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Plant Cell. Sep 2011; 23(9): 3512–3532.
Published online Sep 16, 2011. doi:  10.1105/tpc.111.088229
PMCID: PMC3203443

Nicotiana attenuata LECTIN RECEPTOR KINASE1 Suppresses the Insect-Mediated Inhibition of Induced Defense Responses during Manduca sexta Herbivory[C][W]

Abstract

Nicotiana attenuata has the capacity to respond specifically to herbivory by its natural herbivore, Manduca sexta, through the perception of elicitors in larval oral secretions. We demonstrate that Lectin receptor kinase 1 (LecRK1) functions during M. sexta herbivory to suppress the insect-mediated inhibition of jasmonic acid (JA)–induced defense responses. Gene function analysis performed by reducing LecRK1 expression in N. attenuata by both virus-induced gene silencing and inverted repeated RNA interference (ir-lecRK1 plants) revealed that LecRK1 was essential to mount a full defense response against M. sexta folivory; larvae growing on ir-lecRK1 plants were 40 to 100% larger than those growing on wild-type plants. The insect-induced accumulation of nicotine, diterpene-glucosides, and trypsin protease inhibitors, as well as the expression of Thr deaminase, was severalfold reduced in ir-lecRK1 plants compared with the wild type. The accumulation of JA and JA-Ile was unaffected during herbivory in ir-lecRK1 plants; however, salicylic acid (SA) accumulation was increased by twofold. The expression of nahG in ir-lecRK1 plants prevented the increased accumulation of SA and restored the defense response against M. sexta herbivory. The results suggest that LecRK1 inhibits the accumulation of SA during herbivory, although other mechanisms may also be affected.

INTRODUCTION

Plant species from a wide taxonomical distribution trigger and tailor defense and tolerance responses against lepidopteran larval folivory after perception of components (herbivore-associated elicitors [HAEs]) in the oral secretions (OSs) of the larvae (Tumlinson and Lait, 2005; Schmelz et al., 2009; Bonaventure et al., 2011a). These OS components function as chemical cues that convey sufficient information to the plant to initiate specific responses against the feeding insect (Howe and Jander, 2008; Wu and Baldwin, 2009). Moreover, some HAEs can also counteract the defense response of plants, thus being in this case beneficial to the insect (Musser et al., 2002; Diezel et al., 2009).

The HAEs that act during insect folivory are diverse in structure, ranging from enzymes (e.g., glucose oxidase; Musser et al., 2002) to modified forms of lipids (e.g., fatty acid–amino acid conjugates [FACs]; Alborn et al., 1997; Halitschke et al., 2001), sulfur-containing fatty acids (caeliferins; Alborn et al., 2007), and from fragments of cell walls (e.g., oligogalacturonides) to peptides released from digested plant proteins (e.g., inceptins; Schmelz et al., 2006).

FACs belong to a widely distributed family of HAEs present in the OS of a large number of lepidopteran species, including Manduca sexta (Yoshinaga et al., 2010), and they are necessary and sufficient to elicit herbivory-specific responses in several plant species including maize (Zea mays), soybean (Glycine max), eggplant (Solanum melongena), black nightshade (Solanum nigrum), and wild tobacco (Nicotiana attenuata; Alborn et al., 1997; Schmelz et al., 2009; VanDoorn et al., 2010; Bonaventure et al., 2011a). Some of the most abundant FACs are conjugates of Glu and Gln with unmodified or oxidized derivatives of polyunsaturated fatty acids (Alborn et al., 1997; Spiteller and Boland, 2003; VanDoorn et al., 2010; Yoshinaga et al., 2010). Recently, it has been demonstrated that FACs are essential for the assimilation of nitrogen by developing larvae (Yoshinaga et al., 2008), making it impossible for the caterpillars to feed stealthily without eliciting a defense response by the plant.

N. attenuata is an annual tobacco plant native to the deserts of the Southwestern US, and in its natural habitat it is frequently attacked by folivorous larvae of the specialist M. sexta, which is often responsible for the majority of leaf area lost to herbivores in natural populations. In N. attenuata, responses to M. sexta herbivory and M. sexta OS or FAC elicitation (treatments that simulate herbivory) overlap by >80% and strongly differ from those induced by mechanical damage (Halitschke et al., 2001). These responses include, among others, changes in the expression of >500 genes, 90 proteins, and 170 metabolites and the differential production of jasmonic acid (JA), ethylene (ET), and salicylic acid (SA; Hermsmeier et al., 2001; Giri et al., 2006; Gilardoni et al., 2010; Gaquerel et al., 2010; Kallenbach et al., 2010).

