Nicotiana attenuata MPK4 suppresses a novel jasmonic acid (JA) signaling-independent defense pathway against the specialist insect Manduca sexta, but is not required for the resistance to the generalist Spodoptera littoralis

Authors


Summary

  • How plants tailor their defense responses to attack from different insects remains largely unknown. Here, we studied the role of a mitogen-activated protein kinase (MAPK), MPK4, in the resistance of a wild tobacco Nicotiana attenuata to two herbivores, the specialist Manduca sexta and the generalist Spodoptera littoralis.
  • Stably transformed N. attenuata plants silenced in MPK4 (irMPK4) were generated and characterized for traits important for defense against herbivores.
  • Only the oral secretions (OS) from M. sexta, but not the OS from S. littoralis or mechanical wounding, induced elevated levels of jasmonic acid (JA) in irMPK4 plants relative to the wild-type plants. Moreover, silencing of MPK4 strongly increased the resistance of N. attenuata to M. sexta in a fashion that was independent of COI1 (CORONATINE INSENSITIVE1)-mediated JA signaling. Untargeted metabolomic screening identified several new MPK4-dependent putative defensive compounds against M. sexta. By contrast, silencing of MPK4 did not affect the growth of the generalist insect S. littoralis, and we propose that this was because of the very low levels of fatty acid–amino acid conjugates (FACs) in S. littoralis OS.
  • Thus, MPK4 is likely to be a key signaling element that enables plants to tailor defense responses to different attackers.

Introduction

Plants are challenged by numerous environmental stresses during all stages of their life cycles. Accordingly, they have evolved sophisticated signaling networks to cope with these challenges. These signaling networks quickly convert the extracellular stress stimuli into intracellular defense responses (Jones & Dangl, 2006; Howe & Jander, 2008; Wu & Baldwin, 2009, 2010). Among these signaling systems, the mitogen-activated protein kinase (MAPK) cascades play essential roles (Ichimura et al., 2000; Asai et al., 2002; Teige et al., 2004; Meszaros et al., 2006; Brader et al., 2007). Typically, a MAPK cascade consists of a three-kinase module that is conserved in all eukaryotes. MAPK, the last kinase in the cascade, is activated by the dual phosphorylation of threonine and tyrosine residues in its kinase catalytic activation loop. This phosphorylation is mediated by a MAPK kinase (MAPKK or MEK), which is activated by a MAPKK kinase (MAPKKK or MEKK). Following activation, MAPKs regulate gene expression by phosphorylation of DNA-binding transcription factors or activate directly certain enzymes (Hill & Treisman, 1995; Karin & Hunter, 1995; Hazzalin & Mahadevan, 2002).

There are c. 20 MAPK genes in the Arabidopsis genome (MAPK Group, 2002). However, only the functions of three MAPKs in Arabidopsis stress responses, especially pathogen-induced responses, have been studied intensively. MPK6 and MPK3 are involved in abiotic stress-elicited reactions (Ichimura et al., 2000; Ahlfors et al., 2004; Gudesblat et al., 2007; Xing et al., 2009), and are required for resistance to attack from pathogens (Desikan et al., 2001; Asai et al., 2002; Menke et al., 2004; Ren et al., 2008; Pitzschke et al., 2009). Another Arabidopsis MAPK, MPK4, was identified as a negative regulator of plant immunity to pathogens (Petersen et al., 2000). mpk4 mutants have highly elevated salicylic acid (SA) levels, which result in greatly increased levels of transcripts of PATHOGENESIS-RELATED (PR) genes. In turn, these mutants are strongly resistant to a virulent bacterial pathogen, Pseudomonas syringae pv. tomato DC3000, and a virulent isolate of the oomycete, Hyaloperonospora arabidopsidis (Petersen et al., 2000).

Herbivores pose another biotic stress to plants. Similar to resistance to pathogens, complex defense systems against herbivores have also been found in various plant species (Howe & Jander, 2008; Mithöfer & Boland, 2008; Wu & Baldwin, 2010). Following herbivore attack, plants alter phytohormone levels, including those of JA, SA and ethylene (Howe et al., 1996; Reymond & Farmer, 1998; von Dahl et al., 2007), and reconfigure their transcriptomes and proteomes (Hui et al., 2003; Reymond et al., 2004; Giri et al., 2006). These changes finally lead to enhanced levels of certain secondary metabolites, which confer resistance to herbivores directly or indirectly. The central roles of JA and its amino acid derivative, JA-isoleucine (JA-Ile), in plant–herbivore interactions have been studied intensively (Howe & Jander, 2008; Wu & Baldwin, 2010). JA biosynthesis involves at least eight enzymes located in chloroplasts and peroxisomes, which produce linolenic acid and convert it to JA (Wasternack, 2007). Catalyzed by JAR (JASMONATE RESISTANT) proteins, JA is further conjugated with Ile to form JA-Ile, which binds to the receptor COI1 (CORONATINE INSENSITIVE1) and triggers JA-induced responses (Staswick & Tiryaki, 2004; Chini et al., 2007; Thines et al., 2007). Transgenic (or mutant) plants that are impaired in JA/JA-Ile biosynthesis or signaling have highly decreased levels of defense-related secondary metabolites, and thus show reduced resistance to herbivores (Howe et al., 1996; Reymond et al., 2000; Kessler & Baldwin, 2001; Paschold et al., 2007). Many studies have identified JA-mediated herbivore defense pathways, but still little is known about JA-independent defenses (JIDs) (Howe & Jander, 2008).

Given the central role of JA in plant–herbivore interactions, the signaling networks that control JA biosynthesis and thus activate effective defenses are particularly important for an understanding of the mechanism by which plants regulate their defenses against herbivores. Genetic studies have indicated the involvement of MAPKs in the regulation of wounding- and herbivory-induced JA biosynthesis (Kandoth et al., 2007; Wu et al., 2007). In the wild tobacco Nicotiana attenuata, wounding and herbivory rapidly activate SA-induced protein kinase (SIPK; an ortholog of Arabidopsis MPK6) and wound-induced protein kinase (WIPK; an ortholog of Arabidopsis MPK3) within minutes (Wu et al., 2007). Silencing SIPK and WIPK in N. attenuata and their homologs in tomato greatly compromises wounding- and herbivory-induced JA bursts, demonstrating the critical role of MAPKs in the regulation of JA biosynthesis in plant defense against herbivores (Kandoth et al., 2007; Wu et al., 2007; Meldau et al., 2009).

