig. S3). Both 35s-etr1b plants and 1-MCP-treated wild-type plants are impaired in their ability to perceive ET and are known to synthesize more ET in response to OSMs elicitation than do wild-type plants, whereas ir-aco plants are impaired in the production of the elicited ET burst (von Dahl et al., 2007). These results suggest that residual GOX activity of OSMs (Supplemental Fig. S3) is sufficient to elicit an SA burst as long as the ET burst that is normally elicited by OSMs is either impaired or not perceived. Little is known about GOX activity in tobacco hornworm larval OS. GOX-derived H2O2 plays only a minor role in the elicitation of Manduca spp.-specific plant responses and genes coding enzymes of the JA-signaling cascade are down-regulated in response to H2O2 treatment (Halitschke and Baldwin, 2003). Together with the low levels of GOX activity in OSMs reported here, these results underscore that tobacco hornworm attack elicits predominately JA-based signaling, which is in turn mediated by the FACs in larval OS.

Together, these observations suggest that SA-ET as well as SA-JA cross-signaling is responsible for tuning the responses of N. attenuata plants to the FAC- and GOX-based elicitors of their lepidopteran herbivores (Fig. 9). These differences in the elicited response profiles of the plant can be useful for either the plant or the herbivore, depending on their strategies. The results presented here are consistent with the hypothesis that generalist herbivores, such as beet armyworm, may enhance their fitness by activating the SA pathway concomitantly with the JA pathway so as to weaken JA-mediated resistance (Stotz et al., 2002; Cipollini et al., 2004; Zarate et al., 2007). However, much additional work is required to understand the consequences of this signaling cross-talk for N. attenuata's defense physiology. These results also underscore the value of using transgenic plants impaired in specific parts of their signaling cascades to understand the complex signaling interactions between herbivores and their hosts.

Figure 9.

Figure 9.

Open in a new tab

Model of OS-elicited JA-ET-SA cross-talk in tobacco hornworm and beet armyworm-attacked N. attenuata leaves. When tobacco hornworm larvae feed on N. attenuata plants, FACs and GOX proteins from the larval OS contaminate wounds, which results in the amplification of levels of JA and ET but not SA. OS from beet armyworm larvae, compared to those from tobacco hornworm larvae, contain less of the FACs known to elicit JA and ET production, but more GOX activity, which, together with Glc (•), results in higher H2O2 and SA accumulations. Increased SA levels are known to antagonize the JA burst and the deployment of JA-dependent defenses (Rayapuram and Baldwin, 2007). During tobacco hornworm feeding, perception of the FAC-induced ET burst through ETR1 suppresses SA production. The herbivore-elicited ET burst, therefore, is an important regulator of herbivory-elicited SA-JA cross-talk. ACS, 1-amino-cyclopropane-1-carboxylic acid-synthase; ACO, 1-amino-cyclopropane-1-carboxylic acid oxidase; LOX3, lipoxygenase 3; AOS, allene-oxide synthase, AOC, allene-oxide cyclase; ICS, isochorismate-synthase; PAL, Phe-ammonia lyase.

MATERIALS AND METHODS

Plant Material and Growing Conditions

Wild-type Nicotiana attenuata Torr. ex S. Watson plants were from an inbred line in its twenty-second generation that originated from seeds collected on the DI ranch in Utah in 1988. Seeds of wild-type and genetically transformed plants were germinated on Gamborg's B5 medium (Duchefa) as described previously (Krügel et al., 2002). The transformed plants used were 35s-etr1b plants (A-03 538–1), ectopically expressing the Arabidopsis (Arabidopsis thaliana) mutant ET receptor etr1-1 under the control of a cauliflower mosaic virus 35S constitutive promoter (von Dahl et al., 2007), as well as the transformed as-lox3 (Halitschke and Baldwin, 2003) and the ir-aco line, silenced in the expression of the ET biosynthesis gene ACO (A03-321–10 as described in von Dahl et al., 2007). Briefly, seeds were sterilized and incubated in 0.1 m GA3 (www.carl-roth.de) and 1:50 diluted liquid smoke (v/v) (House of Herbs) before germination on Gamborg's B5 medium at a 26°C/16 h 155 μm s−1 m−2 light: 24°C/8 h dark cycle (Percival). Plants were grown in the glasshouse with a day/night cycle of 16 h (26°C–28°C)/8h (22°C–24°C) h under supplemental light from Master Sun-T PIA Agro 400 or Master Sun-T PIA Plus 600-W sodium lights (Philips).