The biosynthesis of JA and JA-Ile and their perception are essential for the triggering of induced defense responses to chewing arthropods in different plant species (Staswick and Tiryaki, 2004; Wang et al., 2007; Howe and Jander, 2008; VanDoorn et al., 2011). In N. attenuata plants, critical defense responses that are induced during M. sexta folivory include the accumulation of the defense metabolites 17-hydroxygeranyllinalool diterpene glycosides (HGL-DTGs), nicotine, phenylpropanoid-polyamine conjugates, and protease inhibitors (PIs); when N. attenuata plants are engineered to produce reduced amounts of these molecules, they become severely compromised in their capacity to survive M. sexta attack (Steppuhn et al., 2004; Zavala et al., 2004; Jassbi et al., 2008; Kaur et al., 2010). In tomato (Solanum lycopersicum) and N. attenuata, the induction of threonine deaminase (TD) is also a critical induced defense response. TD participates in the deaminaton of Thr to form α-ketobutyrate for Ile biosynthesis, and in N. attenuata, reduced TD expression compromises the levels of JA-Ile formation in leaves and thereby the induction of defense responses such as nicotine and trypsin protease inhibitor (TPI) accumulation (Kang et al., 2006). In tomato, an active form of Sl-TD2 lacking its regulatory domain deaminates Thr in the gut of M. sexta larvae and thereby reduces the ingested levels of this essential amino acid for larval growth (Chen et al., 2005).

Although JA plays a prominent role in the induction of defense responses against chewing insects in plants, SA and ET can modulate either positively or negatively the induction of defense responses stimulated by JA (Doherty et al., 1988; Doares et al., 1995; O’Donnell et al., 1996; Stotz et al., 2002; Zarate et al., 2007; Diezel et al., 2009), and the antagonistic and synergistic effects between JA, SA, and ET signaling pathways have been extensively documented (Niki et al., 1998; Reymond and Farmer, 1998; Gupta et al., 2000; Li et al., 2004; Mur et al., 2006; Diezel et al., 2009; Leon-Reyes et al., 2009). Importantly, the stimulatory effect of JA and the modulation of the JA-mediated responses by SA and ET are the major known determinants of the final response triggered by plants during their interaction with lepidopteran larvae (Howe and Jander, 2008). Also important is the fact that the level of production of JA, SA, and ET in plants depends on the attacking herbivore; thereby, both plants and insects adjust or prevent, respectively, the induction of defense responses (Musser et al., 2002; Howe and Jander, 2008; Diezel et al., 2009).

At present, little is known about the signal transduction components operating specifically during insect herbivory and influencing the modulation of induced defense responses in plants during this biotic stress. Among the known signaling components acting early upon wounding and recognition of FACs or M. sexta OS in Nicotiana tabacum and N. attenuata plants are two mitogen-activated protein kinases (MAPKs), wound-induced protein kinase (WIPK), and salicylate-induced protein kinase (SIPK; Seo et al., 1999; Wu et al., 2007). In N. attenuata, SIPK and WIPK affect JA biosynthesis (Kallenbach et al., 2010) and a large number of induced defense responses (Wu et al., 2007), and in N. tabacum, they affect JA and SA accumulation after wounding (Seo et al., 2007). In tomato, the protein kinase 1b (TPK1b) and the MAPKs MPK1, MPK2, and MPK3 play important roles in mediating JA- and ET-dependent defense responses against folivorous insects (Kandoth et al., 2007; AbuQamar et al., 2008).

With the aim of identifying additional signal transduction components of the pathways mediating defense and tolerance responses against lepidopteran larvae, a SuperSAGE (serial analysis of gene expression) approach combined with 454 sequencing was recently used to quantify the early transcriptional changes elicited by the FAC N-linolenoyl-glutamic acid (18:3-Glu) in N. attenuata plants (Gilardoni et al., 2010). The analysis targeted mRNAs encoding regulatory components: rare transcripts with very rapid FAC-elicited kinetics. Among the 547 differentially expressed transcripts, >25% corresponded to putative regulatory components, including 22 protein kinases (Gilardoni et al., 2010). Here, we demonstrate that the expression of the N. attenuata Lectin receptor kinase 1 (LecRK1) is indispensable during M. sexta herbivory to suppress the insect-mediated inhibition of defense responses and thereby to stimulate the unfettered JA-mediated induction of defense metabolites.

RESULTS

Identification of LecRK1 as a Regulator of the Interaction between N. attenuata and M. sexta

From the SuperSAGE analysis published recently by Gilardoni et al. (2010), the UniTag-5358 was identified as a tag whose abundance was 11-fold enriched in the SuperSAGE library corresponding to 18:3-Glu elicited leaves compared with the library corresponding to wounded leaves. To unravel the role of the gene corresponding to UniTag-5358, we first obtained the full-length cDNA of this Unitag by 5′ and 3′ random amplification of cDNA ends (RACE). The full-length cDNA nucleotide sequence contained an open reading frame of ~2.5 kb encoding for a predicted polypeptide of 830 amino acids (see GenBank accession number in Methods). Analysis of sequence similarity by BLAST using the complete predicted amino acid sequence of UniTag-5358 showed that the protein sequence presented on average >80% sequence similarity to receptor-like kinases of the lectin domain superfamily (see Supplemental Figure 1 online; Bouwmeester and Govers, 2009). The closest homologs with known functions were the lectin receptor kinase protein LRK1 from Nicotiana benthamiana (Kanzaki et al., 2008), Pi-d2 from rice (Oryza sativa; Chen et al., 2006), and RLK1 from Nicotiana glutinosa (Kim et al., 2010; see Supplemental Figure 1 online); the function of these genes has been associated with defense responses against microbial pathogens.