Although increased JA levels are induced by feeding by almost all herbivore species, plants respond to herbivore feeding in a herbivore species-specific manner (Heidel & Baldwin, 2004; De Vos et al., 2005). In analogy with the well-studied pathogen-associated molecular patterns (PAMPs), herbivore-associated molecular patterns (HAMPs) have been proposed to be the main factors used by plants to distinguish different herbivores (Mithöfer & Boland, 2008). However, little is yet known about how plants perceive various HAMPs/elicitors and how these cues activate different downstream signaling systems, such as JA-dependent and -independent pathways, to deploy herbivore-specific defenses. Here, we studied the function of a MAPK in N. attenuata, MPK4, in plant defense against the specialist insect Manduca sexta and the generalist insect Spodoptera littoralis. We show that, compared with wild-type (WT) plants, herbivory (but not wounding) of M. sexta induces increased JA levels in MPK4-silenced plants, indicating that, unlike SIPK and WIPK (Wu et al., 2007), MPK4 is a specific negative regulator of herbivory-induced JA accumulation; furthermore, MPK4 negatively regulates the defense levels of N. attenuata against the specialist M. sexta in a largely JA signaling-independent manner. By contrast, silencing of MPK4 did not alter plant resistance levels to the generalist S. littoralis, demonstrating that MPK4 confers herbivore-specific responses in N. attenuata.

Materials and Methods

Plant growth and sample treatments

Nicotiana attenuata Torr. ex S. Watson (Solanaceae) seeds were from a line maintained in our laboratory that was originally collected in Utah (USA) and inbred for 30 generations in the glasshouse. Seed germination and plant cultivation followed Krügel et al. (2002). Four- to 5-wk-old plants were used for all experiments.

For the collection of M. sexta and S. littoralis oral secretions (OSMs and OSSl), larvae were reared on N. attenuata WT plants until the third to fifth instars. OS were collected on ice as described in Halitschke et al. (2001). To analyze the fatty acid–amino acid conjugate (FAC) contents in the OS, each sample was centrifuged at 16 000 g for 10 min, the supernatants were diluted 1 : 100 with 15% methanol, and analyzed directly on a high-performance liquid chromatograph-mass spectrometer (HPLC-MS) (1200L LC-MS system, Varian, Palo Alto, CA, USA) (Halitschke et al., 2001). For the simulated herbivory treatments, leaves were wounded with a pattern wheel and 20 μl OS were immediately rubbed into the puncture wounds of each wounded leaf (W + OSMs or W + OSSl); for the wounding treatments, leaves were wounded with a pattern wheel, and 20 μl of water were rubbed into the puncture wounds on each wounded leaf (W + W).

Generation of transformed plants

The creation of irMPK4 plants has been described in Hettenhausen et al. (2012). Crossing irCOI1 with irMPK4 plants was performed by removing anthers from flowers of irCOI1 plants before pollen maturation and pollinating the stigmas with pollen from irMPK4 plants.

Analysis of JA and JA-Ile concentrations

One milliliter of ethyl acetate spiked with 200 ng of D2-JA and 40 ng of 13C6-JA-Ile, the internal standards for JA and JA-Ile, respectively, was added to each crushed leaf sample (c. 150 mg). Samples were then ground on a FastPrep homogenizer (Thermo Fisher Scientific, Waltham, MA, USA). After centrifugation at 13 000 g for 10 min at 4°C, the supernatants were transferred to fresh tubes and evaporated to dryness on a vacuum concentrator (Eppendorf, Hamburg, Germany). Each residue was resuspended in 0.5 ml of 70% methanol (v/v) and centrifuged at 13 000 g for 15 min at 4°C to remove particles. The supernatants were analyzed on an HPLC-MS/MS (1200L LC-MS system, Varian).

Herbivore growth bioassays

Freshly hatched M. sexta larvae were placed on 30 replicated plants of each genotype (one larva per plant). To compare herbivore growth rates on WT and irMPK4 plants, larvae were weighed on days 4, 7, 9 and 11; for the comparison of herbivore growth on WT, irMPK4, irCOI1 and irMPK4 × irCOI1 plants, larval masses on days 4, 6 and 8 were recorded.

To measure S. littoralis growth, freshly hatched larvae were grown on an artificial diet for 10 d and then placed on 30 replicate plants of each genotype. Larval masses were recorded 3, 6 and 9 d after transfer to the plants.

To analyze whether S. littoralis was sensitive to the MPK4-dependent and JID compounds, WT and irMPK4 plants were pretreated with W + OSMs or 20 μl of N-linolenoyl-l-glutamine (FAC-A) at 138 ng μl−1 dissolved in an aqueous solution of 0.005% (w/v) Triton X-100, 1 d before S. littoralis larvae were placed on these leaves. Spodoptera littoralis larvae were confined in clip cages to avoid moving to other uninduced leaves and their masses were recorded over time. The same set-up was used to measure S. littoralis performance on irCOI1 and irMPK4 × irCOI1 plants, which had been pretreated with W + OSMs (1 d earlier).

Analyses of trypsin proteinase inhibitor (TPI) activity and contents of nicotine, caffeoylputrescine (CP) and diterpene glycosides

TPI activity was analyzed with a radial diffusion assay described by Van Dam et al. (2001). The accumulation of the direct defenses nicotine, CP and diterpene glycosides were analyzed in samples harvested 3 d after treatments using an HPLC method described in Keinanen et al. (2001).

Profiling of metabolites by ultraperformance liquid chromatography-time of flight-mass spectrometry (LC-ToF-MS)

Leaf tissue from WT, irMPK4, irCOI1 and irMPK4 × irCOI1 plants was harvested after 4 d of M. sexta feeding, and five independent biological replicates per plant genotype were used; nontreated plants served as comparisons. One hundred milligrams of leaf tissue were ground with a Geno/Grinder 2000 (SPEXSamplePrep, Metuchen, NJ, USA) and thoroughly extracted with 1 ml of extraction buffer (40% (v/v) methanol/water and 50 mM acetate buffer, pH 4.8). Homogenized samples were centrifuged at 12 000 g for 15 min at 4°C, the supernatants were transferred into fresh 1.5-ml microcentrifuge tubes, and the samples were centrifuged again under the same conditions. The supernatant (500 μl) was transferred into HPLC vials. Sample separation and MS analysis followed Gaquerel et al. (2010).