Insect Rearing and Feeding Experiments

Tobacco hornworm (Manduca sexta) eggs, purchased from Carolina Biological Supply, were cultured in climate chambers until hatching. Freshly hatched larvae (neonates) were placed onto leaves growing at the +1 nodal position of individual plants in clip cages for feeding experiments. To increase survival rate, beet armyworm (Spodoptera exigua) larvae hatched from eggs supplied by the Plant Protection Centre of Bayer AG were reared on wild-type N. attenuata plants until they reached the second to third instar. One third-instar beet armyworm or two first-instar tobacco hornworm larvae per plant were allowed to feed for 3 d. The number of larvae placed on plants (the two to three tobacco hornworm neonates and the one beet armyworm third-instar larvae) were selected because they produced comparable amounts of damage (data not shown).

For the collection of OS, larvae were reared on N. attenuata wild-type plants until the third to fifth instar. OS was collected on ice as described in Roda et al. (2004).

Plant Treatments

For experiments presented in Figure 3, the three youngest fully expanded leaves (positions +1, +2, and +3) were mechanically wounded with a pattern wheel to produce four puncture rows on each side of the midvein. Fresh wounds were immediately treated with 20 μL of tobacco hornworm OS (diluted 1:1 with water), ethephone (6 mg/mL in 5 mm MES buffer; www.riedeldehaen.de), or 5 mm MES. Control plants remained untreated. To inhibit ET perception, plants were exposed to 1-MCP in a 20-L container for 8 h during the dark phase. Following Kahl et al. (2000), 500 mg of Ethylblock (0.43% 1-MCP; van der Sprong) was weighed into a vial and 10 mL of an alkaline solution (0.75% KOH + NaOH) was added to release 1-MCP.

For all other experiments, one leaf (position +1) was wounded and immediately treated with water, triton—the surfactant used for the dissolution of FACs, FACs (N-linolenoyl-l-Gln and N-linolenoyl-l-Glu at concentrations similar to those found in tobacco hornworm OS). Tobacco hornworm OS (OSMs), beet armyworm OS (OSSe), OS boiled during 10 min at 90°C (OSMsb or OSSeb), OS with 0.01 m Glc (OSMs + g or OSSe + g), OS with 0.5 units GOX (OSMs + GOX; http://www.sigmaaldrich.com), FACs with 0.01 m Glc, and 0.5 units GOX (FAC + g + GOX).

Phytohormone Analysis

Free SA and JA were extracted by homogenizing 200 to 300 mg of leaf material in FastPrep tubes containing 900 mg of lysing matrix (BIO 101; Vista) and 1 mL of ethyl acetate spiked with 200 ng (100 ng during the analysis of caterpillar-attacked leaves) of D4-SA and D4-JA, as internal standards. Samples were homogenized twice by reciprocal shaking at 6.5 m s−1 for 45 s and centrifuged at 13,000 rpm for 20 min at 4°C. Supernatants from two extraction steps were pooled and evaporated until dry. The dried residue was dissolved in 500 μL of 70% of methanol, vortexed, and centrifuged.

