Different lepidopteran elicitors account for cross-talk in herbivory-induced phytohormone signaling

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 (OS(Se) and OS(Ms)) are added to puncture wounds. Fatty acid-amino acid conjugates elicit the JA and ET bursts, but not the SA burst. OS(Se) had enhanced glucose oxidase activity (but not beta-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 OS(Ms)-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 OS(Ms)-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.

Figure 1.

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.

Figure 2.

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 (OS_Ms, gray bars), or beet armyworm OS (OS_Se, 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), OS_Ms (gray bar), or OS_Se (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.

Figure 3.

FAC profiles in tobacco hornworm OS (OS_Ms) and beet armyworm OS (OS_Se). Values are relative concentrations of the most abundant FACs in OS_Ms (black bars) and OS_Se (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.

Figure 4.

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 (OS_Ms), beet armyworm OS (OS_Se), or boiled beet armyworm OS (OS_Seb). 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.

Figure 5.

H₂O₂ levels in fresh or boiled OS of tobacco hornworm and beet armyworm larvae: Glc (g) or GOX supplementation to OS_Ms does not increase levels of H₂O₂. Values are concentrations of H₂O₂ in OS_Ms and OS_Se. 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 OS_Ms.

Figure 6.

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 OS_Ms (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).

Figure 7.

GOX suppresses OS_MS 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 (OS_Ms), boiled tobacco hornworm OS (OS_Msb), tobacco hornworm OS supplemented with GOX, beet armyworm OS (OS_Se), or boiled beet armyworm OS (OS_Seb). 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).

Figure 8.

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 (OS_Ms). 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 (OS_Ms), or ethephone (etp). Also shown are wild-type plants that were previously exposed to 1-MCP (MCP + OS_Ms) to block their ET receptors 120 min after OS_Ms 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.

Figure 9.

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.