Datasets were evaluated from 50 to 550 s in the mass range m/z 90–1400. The raw data files were converted to netCDF format using the export function of Data Analysis version 4.0 software (Bruker Daltonics, Bremen, Germany) and processed using the XCMS package (Tautenhahn et al., 2008) and the R-package CAMERA (http://www.bioconductor.org/biocLite.R), as described previously (Gaquerel et al., 2010). Peak detection was performed using the centWave method (Tautenhahn et al., 2008) and the parameter settings ppm = 20, snthresh = 10 and peak width = 5–20 s. Retention time correction was achieved using the parameter settings minfrac = 1, bw = 60 s, mzwid = 0.1D, span = 1 and missing = extra = 0 (Gaquerel et al., 2010). Metaboanalyst software (Xia et al., 2009) was used to perform multivariate analysis (principal component analysis, PCA). The data were filtered using the coefficient of variation, and normalized using Pareto scaling (Xia et al., 2009; Gaquerel et al., 2010). The public metabolite databases used for analysis were Prime (http://prime.psc.riken.jp/), Metlin (http://metlin.scripps.edu/), MetDAT2 (http://www.sdwa.nus.edu.sg/METDAT2/), KEEG (http://www.genome.jp/kegg/), PubChem (http://pubchem.ncbi.nlm.nih.gov/) and Knapsack (http://kanaya.naist.jp/KNApSAcK/).

Analysis of starch levels

Starch levels were estimated using the anthrone method (Morris, 1948). Soluble sugars were removed with 80% ethanol and, after extraction with perchloric acid, samples were boiled for 8 min with the anthrone reagent (100 mg anthrone in 100 ml 95% H2SO4) and starch concentrations were determined according to the absorbance at 540 nm.

RNA extraction and quantitative real-time PCR (qPCR)

Total RNA was extracted from ground leaf samples using TRIzol reagent (Invitrogen). For qPCR analysis, five replicated biological samples were used and 0.5 μg of total RNA sample was reverse transcribed with oligo(dT) and Superscript II reverse transcriptase (Invitrogen). qPCR was performed on an ABI PRISM 7700 sequence detection system (Life Technologies, Carlsbad, CA, USA) using qPCR Core kits (Eurogentec, Seraing, Belgium). An N. attenuata actin2 gene was employed as the internal standard for the normalization of cDNA concentration variations. qPCR primers were (F, forward primer; R, reverse primer): for actin2: F, 5′-GGTCGTACCACCGGTATTGTG-3′; R, 5′-GTCAAGACGGAGAATGGCATG-3′; for COI1: F, 5′-CAGGGCATCTTCAGCTGGTC-3′; R, 5′-CGGGATGCTCAGCAACGA-3′; for MPK4: F, 5′-TAGGAGCAACTCCGGTGCC; R, 5′-GCAAGGACAACATCTGAGACAGAT-3′.

Statistical analysis

Data were analyzed by analysis of variance (ANOVA) or unpaired t-test using StatView, version 5.0 (SAS Institute, Cary, NC, USA).

Results

Simulated M. sexta herbivory, but not wounding, transiently induces elevated levels of JA in irMPK4 plants

Using an RNAi (RNA interference) vector and Agrobacterium-mediated transformation, we created several independently transformed stable lines of MPK4-silenced N. attenuata (irMPK4) (Hettenhausen et al., 2012); among these, lines 119 and 163 with single T-DNA insertions were selected for this study. Given the central role of JA in plant resistance to herbivores, we first examined whether MPK4 modulates wounding- and herbivory-elicited JA levels. Leaves of rosette-staged WT and irMPK4 plants were wounded with a pattern wheel and 20 μl of M. sexta oral secretions (OSMs) were applied immediately to the wounds (W + OSMs) to mimic herbivory (Halitschke et al., 2001, 2003); for comparison with mechanical wounding, 20 μl of water were applied to wounds (W + W). The JA contents in WT and irMPK4 plants after these treatments were determined by HPLC-MS/MS. No differences in JA levels were detected between WT and irMPK4 plants before and after W + W treatment (Fig. 1a). However, irMPK4 plants showed 65% increased levels of JA 1 h after W + OSMs treatment, compared with the JA levels in similarly treated WT plants (Fig. 1a). Consistently, the contents of JA-Ile similarly increased: 1 h after W + OSMs treatment, JA-Ile contents were 60% higher in irMPK4 plants, whereas JA-Ile levels in W + W-induced WT and irMPK4 plants were the same (Fig. 1b). The antagonistic effect of SA on JA accumulation and signaling has been well documented (Doares et al., 1995; Niki et al., 1998; Kunkel & Brooks, 2002; Spoel et al., 2003). We quantified SA concentrations in order to determine whether the elevated JA contents in W + OSMs-treated irMPK4 plants resulted from attenuated SA levels. WT and irMPK4 plants showed no difference in SA levels before and after either treatment (Supporting Information Fig. S1).

Figure 1.

Silencing of MPK4 specifically elevates simulated Manduca sexta herbivory-induced jasmonic acid (JA) and JA-isoleucine (JA-Ile) levels. Wild-type (WT; white bars) and irMPK4 (line 119 (grey bars) and 163 (black bars)) plants were wounded with a pattern wheel, and 20 μl of water (W + W) or M. sexta oral secretions (OSMs) (W + OSMs) were immediately applied to the wounds. Samples were harvested at the indicated times, and their JA (a) and JA-Ile (b) contents (mean ± SE) were analyzed on a high-performance liquid chromatograph-tandem mass spectrometer (HPLC-MS/MS). Asterisks indicate significant differences between WT and irMPK4 plants (t-test: *, < 0.05; **, < 0.01; n = 5).