SA and JA measurements were conducted on a liquid chromatography-tandem mass spectrometry system (Varian 1200). Fifteen microliters of each sample were injected onto a ProntoSIL column (C18; 5 μm, 50 × 2 mm; Bischoff) attached to a precolumn (C18, 4 × 2mm; Phenomenex). The mobile phase comprised solvent A (0.05% formic acid) and solvent B (0.05% formic acid in acetonitrile) used in a gradient mode (time/concentration [min/%] for B: 0:00/15; 1:30/15; 4:30/98; 12:30/98; 13:30/15; 15:00/15) with a flow of (time/flow [mL/min]: 0:00/0.4; 1:00/0.4; 1:30/0.2; 10:00/0.2; 10:30/0.4; 12:30/0.4; 15:00/0.4). Compounds were detected in the electrospray ionization-negative mode. Molecular ions [M-H](−) at m/z 137 and 209 and 141 and 213 generated from endogenous SA and JA and their internal standards, respectively, were fragmented under 15-V collision energy. The ratios of ion intensities of their respective daughter ions, m/z 93 and 97 and m/z 59 and 63, were used to quantify endogenous SA and JA, respectively.

ET emissions were measured continuously and noninvasively with a photoacoustic spectrometer (INVIVO) as described in von Dahl et al. (2007). The youngest fully expanded leaves of slightly elongated plants were subject to feeding by three neonate tobacco hornworm larvae or one third-instar beet armyworm larvae for 22 h. Leaves were excised at the onset of the experiment and transferred to 250-mL cuvettes and the headspace was allowed to accumulate over the entire feeding period. For experiments requiring mechanical wounding and application of eliciting solutions, leaves were excised directly after treatment and transferred to 250-mL cuvettes, and the headspace allowed to accumulate over a 5-h time period. During measurements, cuvettes were flushed with a flow of purified air at 130 to 150 mL min−1, which had previously passed through a liquid N2 cooling trap to remove CO2 and water.

FAC Analysis

Five microliters of OS were homogenized in 95 μL methanol spiked with 10 ng of N-cis-10-nonadecenoic acid-l-Gln (C19:1-Gln) used as internal standard. Extracts were then centrifuged to remove any particulate matter. Five-microliter aliquots of these solutions were analyzed by liquid chromatography-tandem mass spectrometry using the aforementioned LC settings. Identification of major FACs from tobacco hornworm OS was confirmed by comparison to authentic standards, as described in Halitschke et al. (2001). The structure identified as volicitin (N-17-hydroxylinolenoyl-l-Gln) produced a fragmentation pattern—a dehydrated ion at m/z 404, a doubly dehydrated ion at m/z 378, and a hydroxyl acid-derived ion at m/z 293—identical to that already published by Yoshinaga et al. (2007). Most abundant FACs were detected in the electrospray ionization-negative mode and quantified using parent-ion/daughter-ion selections designed, as follows, from previously published MS/MS spectra by Yoshinaga et al. (2007): 383/145 (N-palmitoyl-l-Gln, C16:0-Gln), 384/255 (N-palmitoyl-l-Glu, C16:0-Glu), 405/145 (N-linolenoyl-l-Gln, C18:3-Gln), 406/277 (N-linolenoyl-l-Glu, C18:3-Glu), 407/145 (N-linoleoyl-l-Gln, C18:2-Gln), 408/279 (N-linoleoyl-l-Glu, C18:2-Glu), 421/145 (volicitin). Collision energy was set to 18.5 V. Fragmentation profiles of FACs containing Gln typically included a common Gln-derived ion.

GOX Assay

GOX activity was measured in OS according to the procedure described in Kelley and Reddy (1988). Briefly, OS were collected from 30 second- to fourth-instar caterpillars. The reaction mixture contained 1.5-mL citrate sodium phosphate buffer (0.1 m; pH 4), 1 mL o-dianisidine (0.31 mm, http://www.sigmaaldrich.com), 0.3 mL d-Glc (1 m), 0.1 mL horseradish peroxidase (60 units/mL, http://www.sigmaaldrich.com). The reaction mixture was incubated in an Ultrospec 3000 (Pharmacia Biotech; http://www4.amershambiosciences.com) spectrophotometer for 5 min to establish a blank activity rate. Aliquots of 0.1 mL of 1:2 diluted OS from tobacco hornworm or beet armyworm were then added to the reaction mixture and the A460 was recorded for 5 min. In parallel, similar incubations were performed by adding a 0.1-mL solution of Aspergillus niger GOX (0.15 units/mL, type VII; Sigma-Aldrich) used as standard. Resulting changes in absorbance were calculated from the initial linear portion of the curve. One activity unit was defined as the amount of enzyme oxidizing 1 μmol o-dianisidine per min at 37°C and pH 4.5.