The amino acid sequence of UniTag-5358 contains a predicted N-terminal extracellular region, a single transmembrane-spanning α-helix, and a C-terminal cytoplasmic region (Figure 1A). The extracellular N-terminal region contains a predicted 22–amino acid secretory pathway signal sequence (amino acids 1 to 22), a lectin domain from amino acids 55 to 177 (e-value 4.08e−16; SMART algorithm; Letunic et al., 2009), an epidermal growth factor-like domain from amino acids 310 to 364 (e-value 6.51e−02), and a PAN_AP (plasminogen-apple-nematode motif) domain from amino acids 364 to 445 (e-value 1.47e−02; Figure 1A). The predicted transmembrane domain extends from amino acids 476 to 490 (e-value 9.9e−02), and the C-terminal cytoplasmatic region contains a predicted Ser/Thr kinase domain from amino acids 519 to 788 (e-value 9.04e−31; Figure 1A; see Supplemental Figure 1 online).

Figure 1.
Na-LecRK1 Predicted Domains and mRNA Expression Pattern.

In Arabidopsis, LecRKs are divided in three classes based on the type of extracellular lectin domain: G-, C-, and L-type LecRKs (Bouwmeester and Govers, 2009). The G-type LecRKs present a G-type lectin motif, one epidermal growth factor-like domain, and PAN_AP domains in the N-terminal region (Bouwmeester and Govers, 2009); domains that were present in UniTag-5358 (Figure 1A; see Supplemental Figure 1 online). The gene corresponding to UniTag-5358 was therefore renamed Na-LecRK1 (for N. attenuata G-type lectin receptor kinase 1).

SIPK and WIPK Are Positive Regulators of LecRK1 Expression, Whereas JAs Have an Inhibitory Role

The kinetics of LecRK1 mRNA induction in leaves as a response to OS elicitation (from M. sexta, Spodoptera littoralis, and grasshopper [Schistocera gregaria]), 18:3-Glu elicitation, and wounding was first evaluated in N. attenuata wild-type plants. Consistent with the SuperSAGE data (Gilardoni et al., 2010), LecRK1 transcript levels were strongly increased (25- and 60-fold at 0.5 and 1 h after 18:3-Glu elicitation, respectively) compared with basal transcript levels in unelicited leaves (Figure 1B). Moreover, the increase was transient and LecRK1 mRNA levels decreased rapidly (within 2 h) to basal levels (Figure 1B). In wounded leaves, LecRK1 mRNA levels were also increased (approximately sevenfold) at 1 h (Figure 1B), showing that mechanical damage also induces the expression of this gene, albeit to levels lower than 18:3-Glu elicitation. Elicitation with M. sexta OS induced a 100-fold increase in LecRK1 transcript levels, and elicitation with S. littoralis OS induced a 50-fold increase (compared with basal levels; Figure 1B). By contrast, elicitation with S. gregaria OS showed an effect similar to wounding (Figure 1B), a result consistent with undetectable levels of FACs in the S. gregaria OS (Schmelz et al., 2009; Schäfer et al., 2011).

Second, we asked whether the induction of LecRK1 expression depended on SIPK and WIPK activities (these MAPKs regulate the earliest known molecular events that trigger defense responses against M. sexta herbivory; Wu et al., 2007). Plants with reduced expression of these two genes (ir-sipk and ir-wipk, respectively; Wu et al., 2007) were elicited with 18:3-Glu, and the LecRK1 mRNA levels were analyzed. The induction of this LecRK1 was delayed and reduced by approximately twofold in ir-sipk plants compared with the wild type, whereas it was completely absent in ir-wipk plants (Figure 1C).

Third, to evaluate whether the induction of LecRK1 mRNA levels was dependent on the endogenous production of JA and JA-Ile, plants with reduced expression of LIPOXYGENASE3 (LOX3) and CORONATINE INSENSITIVE1 (COI1) genes were elicited with 18:3-Glu. These plants, ir-lox3 and ir-coi1, are deficient in JA accumulation (Allmann et al., 2010) and JA signaling (Paschold et al., 2007), respectively. In contrast with ir-sipk and ir-wipk plants, the induction of LecRK1 transcript levels in ir-lox3 and ir-coi1 plants was accelerated and increased to levels threefold higher than in wild-type N. attenuata plants (Figure 1C). Together, these results indicate that, after 18:3-Glu elicitation, SIPK and WIPK are positive regulators of LecRK1 gene expression, whereas JAs have an inhibitory effect.

Finally, the tissue-specific expression of LecRK1 was studied, and based on the transcript levels, LecRK1 was expressed ubiquitously in all tissues analyzed, with higher levels of expression in roots and stems compared with rosette leaves and flower parts (Figure 1D).