To examine whether silencing of MPK4 altered the activity of SIPK and WIPK, we measured their activity before and after W + W and W + OSMs treatment by an in-gel kinase assay (Fig. S2). No differences in SIPK and WIPK activity between WT and irMPK4 plants were found.

We conclude that MPK4 negatively affects M. sexta herbivory-induced JA accumulation, and thus comprises a part of the regulatory network that is required for the normal regulation of JA levels in N. attenuata in response to M. sexta attack.

Silencing of MPK4 enhances N. attenuata's defense against the specialist insect herbivore M. sexta

To further study the function of MPK4 in N. attenuata's defense against herbivores, we performed bioassays on WT and irMPK4 plants to examine whether silencing of MPK4 influences the growth of M. sexta. Freshly hatched M. sexta larvae were placed on WT and irMPK4 plants, and larval masses were recorded over time. From as early as day 4, compared with those on WT plants, larvae on irMPK4 plants showed decreased growth rates; by day 11, the average larval mass on irMPK4 was only one-third of that of the larvae on WT plants (Fig. 2a). Thus, MPK4 strongly suppresses the resistance of N. attenuata to its specialist herbivore, M. sexta.

Figure 2.

Simulated Manduca sexta feeding does not induce large changes in known defensive metabolites. (a) Manduca sexta growth on different plants. Wild-type (WT) and irMPK4 plants (lines 119 and 163) were infested with 30 neonate M. sexta larvae (one larva per plant). Masses of these larvae (mean ± SE) on WT and irMPK4 plants were recorded on days 4, 7, 9 and 11. (b–e) Accumulations of defensive secondary metabolites in WT and irMPK4 plants after wounding and simulated herbivory. WT and irMPK4 plants were wounded with a pattern wheel, and 20 μl of water (W + W) or M. sexta oral secretions (OSMs) (W + OSMs) were immediately applied to the puncture wounds. The activity of trypsin proteinase inhibitor (TPI) (b) and the contents of nicotine (c), caffeoylputrescine (d) and 17-hydroxygeranyllinalool diterpene glucosides (HGL-DTGs) (e) (mean ± SE) were analyzed in samples harvested 3 d after treatment. Asterisks indicate significant differences between the masses of larvae reared on WT and irMPK4 plants (t-test: *, < 0.05; ***, < 0.001; n = 30 for (a) and = 5 for (b–e)).

In N. attenuata, TPIs (Van Dam et al., 2001), nicotine (Steppuhn et al., 2004), CP (Kaur et al., 2010) and 17-hydroxygeranyllinalool diterpene glucosides (HGL-DTGs) (Jassbi et al., 2008; Heiling et al., 2010) are all important direct defenses against herbivores, and the accumulations of these compounds are mainly regulated by JA signaling (Paschold et al., 2007). We sought to determine whether these metabolites were responsible for the increased resistance of irMPK4 plants. Consistent with the transiently increased levels of W + OSMs-induced JA in irMPK4 plants, irMPK4 plants showed 25% higher TPI activity than WT plants after W + OSMs elicitation, but not after W + W treatment (Fig. 2b). WT and irMPK4 plants did not show significant differences in the contents of nicotine, CP and HGL-DTGs (Fig. 2c–e), although irMPK4 tended to have higher HGL-DTG levels after W + OSMs treatments (Fig. 2e; t-test, > 0.19). Therefore, MPK4 has little influence on the levels of secondary metabolites that are known to function as direct defenses against M. sexta.

Wounding and herbivory elicit a release of green leaf volatiles (GLVs), which can attract predators of herbivores or increase herbivore loads in nature (Halitschke et al., 2008; Meldau et al., 2009; Dicke & Baldwin, 2010). In addition, GLVs also function as feeding stimulants for M. sexta larvae on N. attenuata (Halitschke et al., 2004; Meldau et al., 2009). The quantities of GLVs released from WT and irMPK4 plants after either W + W or W + OSMs treatment were similar (Fig. S3), excluding the possibility that the reduced M. sexta larval growth resulted from impaired GLV emissions in irMPK4 plants.

MPK4 negatively affects an important JA signaling-independent defense pathway against M. sexta

In N. attenuata, the deployment of all known inducible direct and indirect defenses requires JA signaling (Paschold et al., 2007). Although irMPK4 plants show increased JA levels after OSMs elicitation, only one of the known direct defensive compounds, TPI, showed moderately elevated levels (Fig. 2b). Given the dramatically enhanced resistance levels of irMPK4 plants against M. sexta attack, we speculated that MPK4 may also regulate a novel defense pathway that is independent of JA signaling.

To test this hypothesis, an N. attenuata line silenced in COI1 (irCOI1 plants, deficient in JA perception) (Paschold et al., 2007) was crossed with irMPK4 (line 119) to obtain irMPK4 × irCOI1 plants, which were silenced in both MPK4 and COI1 (Fig. S4a). During the rosette stage, when all experiments were performed, no obvious morphological differences were found among WT, irMPK4, irCOI1 and irMPK4 × irCOI1 plants. We analyzed the secondary metabolites that function as defenses against M. sexta, and confirmed that irMPK4 × irCOI1 plants showed substantially diminished levels of TPI, nicotine, CP and HGL-DTGs owing to the defect in JA signaling (Fig. S4b). Manduca sexta neonates were placed on WT, irCOI1, irMPK4 (line 119) and irMPK4 × irCOI1 plants, and herbivore growth was recorded over 8 d (Fig. 3). After 8 d, compared with those grown on WT plants (260 mg), M. sexta larvae gained c. 200% more average mass on irCOI1 (780 mg) and 50% less average mass on irMPK4 (120 mg) plants; importantly, silencing of COI1 in irMPK4 plants only resulted in a one-fold increase in M. sexta masses, which were similar to the masses of the larvae on WT plants, but were only one-third of the caterpillar masses on irCOI1 plants (Fig. 3). Similar results were obtained in two more independently repeated bioassays.

Figure 3.

Silencing of MPK4 enhances Nicotiana attenuata's resistance to Manduca sexta in a largely jasmonic acid (JA) signaling-independent manner. Wild-type (WT), irMPK4 (line 119), irCOI1 and irMPK4 × irCOI1 plants were infested with 30 neonate M. sexta larvae (one larva per plant). Masses of these larvae (mean ± SE) were recorded on days 4, 6 and 8. Asterisks indicate significant differences between the masses of larvae reared on WT and irMPK4, irCOI1 or irMPK4 × irCOI1 plants (t-test: ***, < 0.001; n = 30).