H2O2 Assays

Concentrations of H2O2 in OS were determined using the procedure described in the Amplex red hydrogen peroxide/peroxidase assay kit (A22188; http://www.invitrogen.com). In the presence of peroxidase, the amplex red reagent (10-acetyl-3,7-dihydroxyphenoxazine) reacts with H2O2 to produce resofurin, a red fluorescent oxidation product. Resofurin has an absorption maximum at 585 nm. For each measurement, 1 μL sample, 49 μL reaction buffer (0.05 m sodium phosphate, pH 7.4), and 50 μL working solution (10 μm Amplex Red reagent and 0.2 units/mL horseradish peroxidase in reaction buffer) were combined in a 96-well microplate under exclusion of light. For the H2O2 standard curve, 50 μL of each standard (concentrations: 0.01 μm, 0.1 μm, 1 μm, and 2 μm) were added to the working solution. Measurements were conducted with a TECAN infinite M200 plate reader (http://www.tecan.de). Fluorescence was measured 30 min after incubation with an excitation of 540 nm and detected at 590 nm. Each measurement was corrected for background fluorescence by subtracting the value derived from a no-H2O2 control.

Statistical Analysis

Data were analyzed with Statview 5.0 (SAS Institute). Data were transformed if they did not meet the assumption of homoschedacity.

Supplemental Data

The following materials are available in the online version of this article.

  • Supplemental Figure S1. FACs are the elicitors of the JA burst but not the elicitors of the SA burst in wild-type plants.

  • Supplemental Figure S2. β-Glucosidase is not the elicitor of the SA burst.

  • Supplemental Figure S3. GOX activity in OSMs and OSSe.

  • Supplemental Figure S4. Fresh OS of tobacco hornworm and beet armyworm do not quench H2O2 production when the OS of either species are supplemented with Glc or GOX.

  • Supplemental Figure S5. Unwounded plants and plants that were wounded following a 1-MCP treatment do not differ in their SA levels from those of 1-MCP-free plants, but 1-MCP treatment of OSMs-elicited plants resulted in significantly elevated SA levels.

Supplementary Material

[Supplemental Data]
pp.109.139550_index.html (1.2KB, html)

Acknowledgments

We thank Dr. Tamara Krügel, Andreas Weber, and Andreas Schünzel for growing the plants in the glasshouse, Dr. Matthias Schöttner and Eva Rothe for invaluable technical assistance, Danny Kessler for providing the insects, Dr. Channabasavangowda Rayapuram for assistance with the ET measurements, the reviewers for improving the manuscript, and Emily Wheeler for editorial assistance.

1

This work was supported by the Max Planck Society.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Ian T. Baldwin (baldwin@ice.mpg.de).

[W]

The online version of this article contains Web-only data.

[OA]

Open Access articles can be viewed online without a subscription.