Silencing of LecRK1 Expression Transiently by VIGS and Stably by Inverted-Repeat RNA Interference Reveals a Critical Function of This Gene in N. attenuata Defense Responses against M. sexta Folivory

The function of LecRK1 in the response of N. attenuata to M. sexta herbivory was first evaluated by transiently reducing the expression of LecRK1 by virus-induced gene silencing (VIGS). An 86-bp fragment corresponding to the 3′-untranslated region of the LecRK1 mRNA was used to reduce its expression via the tobacco rattle virus system (Ratcliff et al., 2001) and Agrobacterium tumefaciens leaf infiltration (Saedler and Baldwin, 2004). Fifteen days after leaf infiltration, the LecRK1 mRNA levels were analyzed in newly emerged leaves of plants infected to reduce LecRK1 expression (NaLecRK1-VIGS) and control plants infected with the empty vector (EV-VIGS). LecRK1 transcript levels were reduced between 85 and 90% after wounding and 18:3-Glu and M. sexta OS elicitation in NaLecRK1-VIGS plants compared with EV-VIGS plants (see Supplemental Figure 2A online).

Second, freshly hatched M. sexta neonates were placed on these plants, and the larval masses were determined at 4, 7, and 11 d after the start of the experiment. Larvae feeding on NaLecRK1-VIGS plants gained ~50 and 100% more mass after 7 and 11 d, respectively, compared with larvae feeding on EV-VIGS plants (see Supplemental Figure 2B online). These results suggested that LecRK1 plays an important role in the regulation of the interaction between N. attenuata and M. sexta.

To examine in further detail the function of LecRK1, stably transformed N. attenuata plants with reduced expression of LecRK1 were generated by inverted repeat–mediated RNA interference (see Methods for a detailed description of the generation of these plants). Two homozygous independently transformed lines named ir-lecRK1-378 and ir-lecRK1-380, respectively, were selected and used for all the experiments described below (Figure 2). These lines carried a single T-DNA insertion in their genomes (Figure 2A), and the levels of LecRK1 mRNA were reduced on average by 95% compared with wild-type plants after 18:3-Glu elicitation (Figure 2B). The growth and morphology of ir-lecRK1 plants were indistinguishable from those of wild-type plants at all stages of development (Figure 2C).

Figure 2.
Silencing Efficiency in ir-lecRK1 Plants and Growth Morphology.

To confirm the results observed with NaLecRK1-VIGS plants, freshly hatched M. sexta larvae were placed on wild-type, ir-lecRK1-378, and ir-lecRK1-380 plants, and the mass of the larvae was determined at 4, 7, and 11 d after the start of the experiment. Larvae growing on ir-lecRK1 plants gained 30 to 35% more mass after 11 d of feeding than larvae growing on wild-type plants (Figures 3A and and3B).3B). Estimation of the leaf area consumed after 11 d showed that ir-lecRK1 plants lost on average 2 times the leaf area of wild-type plants (Figure 3A, inset). The levels of total protein and starch in leaves were similar between ir-lecRK1 and wild-type plants (see Supplemental Figure 3 online). In conclusion, the stable reduction of LecRK1 expression in transgenic plants was consistent with the function of this gene in the regulation of defense responses in N. attenuata against M. sexta herbivory.

Figure 3.
ir-lecRK1 Plants Are More Susceptible to M. sexta Herbivory.

Reduction of LecRK1 Expression Increases the Accumulation of SA after Simulated Herbivory

To evaluate whether the reduced expression of LecRK1 affected the production of JA, JA-Ile, SA, and ET, the levels of these phytohormones were quantified in ir-lecRK1 and wild-type plants at different times after wounding and M. sexta OS elicitation. The accumulation of JA was similar between wild-type and ir-lecRK1 plants after both treatments (Figure 3C; see Supplemental Figure 4A online). By contrast, 1 h after OS elicitation, JA-Ile levels were reduced by 34 and 26% in ir-lecRK1-378 and ir-lecRK1-380 plants, respectively, compared with wild-type plants (Figure 3D). At 1 and 3 h after wounding, the levels of JA-Ile were similar between wild-type and ir-lecRK1 plants (see Supplemental Figure 4B online). Interestingly, 3 h after OS elicitation, the levels of SA were increased by two- and 2.5-fold in ir-lecRK1-378 and ir-lecRK1-380 plants, respectively, remaining 1.4-fold higher at 4 h compared with wild-type plants (Figure 3E). After wounding, the SA levels were similar between wild-type plants and ir-lecRK1 plants (see Supplemental Figure 4C online). Finally, the levels of ET produced by wild-type and ir-lecRK1 plants after wounding and 18:3-Glu or M. sexta OS elicitation were also similar (see Supplemental Figure 5 online).

To investigate if the changes in SA accumulation after M. sexta OS elicitation in ir-lecRK1 plants were associated with changes in the expression of genes related to SA biosynthesis or signaling, the transcript levels of isochorismate synthase (ICS) and non-expressor of PR-1 (NPR1) were quantified after this treatment. The mRNA levels of ICS were rapidly decreased after the treatment in both wild-type and ir-lecRK1 plants; however, the repression was slower in the latter, with ICS mRNA levels remaining six- and twofold higher than in wild-type plants at 0.5 and 1 h, respectively (see Supplemental Figure 6A online). The mRNA levels of NPR1 were induced by fourfold at 1 h after M. sexta OS elicitation, whereas they were reduced to basal levels at 2 h in wild-type plants, and they remained threefold higher in ir-lecRK1 plants (see Supplemental Figure 6 online). Finally, expression of phenylalanine ammonia lyase 2, an SA-responsive gene, was increased by threefold at 1 h after the treatment in ir-lecRK1 plants compared with the wild type (see Supplemental Figure 6C online).