Very likely, certain compounds, which are regulated by MPK4 but not by JA signaling, accumulate to high levels in irMPK4 plants and strongly retard the growth of M. sexta larvae.

Unbiased metabolomic analysis reveals JA signaling-dependent and -independent changes in M. sexta feeding-induced secondary metabolites

To identify the potential MPK4-regulated anti-M. sexta metabolites, the differential accumulation of metabolites in M. sexta feeding-elicited leaves from irMPK4, irCOI1, irMPK4 × irCOI1 and WT plants was profiled by LC-ToF-MS analysis. Herbivory-elicited leaves were harvested after 4 d of M. sexta feeding, and polar metabolites were analyzed using a method described previously (Gaquerel et al., 2010; Gilardoni et al., 2011). Metabolites were ionized using the electrospray ionization (ESI) interface in both positive and negative ionization modes, and those metabolites that eluted from the column between 50 and 500 s with mass-to-charge (m/z) values ranging from 90 to 1400 were selected for analysis.

Totals of 2516 and 3256 ions were detected in the negative and positive modes, respectively. To obtain an overview of the differences among genotypes, the dataset corresponding to the differentially accumulated ions was analyzed after Pareto scaling by PCA. Although the ions detected in the positive mode could not separate the individual lines (Fig. S5a), they were clearly distinguished in the negative mode, indicating pronounced metabolomic changes resulting from the silencing of MPK4. The first and third principal components (PCs) together explained 64.4% of the variation within this dataset, and PC1 and PC3 clearly separated the samples based on their genotypes (Fig. 4). PC1 separated irCOI1 and irMPK4 × irCOI1 from WT and irMPK4, indicating that ions within this group required JA signaling. PC3 separated irMPK4 and irMPK4 × irCOI1 from WT and irCOI1, underscoring the ability of MPK4 to sculpt the secondary metabolite composition of leaves. As MPK4 mediates a JID against M. sexta, we expected that these unknown defensive compounds should have greater concentrations in irMPK4 than in WT and irCOI1 plants, and that their concentrations would not be strongly decreased in irMPK4 × irCOI1 plants. To identify the ions that were specifically up-regulated by silencing of MPK4, individual ions, whose abundances were at least 1.5-fold (ANOVA; < 0.05) greater in irMPK4 than in WT and irCOI1, and were also at least 1.5-fold (ANOVA; < 0.05) greater in irMPK4 × irCOI1 than in WT and irCOI1, were selected. Ten and five ions in negative and positive mode, respectively, accumulated to significantly (< 0.05) higher levels in irMPK4 plants throughout all four comparisons (Table S1). To define the identities of these ions, a search in the public metabolite database and in home-built databases (Gaquerel et al., 2010) was performed using the m/z values. Approximately 73% of the ions did not match to any of the metabolites in the databases. Notably, the compounds which could be identified all belonged to the group of phenylpropanoid metabolites, and some members of this group have been shown to confer resistance to M. sexta (Kaur et al., 2010).

Figure 4.

Principal component analysis (PCA) of the secondary metabolites detected by ultraperformance liquid chromatography-time of flight-mass spectrometry (UPLC-ToF-MS). PCA obtained after Pareto scaling revealed a clear differentiation of metabolite profiles in the Manduca sexta-elicited leaves of wild-type (WT), irMPK4, irCOI1 and irMPK4 × irCOI1 plants. Herbivory-elicited WT, irMPK4, irCOI1 and irMPK4 × irCOI1 leaves were harvested after 4 d of M. sexta feeding. Polar metabolites were extracted and analyzed by UPLC-ToF-MS in negative ion detection mode, as described in the Materials and Methods section. Ellipses delimit the 95% statistical confidence areas for each biological group in the score plot.

In contrast with M. sexta feeding-elicited leaves, untreated WT and irMPK4 samples were not separated by PCA, indicating very similar metabolite profiles (Fig. S5b,c). From the analysis performed in the negative mode, no ions were found to be significantly different between untreated WT and irMPK4 plants, whereas 20 ions, all detected in the positive ionization mode, accumulated to at least 1.5-fold (ANOVA; < 0.05) greater levels in untreated irMPK4 than in WT, but, importantly, none of these ions was identical to those identified from M. sexta-induced plants (Table S2). Thus, the anti-M. sexta JID metabolites are probably induced by M. sexta feeding in irMPK4 plants, but are not constitutively controlled by MPK4.

irMPK4 plants have elevated photosynthetic rates (Hettenhausen et al., 2012) and it is possible that these may affect the nutritional contents of irMPK4. We measured both the contents of total proteins and starch (representing the sugars) in WT and irMPK4 plants; no differences were found between the protein contents of samples harvested before and after M. sexta feeding, whereas, in line with the elevated photosynthesis, irMPK4 contained c. 50% more starch than did WT plants (Fig. S6). These results argue against the possibility that the decreased M. sexta growth rates on irMPK4 plants resulted from certain indirect effects of silencing of MPK4, such as altered photosynthesis capacity.

MPK4 is not required for defense against the generalist herbivore S. littoralis

To examine whether MPK4 is also important for defense against the generalist herbivore S. littoralis, S. littoralis larvae were allowed to feed on WT and irMPK4 plants for 9 d. In contrast with the specialist M. sexta, the generalist S. littoralis did not show different growth rates on WT and irMPK4 plants (Fig. 5).

Figure 5.

Spodoptera littoralis grows similarly on wild-type (WT) and irMPK4 plants. WT (diamonds) and irMPK4 (lines 119 (squares) and 163 (triangles)) plants were infested with 30 S. littoralis larvae (one larva per plant). Masses of these larvae (mean ± SE) were recorded on days 3, 6 and 9. No statistical differences were found between the masses of S. littoralis grown on WT and irMPK4 plants (t-test).