References

  1. Alborn HT, Turlings TCJ, Jones TH, Stenhagen G, Loughrin JH, Tumlinson JH (1997) An elicitor of plant volatiles from beet armyworm oral secretion. Science 276 945–949 [Google Scholar]
  2. Baldwin IT (1988) The alkaloidal responses of wild tobacco to real and simulated herbivory. Oecologia 77 378–381 [DOI] [PubMed] [Google Scholar]
  3. Baldwin IT, Halitschke R, Kessler A, Schittko U (2001) Merging molecular and ecological approaches in plant-insect interactions. Curr Opin Plant Biol 4 351–358 [DOI] [PubMed] [Google Scholar]
  4. Baldwin IT, Zhang ZP, Diab N, Ohnmeiss TE, McCloud ES, Lynds GY, Schmelz EA (1997) Quantification, correlations and manipulations of wound-induced changes in jasmonic acid and nicotine in Nicotiana sylvestris. Planta 201 397–404 [Google Scholar]
  5. Cipollini D, Enright S, Traw MB, Bergelson J (2004) Salicylic acid inhibits jasmonic acid-induced resistance of Arabidopsis thaliana to Spodoptera exigua. Mol Ecol 13 1643–1653 [DOI] [PubMed] [Google Scholar]
  6. De Vos M, Van Zaanen W, Koornneef A, Korzelius JP, Dicke M, Van Loon LC, Pieterse CMJ (2006) Herbivore-induced resistance against microbial pathogens in Arabidopsis. Plant Physiol 142 352–363 [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Doares SH, Narvaezvasquez J, Conconi A, Ryan CA (1995) Salicylic-acid inhibits synthesis of proteinase-inhibitors in tomato leaves induced by systemin and jasmonic acid. Plant Physiol 108 1741–1746 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Doherty HM, Selvendran RR, Bowles DJ (1988) The wound response of tomato plants can be inhibited by aspirin and related hydroxybenzoic acids. Physiol Mol Plant Pathol 33 377–384 [Google Scholar]
  9. Eichenseer H, Mathews MC, Bi JL, Murphy JB, Felton GW (1999) Salivary glucose oxidase: multifunctional roles for Helicoverpa zea? Arch Insect Biochem Physiol 42 99–109 [DOI] [PubMed] [Google Scholar]
  10. Gaquerel E, Weinhold A, Baldwin IT (2009) Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata. VIII. An unbiased GCxGC-ToFMS analysis of the plant#s elicited volatile emissions. Plant Physiol 149 1408–1423 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Giri AP, Wuensche H, Mitra S, Zavala JA, Muck A, Svatos A, Baldwin IT (2006) Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata. VII. Changes in the plant's proteome. Plant Physiol 142 1621–1641 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Glazebrook J (2005) Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens. Annu Rev Phytopathol 43 205–227 [DOI] [PubMed] [Google Scholar]
  13. Gupta V, Willits MG, Glazebrook J (2000) Arabidopsis thaliana EDS4 contributes to salicylic acid (SA)-dependent expression of defense responses: evidence for inhibition of jasmonic acid signaling by SA. Mol Plant Microbe Interact 13 503–511 [DOI] [PubMed] [Google Scholar]
  14. Halitschke R, Baldwin IT (2003) Antisense LOX expression increases herbivore performance by decreasing defense responses and inhibiting growth-related transcriptional reorganization in Nicotiana attenuata. Plant J 36 794–807 [DOI] [PubMed] [Google Scholar]
  15. Halitschke R, Baldwin IT (2005) Jasmonates and related compounds in plant-insect interactions. J Plant Growth Regul 23 238–245 [Google Scholar]
  16. Halitschke R, Gase K, Hui D, Schmidt DD, Baldwin IT (2003) Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata. VI. Microarray analysis reveals that most herbivore-specific transcriptional changes are mediated by fatty acid-amino acid conjugates. Plant Physiol 131 1894–1902 [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Halitschke R, Schittko U, Pohnert G, Boland W, Baldwin IT (2001) 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 herbivores. Plant Physiol 125 711–717 [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Halitschke R, Ziegler J, Keinanen M, Baldwin IT (2004) Silencing of hydroperoxide lyase and allene oxide synthase reveals substrate and defense signaling crosstalk in Nicotiana attenuata. Plant J 40 35–46 [DOI] [PubMed] [Google Scholar]