Metabolic Profiling of ir-lecRK1 Plants Reveals Strong Changes in the Accumulation of Defense Metabolites

Because defense responses of N. attenuata against M. sexta herbivory rely strongly on the de novo synthesis of defense metabolites and thereby on changes in primary and secondary metabolism, the differential accumulation of metabolites in OS-elicited leaves from ir-lecRK1 and wild-type plants was profiled by liquid chromatography–time-of-flight–mass spectrometry (LC-ToF-MS) analysis. OS-elicited leaves were harvested at 12 h and 3 and 6 d after the treatment, and polar metabolites were analyzed using a modified version of a previously described LC-ToF-MS method (Gaquerel et al., 2010; see Methods for a detailed description of the method used). Positively charged metabolites were selected using the electrospray ionization (ESI) interface in the positive ion mode, and those metabolites eluting from the column between 125 and 550 s and having mass-to-charge (m/z) values ranging from 90 to 1400 were selected for analysis (see Methods for a detailed description of the analysis). Using the conditions mentioned above and after data processing, a total of 2831 ions were identified (see Supplemental Data Set 1 online). To identify the ions that accumulated differentially in wild-type and ir-lecRK1-378 plants, the fold changes in ion abundance between these two genotypes were calculated. Ion abundances that differed statistically (P value ≤ 0.05) and had fold changes larger than 1.5 and smaller than 0.67 (ir-lecRK1-378 versus the wild type) were considered as up- and downregulated, respectively. Although this selection was arbitrary, it has proved useful for the identification of differentially regulated metabolites in N. attenuata plants during M. sexta herbivory (Gaquerel et al., 2010). Using these conditions, a total of 148 ions accumulated differentially after OS elicitation in ir-lecRK1-378 versus wild-type plants with 57 up- and 91 downregulated (see Supplemental Data Set 2 online). Most of these ions accumulated specifically at particular times with only seven ions accumulating differentially after both 12 h and 3 d of the treatment and two ions after both 12 h and 6 d after the treatment (Figure 4A).

Figure 4.
Leaf Metabolic Profiling of ir-lecRK1 and Wild-Type Plants.

To facilitate the graphical interpretation of the differences among genotypes and times of treatments, the data set corresponding to the differentially accumulating ions was first analyzed by principal component analysis (PCA). The first and second principal components (PCs) together explained 64.7% of the variation within this data set, and PC1 and 2 clearly separated the samples based on genotype and times of the treatments (see Supplemental Figure 7A online). A supervised method, partial least squares discriminant analysis (PLSDA), was then used to help with the identification of those ions with stronger effects on the separation of samples. The first and second PCs of the PLSDA analysis explained 62.4% of the variation in the data set, and, similar to PCA analysis, these two PCs clearly separated ir-lecRK1-378 samples from wild-type samples at the different times of the experiment (Figure 4B). The PLSDA analysis was validated by a permutation test as previously described (Westerhuis et al., 2008; see Supplemental Figure 7B online) and was used to calculate the variable importance in the projection (VIP) value to estimate and rank the influence of individual ions on the separation of the samples by the PLSDA model. VIP values equal to or larger than 1.0 were considered significant, and the higher the VIP value was, the stronger was its influence on the separation of the samples (Xie et al., 2008; Qiu et al., 2009). A total of 42 ions presented VIP values larger or equal to 1.0 and 16 of these ions were downregulated and 26 upregulated at specific times (see Supplemental Data Set 3 online). The fold change in the abundance of these 42 ions ranged from 0.0025 to 0.67 for the downregulated ions and from 1.5 to 146 for the upregulated ions (ir-lecRK1 versus the wild type; see Supplemental Data Set 3 online).

To define the identity of the 148 differentially accumulating ions, a search in the public metabolite database and in custom databases (Gaquerel et al., 2010; see Methods) was performed using the m/z values (with an error of ±0.02) and the retention times. Approximately 87% of the ions did not match significantly to any of the metabolites in the databases, and for some of these metabolites, a theoretical elemental molecular formula could be assigned with a significant degree of confidence (Table 1; see Supplemental Data Sets 2 and 3online) . Importantly, among the identified metabolites, several were involved in defense responses against M. sexta herbivory. Different ions corresponding to nicotine and HGL-DTGs were found significantly downregulated at different times after OS elicitation in ir-lecRK1-378 compared with wild-type plants (Table 1). N. attenuata accumulates 11 different HGL-DTG forms differing from each other in the sugar moiety they carry and in the number of malonyl ester groups in the sugar moieties (Heiling et al., 2010). Among the 11 HGL-DTG forms, Lyciumoside IV and Attenoside were reduced in ir-lecRK1-378 at 12 h and 3 d after OS elicitation, while the monomalonylated HGL-DTGs Nicotianoside I, Nicotianoside IV, and Nicotianoside VI and the dimalonylated HGL-DTGs Nicotianoside II were reduced at 6 d after the treatment (Table 1; see Supplemental Data Set 2 online). Nicotine and Nicotianoside II were among the metabolites with VIPs greater or equal to 1.0 (Figure 4C; see Supplemental Data Set 3 online), indicating that they strongly contributed to the separation of samples observed in the PLSDA analysis (Figure 4B).