The FACs in OSMs are necessary and sufficient to trigger M. sexta feeding-specific responses in N. attenuata, including the rapid activation of SIPK and WIPK and the initiation of JA and JA-Ile biosynthesis (Halitschke et al., 2001, 2003; Wu et al., 2007). Given the important role of FACs in the activation of herbivore defense-related responses, including the activation of SIPK and WIPK signaling in N. attenuata, the FAC contents in OSSl were analyzed. Compared with those in OSMs, substantially lower levels (c. 500 times lower) of all FACs were found in OSSl (Fig. 6a).We next explored whether the concentrations of FACs were important for W + OS-induced JA levels in irMPK4 plants. The application of OSMs (W + OSMs) or FACs in a concentration similar to that in OSMs (W + FACMs) to wounds both elicited c. 60% more JA in irMPK4 than in WT plants; by contrast, W + OSSl did not induce different JA levels in WT and irMPK4 plants, although FACs in OSSl were still sufficient to elicit high levels of JA (c. 2.5 μg g−1 FM), which were > 1.6-fold greater than those induced by W + W (0.95 μg g−1 FM) (Fig. 6b). The treatment of irMPK4 plants with OSSl, which had been supplemented with FAC-A (Halitschke et al., 2001) to mimic OSMs (W + OSSl + FAC), elevated the JA levels to those in W + OSMs-elicited plants; furthermore, compared with OSMs, 500-fold-diluted OSMs showed reduced ability to elicit JA accumulation in irMPK4 plants (Fig. 6b). A FAC titration assay was also performed to examine the effect of FAC concentrations on the elicitation of JA accumulation in WT and irMPK4 plants; it was found that, only when the FAC-A concentration was at least 13.8 ng μl−1 (similar to that in 10-fold-diluted OSMs), were JA levels in irMPK4 significantly greater than in WT plants (Fig. 6c). FACs in OSSl had very low concentration and therefore S. littoralis feeding could not induce greater levels of JA in irMPK4 than in WT plants.

Figure 6.

Fatty acid–amino acid conjugates (FAC) in low concentrations do not induce different jasmonic acid (JA) levels in wild-type (WT) and irMPK4 plants. (a) Comparison of the FAC contents in the oral secretions (OS) of Manduca sexta and Spodoptera littoralis. (b) JA induced by wounding and simulated herbivory. WT and irMPK4 (line 119) plants were wounded with a pattern wheel, and 20 μl of water (W + W), M. sexta OS (W + OSMs), S. littoralis OS (W + OSSl), FAC-A at concentrations similar to those in S. littoralis OS (W + FACSl) and M. sexta OS (W + FACMs), S. littoralis OS supplied with FAC-A (OSSl + FAC) and 1–500-diluted M. sexta OS were immediately applied to the wounds and the JA contents were measured in samples collected 1 h after treatments. Nontreated plants served as controls. (c) FAC titration assay. Plants were wounded with a pattern wheel, 20 μl of FAC-A in a serial dilution were applied to the wounds and the JA contents were measured in samples collected 1 h after treatments (FACMs represents FAC-A in a concentration similar to that in OSMs, 138 ng μl−1). Asterisks indicate significant differences between WT and irMPK4 plants (t-test: **, < 0.01; = 5).

The similar levels of JA in S. littoralis-induced WT and irMPK4 plants were consistent with the equal growth rates of S. littoralis on WT and irMPK4 plants. The possibility that the contents of FACs in OSSl were too low to activate the JID pathway in which MPK4 plays a negative role, or that S. littoralis can tolerate this JID, could not be ruled out.

The MPK4-regulated JA signaling-independent defense pathway in irMPK4 does not inhibit the growth of S. littoralis

Our genetic analysis indicated that the highly increased resistance to M. sexta in irMPK4 plants was largely contributed by certain defensive compounds regulated by a novel JID pathway (Fig. 3), but silencing of MPK4 did not change the resistance levels of N. attenuata against S. littoralis. We speculated that N. attenuata evolved the JID pathway (which is suppressed by MPK4) to counteract the specialist M. sexta, but not the generalist S. littoralis; thus, it is likely that these compounds have no effect on S. littoralis growth. To examine this possibility, we treated WT and irMPK4 with W + OSMs to ensure the accumulation of the JA-independent metabolites. One day after W + OSMs treatment, S. littoralis larvae were placed on these plants and their masses were recorded. By day 4, S. littoralis grown on WT plants had an average mass of 54 mg, but those on irMPK4 plants reached only c. 28 mg (Fig. 7). As W + OSMs treatment induces JA-dependent defense (such as TPI) and JID, caterpillar growth was monitored in parallel on irCOI1 and irMPK4 × irCOI1, which were also treated with W + OSMs 1 d before the bioassay: S. littoralis average masses increased equally to c. 120 mg on both irCOI1 and irMPK4 × irCOI1 (Fig. 7). Similar results were obtained from an independently repeated experiment, in which the S. littoralis bioassay was performed on plants that had been pretreated with W + FAC-A (138 ng μl−1, similar to the levels in OSMs) (Fig. S7a). Given that, after W + OSMs treatment, only TPI showed elevated levels in irMPK4 plants, we examined S. littoralis growth on irTPI, which were silenced in TPI transcripts (Steppuhn et al., 2004), and on irMPK4 × irTPI plants, which had been pretreated with W + OSMs. The results confirmed that the increased TPI activity in irMPK4 plants after W + FAC-A treatment was responsible for the decreased growth of S. littoralis (Fig. S7b).

Figure 7.

The jasmonic acid (JA)-independent defense pathway does not inhibit the growth of Spodoptera littoralis. Wild-type (WT), irMPK4 (line 119), irCOI1 and irMPK4 × irCOI1 plants were wounded with a pattern wheel, and 20 μl of M. sexta oral secretions (OS) were applied to the wounds immediately. After 1 d, each plant type was infested with 30 S. littoralis larvae (one larva per plant). The masses of these larvae (mean ± SE) were recorded after 3 d of feeding. Different lower case letters represent statistical differences (ANOVA:< 0.05; n = 30).

Thus, S. littoralis is tolerant to the anti-M. sexta compounds that are regulated by the JA-independent pathway in irMPK4 plants, but its growth is negatively affected by defenses elicited by JA signaling.