  19. Kahl J, Siemens DH, Aerts RJ, Gäbler R, Kühnemann F, Preston CA, Baldwin IT (2000) Herbivore-induced ethylene suppresses a direct defense but not a putative indirect defense against an adapted herbivore. Planta 210 336–342 [DOI] [PubMed] [Google Scholar]
  20. Kaloshian I, Walling LL (2005) Hemipterans as plant pathogens. Annu Rev Phytopathol 43 491–521 [DOI] [PubMed] [Google Scholar]
  21. Kelley RL, Reddy CA (1988) Glucose oxidase of Phanerochaete chrysoporium. Methods Enzymol 161 307–316 [DOI] [PubMed] [Google Scholar]
  22. Kloek AP, Verbsky ML, Sharma SB, Schoelz JE, Vogel J, Klessig DF, Kunkel BN (2001) Resistance to Pseudomonas syringae conferred by an Arabidopsis thaliana coronatine-insensitive (coi1) mutation occurs through two distinct mechanisms. Plant J 26 509–522 [DOI] [PubMed] [Google Scholar]
  23. Koornneef A, Pieterse CMJ (2008) Cross-talk in defense signaling. Plant Physiol 146 839–844 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Krügel T, Lim M, Gase K, Halitschke R, Baldwin IT (2002) Agrobacterium-mediated transformation of Nicotiana attenuata, a model ecological expression system. J Chem Ecol 12 177–183 [Google Scholar]
  25. Leon-Reyes A, Spoel SH, De Lange ES, Abe H, Kobayashi M, Tsuda S, Millenaar FF, Welschen RAM, Ritsema T, Pieterse CMJ (2009) Ethylene modulates the role of nonexpressor of pathogenesis-related genes1 in cross-talk between salicylate and jasmonate signaling. Plant Physiol 149 1797–1809 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Li J, Brader G, Palva ET (2004) The WRKY70 transcription factor: a node of convergence for jasmonate-mediated and salicylate-mediated signals in plant defense. Plant Cell 16 319–331 [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Maffei M, Bossi S, Spiteller D, Mithofer A, Boland W (2004) Effects of feeding Spodoptera littoralis on lima bean leaves. I. Membrane potentials, intracellular calcium variations, oral secretions, and regurgitate components. Plant Physiol 134 1752–1762 [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mattiacci L, Dicke M, Posthumus MA (1995) β-Glucosidase: an elicitor of herbivore-induced plant odor that attracts host-searching parasitic wasps. Proc Natl Acad Sci USA 92 2036–2040 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Merkx-Jaques M, Bede JC (2004) Caterpillar salivary enzymes: “eliciting” a response. Phytoprotection 85 33–37 [Google Scholar]
  30. Mithofer A, Boland W (2008) Recognition of herbivory-associated molecular patterns, HAMPs. Plant Physiol 146 825–831 [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Moran PJ, Cheng YF, Cassell JL, Thompson GA (2002) Gene expression profiling of Arabidopsis thaliana in compatible plant-aphid interactions. Arch Insect Biochem Physiol 51 182–203 [DOI] [PubMed] [Google Scholar]
  32. Mori N, Alborn HT, Teal PE, Tumlinson JH (2001) Enzymatic decomposition of elicitors of plant volatiles in Heliothis virescens and Helicoverpa zea. J Insect Physiol 47 749–757 [DOI] [PubMed] [Google Scholar]
  33. Mur LAJ, Kenton P, Atzorn R, Miersch O, Wasternack C (2006) The outcomes of concentration-specific interactions between salicylate and jasmonate signaling include synergy, antagonism, and oxidative stress leading to cell death. Plant Physiol 140 249–262 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Musser RO, Cipollini DF, Hum-Musser SM, Williams SA, Brown JK, Felton GW (2005) Evidence that the caterpillar salivary enzyme glucose oxidase provides herbivore offense in solanaceous plants. Arch Insect Biochem Physiol 58 128–137 [DOI] [PubMed] [Google Scholar]