Table 1.
List of Ions Corresponding to Defense Metabolites Differentially Accumulating in Leaves of ir-lecRK1 Plants after M. sexta OS Elicitation

LecRK1 Expression Affects the M. sexta OS-Mediated Induction of Nicotine and HGL-DTG Levels

Previous studies have shown that M. sexta larvae fed on N. attenuata plants with reduced levels of nicotine or HGL-DTGs gained severalfold more mass than larvae fed on wild-type plants (Steppuhn et al., 2004; Jassbi et al., 2008). Based on the reductions in nicotine and HGL-DTGs levels detected in ir-lecRK1-378 plants by metabolic profiling, nicotine and total HGL-DTGs were quantified by HPLC in the wild type and the two ir-lecRK1 lines (378 and 380) at different times after wounding or M. sexta OS elicitation. The nicotine levels in unelicited and wounded leaves were similar between wild-type and ir-lecRK1 plants (Figure 5A; see Supplemental Figure 8A online). By contrast, at 3 and 6 d after OS elicitation, the induced nicotine levels in ir-lecRK1-378 and ir-lecRK1-380 plants were on average 30 and 40% reduced, respectively, compared with wild-type plants (Figure 5A).

Figure 5.
ir-lecRK1 Plants Induce Reduced Levels of Nicotine and HGL-DTGs after M. sexta OS Elicitation.

In unelicited and wounded leaves, total HGL-DTG levels were similar between wild-type and ir-lecRK1 plants (Figure 5B; see Supplemental Figure 8B online); however, the levels of induced HGL-DTGs were reduced on average by 60 and 40% at 3 and 6 d, respectively, after OS elicitation in ir-lecRK1 plants compared with wild-type plants (Figure 5B). Analysis of the different forms of HGL-DTGs revealed that the amounts of the precursor molecules Lyciumoside I and Lyciumoside II were similar between wild-type and ir-lecRK1-378 plants at the different times of the experiment; however, the core molecules Lyciumoside IV (the most abundant HGL-DTG form) and Nicotianoside III were reduced by 32 and 43%, respectively, at 3 d after the treatment in ir-lecRK1 plants compared with wild-type plants (Figures 5C and and5D).5D). At day 6 after the treatment, the reduction in Lyciumoside IV amounts was of 60% (Figure 5D). The levels of the monomalonylated HGL-DTGs Nicotianoside I and Nicotianoside IV were reduced by 70 and 24%, respectively, in ir-lecRK1 plants compared with wild-type plants at 6 d after OS elicitation (Figure 5D). The levels of the dimalonylated HGL-DTGs Nicotianoside II, Nicotianoside V, and Nicotianoside VII were reduced by 85, 86, and 78%, respectively, in ir-lecRK1 plants compared with wild-type plants at 6 d after the treatment (Figure 5D). In summary, the accumulation of most of the core and mono- and dimalonylated forms of HGL-DTG molecules was reduced in ir-lecRK1 plants after M. sexta OS elicitation.

The transcript levels of the plastidial geranylgeranyl pyrophosphate synthase, the enzyme involved in the biosynthesis of HGL-DTGs (Jassbi et al., 2008), were reduced twofold in leaves of ir-lecRK1 compared with wild-type plants at 24 and 72 h after OS elicitation (see Supplemental Figure 9A online), suggesting that LecRK1 affects (at least partially) the induced levels of HGL-DTGs by affecting the expression of the biosynthetic enzyme geranylgeranyl pyrophosphate synthase.

The phenyl-propanoid-polyamine conjugates caffeoylputrescine and dicaffeoylspermidine and the phenyl-propanoid derivatives chlorogenic acid and rutin are metabolites whose accumulation has been associated with defense against M. sexta herbivory in N. attenuata (Kaur et al., 2010). The amounts of these four metabolites were quantified in ir-lecRK1 and wild-type plants at 3 d after both wounding and M. sexta OS elicitation, and their levels did not differ between wild-type and ir-lecRK1 plants (see Supplemental Figure 10 online). These results are consistent with the metabolic profiling analysis that showed no significant differences in the accumulation of these metabolites (see Supplemental Data Set 1 online) and indicate that LecRK1 specifically affects the accumulation of some defense metabolites in N. attenuata.