Discussion

The deactivation of a physiological process is equally as important as its activation. However, much less is known about proteins that play negative regulatory roles in plant physiology than about those that play positive roles. Using a reverse genetic approach, we examined the function of a MAPK, MPK4, in plant resistance to insect attack. Unlike SIPK and WIPK, two MAPKs that play positive roles in mediating JA levels, MPK4 negatively affected JA accumulation elicited by M. sexta feeding (but was not involved in S. littoralis-induced JA accumulation). Importantly, MPK4 also suppressed an essential anti-M. sexta pathway that was independent of JA signaling. By contrast, the MPK4-suppressed defense pathways were not required for the N. attenuata–S. littoralis interaction. In addition to being a novel negative MAPK regulator of FAC-induced JA, we further demonstrated that MPK4 probably represents a node in the signaling network that confers herbivore species-specific responses.

Concentrations of FACs are important in activating MPK4-dependent JA accumulations

Although the mechanisms of perception remain unclear, some plants use FACs as HAMPs and deploy specific defense reactions (Mithöfer & Boland, 2008; Bonaventure et al., 2011). OSMs contain FACs at millimolar concentrations (Halitschke et al., 2001); however, Spodoptera spp. appear to have much less FACs in their OS, as FACs in OSSl are c. 500 times lower in concentration than those in OSMs, and similarly low contents of FACs were also found in S. exigua (Diezel et al., 2009). Consistent with the finding that FACs in low concentrations can still effectively activate SIPK and WIPK (Wu et al., 2007), W + OSSl induced greater JA levels than did W + W in N. attenuata. However, using OSMs, OSSl, supplementation of FAC-A to OSSl and FAC-A in different concentrations, we found that FAC concentrations play a critical role in eliciting MPK4-dependent JA accumulation. Furthermore, our titration assay using FAC-A at different concentrations confirmed that relatively high concentrations of FACs are required to induce high JA levels in irMPK4 plants (Fig. 6c). We propose that it is likely that, similar to SIPK and WIPK, MPK4 is also located downstream of the putative FAC receptor, and that N. attenuata uses FAC concentrations to recognize different insect herbivores (such as the specialist M. sexta and generalist S. littoralis) and activates herbivore-specific reactions, including the MPK4-mediated responses (Fig. 8). It is possible that the different concentrations of FACs in the OS of the specialist M. sexta and the generalist S. littoralis not only account for the different levels of herbivory-induced JA, and thus the JA-dependent defenses, but also the JIDs. This possibility should be examined after the identities of the JA-independent metabolites have been determined.

Figure 8.

A hypothetical working model summarizing Manduca sexta- and Spodoptera littoralis-induced mitogen-activated protein kinase (MAPK) signaling in Nicotiana attenuata. The fatty acid–amino acid conjugates (FACs) in M. sexta oral secretions (OS) are perceived by a putative FAC receptor in N. attenuata and thereby activate at least MEK2 (Heinrich et al., 2011) and its downstream MAPKs, salicylic acid-induced protein kinase (SIPK) and wound-induced protein kinase (WIPK), which positively regulate the accumulation of jasmonic acid (JA). Highly increased JA levels induce the biosynthesis of defensive compounds, such as trypsin proteinase inhibitor (TPI), 17-hydroxygeranyllinalool diterpene glucosides (HGL-DTGs) and caffeoylputrescine. The perception of FACs in M. sexta OS might also activate MPK4 (probably through activation of SIPKK; Gomi et al., 2005), which suppresses JA levels and, more importantly, the accumulation of unknown anti-M. sexta compounds in a JA signaling-independent manner. It is unclear which signaling pathway positively regulates the production of these unknown compounds. Compared with those in M. sexta OS, FACs in S. littoralis OS occur at much lower concentrations and thus do not activate the MPK4 suppressor pathway, but are still sufficiently abundant to activate an amplification of SIPK and WIPK activity compared with mechanical wounding and to induce JA-dependent defenses.

FACs are thought to function in the nitrogen metabolism of certain insects (Yoshinaga et al., 2008) and are probably essential for insect physiology, as they have been found not only in many caterpillars, but also in crickets (Teleogryllus taiwanemma) and fruit flies (Drosophila melanogaster) (Yoshinaga et al., 2007). In the evolutionary arms races between N. attenuata and generalist and specialist insects, FACs in the OS of insects probably play a critical role in plant–insect interactions: plants have evolved recognition systems to deploy herbivore-specific defenses to achieve optimal growth and defense output and/or insects may also have evolved to alter the components of their OS, for example, FAC contents, to minimize the risks of triggering strong defenses from their hosts.

JA biosynthesis takes place in chloroplasts and peroxisomes. SIPK, WIPK and MPK4 are mainly located in the cytoplasm and nucleus (Ahlfors et al., 2004; Hettenhausen et al., 2012), and there is no evidence that they are also located in or can translocate into chloroplasts and/or peroxisomes. The enzymatic reactions in JA biosynthesis have been studied intensively (Delker et al., 2006). However, little is known about the mechanisms underlying the regulation of JA biosynthesis – how plants rapidly activate JA biosynthesis. To further study the mechanisms by which these MAPKs influence JA biosynthesis, new methods are required to determine the in vivo activity of the biosynthetic enzymes (at least eight enzymes) and to quantify the intermediate products (precursors) of JA, all of which have high turnover rates. The catabolism of JA remains unclear. The possibility that irMPK4 plants have decreased catabolic rates of JA cannot be ruled out.

MPK4 suppresses a novel JA-independent defense pathway against the specialist insect herbivore M. sexta, but is not important in the resistance to the generalist S. littoralis

Several studies have shown that wounding not only activates the JA pathway, but also the JA-independent responses. For example, in Arabidopsis, JA and wounding induce overlapping, yet different, sets of genes, and, after wounding, several genes are still induced in coi1 mutants (Titarenko et al., 1997); many wounding-induced genes are also highly elevated in Arabidopsis jar1 mutants, which are deficient in JA-Ile (Suza & Staswick, 2008). It is possible that wounding- and herbivory-induced JA-independent pathways are mediated by other phytohormones, such as ethylene, SA and abscisic acid signaling (Erb et al., 2012). Similarly, JA-independent defenses are also involved in Arabidopsis–Botrytis cinerea (a necrotrophic fungus) interactions (Rowe et al., 2010).