  35. Musser RO, Hum-Musser SM, Eichenseer H, Peiffer M, Ervin G, Murphy JB, Felton GW (2002) Caterpillar saliva beats plant defences. Nature 416 599–600 [DOI] [PubMed] [Google Scholar]
  36. Niki T, Mitsuhara I, Seo S, Ohtsubo N, Ohashi Y (1998) Antagonistic effect of salicylic acid and jasmonic acid on the expression of pathogenesis-related (PR) protein genes in wounded mature tobacco leaves. Plant Cell Physiol 39 500–507 [Google Scholar]
  37. O'Donnell PJ, Calvert C, Atzorn R, Wasternack C, Leyser HMO, Bowles DJ (1996) Ethylene as a signal mediating the wound response of tomato plants. Science 274 1914–1917 [DOI] [PubMed] [Google Scholar]
  38. Pandey SP, Shahi P, Gase K, Baldwin IT (2008) Herbivory-induced changes in the small-RNA transcriptome and phytohormone signaling in Nicotiana attenuata. Proc Natl Acad Sci USA 105 4559–4564 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Pegadaraju V, Knepper C, Reese J, Shah J (2005) Premature leaf senescence modulated by the Arabidopsis phytoalexin deficient4 gene is associated with defense against the phloem-feeding green peach aphid. Plant Physiol 139 1927–1934 [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Peiffer M, Felton GW (2005) The host plant as a factor in the synthesis and secretion of salivary glucose oxidase in larval Helicoverpa zea. Arch Insect Biochem Physiol 58 106–113 [DOI] [PubMed] [Google Scholar]
  41. Peiffer M, Felton GW (2009) Do caterpillars secrete “oral secretions”? J Chem Ecol 35 326–335 [DOI] [PubMed] [Google Scholar]
  42. Péna-Cortes H, Albrecht T, Prat S, Weiler EW, Willmitzer L (1993) Aspirin prevents wound-induced gene-expression in tomato leaves by blocking jasmonic acid biosynthesis. Planta 191 123–128 [Google Scholar]
  43. Pieterse CMJ, van Wees SCM, van Pelt JA, Knoester M, Laan R, Gerrits N, Weisbeek PJ, van Loon LC (1998) A novel signaling pathway controlling induced systemic resistance in Arabidopsis. Plant Cell 10 1571–1580 [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Pohnert G, Jung V, Haukioja E, Lempa K, Boland W (1999) New fatty acid amides from regurgitant of lepidopteran (Noctuidae, Geometridae) caterpillars. Tetrahedron 55 11275–11280 [Google Scholar]
  45. Potlakayala SD, Reed DW, Covello PS, Fobert PR (2007) Systemic acquired resistance in canola is linked with pathogenesis-related gene expression and requires salicylic acid. Phytopathology 97 794–802 [DOI] [PubMed] [Google Scholar]
  46. Rayapuram C, Baldwin IT (2007) Increased SA in NPR1-silenced plants antagonizes JA and JA-dependent direct and indirect defenses in herbivore-attacked Nicotiana attenuata in nature. Plant J 52 700–715 [DOI] [PubMed] [Google Scholar]
  47. Reymond P, Farmer EE (1998) Jasmonate and salicylate as global signals for defense gene expression. Curr Opin Plant Biol 1 404–411 [DOI] [PubMed] [Google Scholar]
  48. Roda A, Halitschke R, Steppuhn A, Baldwin IT (2004) Individual variability in herbivore-specific elicitors from the plant's perspective. Mol Ecol 13 2421–2433 [DOI] [PubMed] [Google Scholar]
  49. Ross AF (1961) Systemic acquired resistance induced by localized virus infections in plants. Virology 14 340–358 [DOI] [PubMed] [Google Scholar]
  50. Ryals JA, Neuenschwander UH, Willits MG, Molina A, Steiner HY, Hunt MD (1996) Systemic acquired resistance. Plant Cell Environ 8 1809–1819 [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Schittko U, Preston CA, Baldwin IT (2000) Eating the evidence? Manduca sexta larvae cannot disrupt specific jasmonate induction in Nicotiana attenuata by rapid consumption. Planta 210 343–346 [DOI] [PubMed] [Google Scholar]