LecRK1 Expression Affects the M. sexta OS-Mediated Induction of TPI Activity

TPIs are induced during M. sexta herbivory in N. attenuata plants, and they play a critical role as direct defenses by inhibiting the hydrolysis of ingested proteins in the digestive system of M. sexta larvae; N. attenuata plants with reduced expression of TPI are compromised in their defense against M. sexta herbivory (Zavala et al., 2004). To evaluate whether LecRK1 affected the levels of TPI activity, leaves from wild-type and ir-lecRK1 plants were elicited with M. sexta OS, and TPI activity was quantified at 2 and 3 d after the treatment. In unelicited leaves, TPI activity was similar between wild-type and ir-lecRK1 plants (Figure 6), and the induced levels did not differ between genotypes after wounding (see Supplemental Figure 8C online). By contrast, after OS elicitation, the induced TPI activity was reduced by 40% in ir-lecRK1 plants compared with the wild type (Figure 6). In agreement, the induction of TPI transcript levels was reduced by ~50% in ir-lecRK1-378 plants compared with wild-type plants at 24 h after OS elicitation (see Supplemental Figure 9B online). Thus, not only nicotine and HGL-DTGs were induced to lower levels than in wild-type in ir-lecRK1 plants but also TPI expression and activity, indicating that, although LecRK1 affects the specific accumulation of some defense metabolites, it may have a broader effect on the induction of defense responses against herbivores.

Figure 6.
ir-lecRK1 Plants Induce Reduced Levels of TPIs.

The Expression of TD Is Strongly Reduced in Leaves of ir-lecRK1 Plants after OS Elicitation

To gain further insights into the mechanisms affected in ir-lecRK1 plants, changes in gene expression at 1 h after M. sexta OS elicitation were evaluated by microarray analysis. Genes were considered to be differentially regulated when log2(fold changes [FCs]) were larger or equal to 1.3 or smaller or equal to −1.3 (ir-lecRK1-378 versus the wild type) and q-values were lower than 0.05 (corresponding to a 4% false discovery rate [FDR]; see Methods). Using these conditions, transcripts corresponding to 78 genes were identified as differentially regulated in ir-lecRK1-378 compared with the wild type, with 41 down- and 37 upregulated (see Supplemental Data Set 4 online). Thirty of these 78 genes did not match significantly with any of the protein entries in the nonredundant GenBank protein database and six matched with hypothetical proteins (see Supplemental Data Set 4 online).

Among the most strongly downregulated genes was, as expected, LecRK1 [log2(FC) = −3.1] and, interestingly, TD [log2(FC) = −2.3; see Supplemental Data Set 4 online). Additional genes downregulated in ir-lecRK1 plants and potentially associated with defense responses were N. attenuata homologs of a putative protease inhibitor-I from Nicotiana sylvestris and of a protein from N. tabacum able to induce hypersensitive response–like lesions. In the group of upregulated genes with a predicted function in defense responses, a putative PR-10–type pathogenesis-related protein was sixfold upregulated in ir-lecRK1 compared with the wild type (see Supplemental Data Set 4 online).

Among the remaining genes that changed expression were N. attenuata homologs of genes involved in the regulation of gene expression (e.g., Histone H2A variant 1 and Parafibromin 1), metabolic enzymes (e.g., Choline kinase 1, cellulose synthase-like D2, and thiamine biosynthesis protein ThiC), and signal transduction components (e.g., phytochrome F and NUCLEOSOME ASSEMBLY PROTEIN 1;2; see Supplemental Data Set 4 online and Discussion).

TD Downregulation in ir-lecRK1 Plants Affects the Levels of Thr Ingested by M. sexta Larvae

Based on the microarray results and knowing that TD plays important roles in defense responses against lepidopteran larvae in both tomato (Chen et al., 2005) and N. attenuata (Kang et al., 2006), we reasoned that the reduced levels of expression of this gene in ir-lecRK1 plants could contribute to the reduced defense response observed in these plants during M. sexta herbivory. To validate the microarray results, the transcript levels of TD were first quantified by quantitative PCR (qPCR) in leaves of ir-lecRK1 and wild-type plants at different times after M. sexta OS elicitation. TD mRNA levels were reduced by 75 and 50% at 30 and 60 min after the treatment, respectively, in ir-lecRK1 compared with wild-type plants (Figure 7A).

Figure 7.
Reduced TD Expression in ir-lecRK1 Plants Affects Thr Accumulation in the Midgut of M. sexta Larvae.

Second, the levels of Thr were quantified in the midgut content and tissue of M. sexta larvae fed for 11 continuous days on either wild-type, ir-lecRK1, or N. attenuata plants with reduced levels of TD (as-tdm2; Kang et al., 2006). The larvae were dissected to isolate the midgut and to separate the midgut tissue from its contents. In parallel, the leaves of the plants were also harvested after 11 d of larval feeding and used to quantify Thr amounts. The Thr levels in the midgut tissue of M. sexta larvae fed on ir-lecRK1-378 and as-tdm2 plants were 34 and 50% higher, respectively, than the Thr levels in the midgut tissue of larvae fed on wild-type plants (Figure 7B). The Thr levels in the midgut content of the larvae fed on ir-lecRK1-378 and as-tdm2 plants were 70 and 60% higher, respectively, than the Thr levels found in the midgut content of larvae fed on wild-type plants (Figure 7C). Due to the high water content in the midgut content, in this case Thr levels were expressed as mol % of the levels of all amino acids (Figure 7C). In leaves, the levels of Thr and JA-Ile were similar between wild-type, as-tdm2, and ir-lecRK1-378 plants (Figures 7D and and7E),7E), whereas the levels of SA were 100% higher in ir-lecRK1-378 plants compared with wild-type and as-tdm2 plants (Figure 7F).