In bioassays, we found that irMPK4 plants show high levels of resistance to M. sexta. Using irMPK4 × irCOI1 plants, which are deficient in both MPK4 and COI1, we examined the contribution of JA signaling. Strikingly, despite highly compromised levels of all known anti-herbivore compounds, irMPK4 × irCOI1 plants still largely retained their elevated resistance levels compared with irCOI1. Clearly, MPK4 suppresses certain defensive compounds in a JA signaling-independent manner (Fig. 3). Given that silencing of COI1 in irMPK4 highly compromised the accumulation of CP, HGL-DTGs and TPIs (Fig. S4b), this JID pathway in irMPK4 plants is remarkably effective against M. sexta. By contrast, S. littoralis grew similarly on WT and irMPK4 plants, demonstrating that MPK4 is not important in the interaction between N. attenuata and S. littoralis larvae.

We found that only high levels of FACs (such as those in OSMs) elicited greater levels of JA in irMPK4 than in WT plants (Fig. 6). Most probably as a result of the low FAC concentrations in OSSl, S. littoralis feeding did not elicit greater levels of JA in irMPK4 than in WT N. attenuata, and thus MPK4 is not important for the JA-dependent defenses in S. littoralisN. attenuata interactions. Given the low FAC contents in OSSl, it is possible that S. littoralis feeding does not elicit the JID pathway, and this resulted in the equal growth of S. littoralis on irMPK4 and WT plants; another possibility is that S. littoralis feeding activates the accumulation of JA-independent defensive metabolites, but S. littoralis tolerates these metabolites. By applying OSMs to ensure the production of JA-independent metabolites, we provided in planta evidence that, despite their strong inhibitory effects on M. sexta growth, these compounds do not interfere with the growth of S. littoralis, indicating the tolerance of S. littoralis larvae. Using a large-scale metabolomic analysis, we identified 15 putative metabolites that were up-regulated after M. sexta feeding in MPK4-silenced plants compared with those in WT plants, and it is probable that some of these metabolites play important roles in the interaction between M. sexta and N. attenuata. The identity of most of the corresponding metabolites could not be determined unambiguously and the characterization of these compounds will be the focus of future work. Comparisons between the metabolic profiles of untreated and M. sexta-induced samples indicated that several JA-independent metabolites are induced by M. sexta feeding in irMPK4 plants (Fig. 4 and Table S1). It will be interesting to study whether low concentrations of FACs (such as those in OSSl) can also elicit the accumulation of these JA-independent metabolites in irMPK4 plants. As MS-based metabolic screening only identifies compounds that can be ionized, it is possible that there are certain other undetected MPK4-regulated JA-independent metabolites. In tomato, threonine deaminase (TD) plays a defensive role against M. sexta by degrading the essential amino acids arginine and threonine in the M. sexta midgut (Chen et al., 2005). Whether proteins are a part of the JIDs remains unknown.

In N. tabacum, wounding transiently enhances the activity of MPK4 (Gomi et al., 2005). Thus, we speculate that M. sexta herbivory (and FACs in high concentrations) might also activate MPK4, and this might lead to the suppression of JA biosynthesis and the JIDs (Fig. 8). In addition, it is possible that another pathway, which is also activated by the perception of FACs in M. sexta OS, positively regulates the concentrations of these JA-independent defensive compounds (Fig. 8). After M. sexta feeding, FACs activate both positive and negative (MPK4) pathways in a concerted (e.g. different timings and scales) manner, and thereby N. attenuata responds to M. sexta feeding with the accumulation of a particular blend of defensive metabolites, including the JA-independent ones (Fig. 8). However, the possibility that MPK4 is deactivated (dephosphorylated) by M. sexta feeding or FAC treatment cannot be ruled out. We hypothesize that, when S. littoralis attacks N. attenuata, the FACs in OSSl are probably too low to activate this positive regulatory pathway (and the suppressor MPK4) to increase the levels of JIDs. The identification of the JA-independent metabolites is necessary to rigorously test these hypotheses and to study how MPK4 suppresses their accumulation and which signaling pathway in N. attenuata positively regulates these metabolites, and whether generalist S. littoralis feeding or FACs in low concentrations induce the JIDs.

Unlike SIPK and WIPK, which are important in wounding- and generalist and specialist herbivore-induced responses, MPK4 is only required in the interaction between N. attenuata and M. sexta, but not S. littoralis. Furthermore, our bioassays using generalist and specialist insects and genetically modified plants also revealed herbivore-specific effectiveness of different secondary metabolites. These findings are consistent with the expectations of an evolutionary arms race between plants and herbivores (Becerra et al., 2009; Ali & Agrawal, 2012). Our previous study has indicated that MPK4 plays a key role in controlling various ecologically important traits. irMPK4 plants are very sensitive to drought stress and deficient in stomatal closure responses because of their insensitivity to abscisic acid; furthermore, they are also highly compromised in guard cell-based resistance to surface-deposited bacterial pathogens (Hettenhausen et al., 2012). Here, we show that MPK4 is likely to be part of a signaling network that is important for N. attenuata to recognize different types of predators: it negatively affects plant defense against the specialist herbivore, M. sexta, but not against S. littoralis. Thus, in N. attenuata, MPK4 has both positive and negative functions in plant interactions with various environmental factors. Large-scale transcriptome analysis and phosphorylation target screening will shed further light on the molecular basis of MPK4 function, such as the activation of transcription factors that are downstream of MPK4. The identification of the specific anti-M. sexta compounds (JIDs) in irMPK4, which are regulated by the JA-independent pathway, and elucidation of their biosynthesis pathways, will enrich our understanding of this new form of herbivore defense. It is also important to discover the positive signaling components that influence their biosynthesis, which could be other MAPKs.

Acknowledgements

We thank Dr Mario Kallenbach for synthesizing FACs, Dr Klaus Gase, Susan Kutschbach and Wibke Kröber for technical assistance, Dr Tamara Krügel, Andreas Schünzel and Andreas Weber for plant cultivation, a European Research Council (ERC) advanced grant ClockworkGreen (no. 293926) to I.T.B. and the Max Planck Society for financial support.

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