  52. Schmelz EA, Carroll MJ, LeClere S, Phipps SM, Meredith J, Chourey PS, Alborn HT, Teal PEA (2006) Fragments of ATP synthase mediate plant perception of insect attack. Proc Natl Acad Sci USA 103 8894–8899 [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Schmelz EA, LeClere S, Carroll MJ, Alborn HT, Teal PE (2007) Cowpea chloroplastic ATP synthase is the source of multiple plant defense elicitors during insect herbivory. Plant Physiol 144 793–805 [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Spiteller D, Boland W (2003) N-(15,16-dihydroxylinoleoyl)-glutamine and N-(15,16-epoxylinoleoyl)-glutamine isolated from oral secretions of lepidopteran larvae. Tetrahedron 59 135–139 [Google Scholar]
  55. Steppuhn A, Gase K, Krock B, Halitschke R, Baldwin IT (2004) Nicotine's defensive function in nature. PLoS Biol 2 1074–1080 [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Stotz HU, Koch T, Biedermann A, Weniger K, Boland W, Mitchell-Olds T (2002) Evidence for regulation of resistance in Arabidopsis to Egyptian cotton worm by salicylic and jasmonic acid signaling pathways. Planta 214 648–652 [DOI] [PubMed] [Google Scholar]
  57. Stout MJ, Fidantsef AL, Duffey SS, Bostock RM (1999) Signal interactions in pathogen and insect attack: systemic plant-mediated interactions between pathogens and herbivores of the tomato, Lycopersicon esculentum. Physiol Mol Plant Pathol 54 115–130 [Google Scholar]
  58. Thomma B, Tierens KFM, Penninckx I, Mauch-Mani B, Broekaert WF, Cammue BPA (2001) Different micro-organisms differentially induce Arabidopsis disease response pathways. Plant Physiol Biochem 39 673–680 [Google Scholar]
  59. van Loon LC, Bakker P, Pieterse CMJ (1998) Systemic resistance induced by rhizosphere bacteria. Annu Rev Phytopathol 36 453–483 [DOI] [PubMed] [Google Scholar]
  60. Vijayan P, Shockey J, Levesque CA, Cook RJ, Browse J (1998) A role for jasmonate in pathogen defense of Arabidopsis. Proc Natl Acad Sci USA 95 7209–7214 [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Voelckel C, Baldwin IT (2004) Generalist and specialist lepidopteran larvae elicit different transcriptional responses in Nicotiana attenuata, which correlate with larval FAC profiles. Ecol Lett 7 770–775 [Google Scholar]
  62. von Dahl CC, Baldwin IT (2007) Deciphering the role of ethylene in plant-herbivore interactions. J Plant Growth Regul 26 201–209 [Google Scholar]
  63. von Dahl CC, Winz R, Halitschke R, Kühnemann F, Gase K, Baldwin IT (2007) Tuning the herbivore-induced ethylene burst: the role of transcripts accumulation and ethylene perception in Nicotiana attenuata. Plant J 51 293–307 [DOI] [PubMed] [Google Scholar]
  64. White JW, Subers MH (1963) Studies on honey inhibine 2. A chemical assay. J Apic Res 2 93–100 [Google Scholar]
  65. Winz RA, Baldwin IT (2001) 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 125 2189–2202 [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Wu J, Hettenhausen C, Meldau S, Baldwin IT (2007) Herbivory rapidly activates MAPK signaling in attacked and unattacked leaf regions but not between leaves of Nicotiana attenuata. Plant Cell 19 1096–1122 [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Yoshinaga N, Aboshi T, Ishikawa C, Fukui M, Shimoda M, Nishida R, Lait CG, Tumlinson JH, Mori N (2007) Fatty acid amides, previously identified in caterpillars, found in the cricket Teleogryllus taiwanemma and fruit fly Drosophila melanogaster larvae. J Chem Ecol 33 1376–1381 [DOI] [PubMed] [Google Scholar]
  68. Zarate SI, Kempema LA, Walling LL (2007) Silverleaf whitefly induces salicylic acid defenses and suppresses effectual jasmonic acid defenses. Plant Physiol 143 866–875 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

[Supplemental Data]
pp.109.139550_index.html (1.2KB, html)
pp.109.139550_139550Figure_S1.doc (33.1KB, doc)
pp.109.139550_139550Figure_S2.doc (24.6KB, doc)
pp.109.139550_139550Figure_S3.doc (20.3KB, doc)
pp.109.139550_139550Figure_S4.doc (27.3KB, doc)
pp.109.139550_139550Figure_S5.doc (32.8KB, doc)

Articles from Plant Physiology are provided here courtesy of Oxford University Press

RESOURCES