Expression of nahG in ir-lecRK1 Plants Restores the Defense Response against M. sexta Herbivory

The results presented in the previous section showed that, during continuous M. sexta larval feeding, similar levels of JA-Ile accumulate in wild-type and ir-lecRK1 plants but increased SA levels in the latter. Based on these results and the increased accumulation of SA in ir-lecRK1 plants upon M. sexta OS elicitation (Figure 3E), we reasoned that the increased performance of M. sexta larvae on ir-lecRK1 plants could be the result of an SA-mediated suppression of induced defense responses. To test this hypothesis, transgenic N. attenuata plants ectopically expressing the Pseudomonas putida nahG gene under the control of the cauliflower mosaic virus 35S promoter (ov-nahG; see Supplemental Figure 11 online) were generated and crossed with ir-lecRK1-378 plants to generate ir-lecRK1xov-nahG plants. As a control, ir-lecRK-378 plants were also crossed with wild-type plants to generate ir-lecRK1xWT plants. It is important to note at this point that the basal levels of SA in ov-nahG and wild-type plants were similar; however, after a treatment that strongly induces SA levels (e.g., leaf infiltration with pathogenic bacteria), the accumulation of SA was strongly suppressed in ov-nahG plants (see Supplemental Figure 11 online), indicating that the ectopic expression of nahG efficiently prevented the accumulation of induced levels of SA. Also important, the ectopic expression of nahG did not affect the accumulation of JA, JA-Ile, or ET after M. sexta OS elicitation (see Supplemental Figure 12 online).

The levels of SA were first quantified in wild-type, ir-lecRK1-378, ov-nahG, ir-lecRK1xov-nahG, and ir-lecRK1xWT plants at 3 h after OS elicitation (Figure 8A). SA levels were twofold higher in ir-lecRK1 and in ir-lecRK1xWT plants compared with the wild type and similar to wild-type levels in ov-nahG and ir-lecRK1xov-nahG plants (Figure 8A). The levels of SA were also quantified in leaves of these plants at 11 d after larval feeding, and the results were similar to those observed after OS elicitation (see Supplemental Figure 13A online). Thus, the ectopic expression of nahG in ir-lecRK1 plants suppressed the OS- and M. sexta–elicited SA burst. The levels of JA and JA-Ile were also quantified in leaves at 11 d after M. sexta larval feeding, and the accumulation of these two molecules was similar between the genotypes (see Supplemental Figures 13B and 13C online).

Figure 8.
Suppression of SA Accumulation in ir-lecRK1 Plants Recovers the Defense Response against M. sexta Herbivory.

Freshly hatched M. sexta neonates were placed on the five genotypes mentioned above and the larval masses were determined at 4, 7, and 11 d after the start of the experiment. Strikingly, larvae feeding on ir-lecRK1xov-nahG gained similar weight to larvae feeding on wild-type and ov-nahG plants and comparatively less weight than larvae feeding on ir-lecRK1-378 and ir-lecRK1xWT plants (Figure 8B).

The induced levels of total HGL-DTGs in ir-lecRK1xov-nahG plants were similar to those in ov-nahG and wild-type plants and twofold higher compared with the levels in ir-lecRK1-378 and ir-lecRK1xWT plants (Figure 8C). The levels of induced TPI activity in lecRK1xov-nahG and ov-nahG plants were statistically similar to wild-type plants and higher than in ir-lecRK1 and ir-lecRK1xWT plants (Figure 8D). Induced nicotine levels were increased by 15% in ir-lecRK1xov-nahG compared with ir-lecRK1 plants but remained 14% lower than in the wild type (Figure 8E). Finally, TD transcript levels were increased threefold in ir-lecRK1xov-nahG compared with ir-lecRK1 plants; however, the levels remained twofold lower than in wild-type plants (Figure 8F). In summary, the suppression of SA accumulation in ir-lecRK1 plants restored the induced levels of HGL-DTGs and TPI activity to wild-type levels, while induced nicotine levels and TD expression were partially restored.

DISCUSSION

Na-LecRK1 Encodes a G-Type Lectin Receptor Kinase

Based on amino acid sequence similarity and according to the proposed classification of lectin receptor-like kinases (LRKs; Bouwmeester and Govers, 2009), Na-LecRK1 belongs to the G-type LRK family, and the best-studied members of this subfamily are the S-locus receptor kinases that function in self-incompatibility mechanisms during pollination (Takayama and Isogai, 2005). Recently, some G-type LecRKs have also been associated with defense responses against pathogenic microorganisms. For example, the rice Pi-d2 LecRK protein participates in the resistance mechanisms against Magnaporthe grisea (Chen et al., 2006) and RLK1 from N. glutinosa participates in the resistance mechanisms against Phytophthora capsici (Kim et al., 2010). In the group of L-type LecRKs, LRK1 from N. benthamiana interacts with the Phytophthora infestans elicitin INF1 and triggers INF1-induced cell death (Kanzaki et al., 2008), and the Arabidopsis thaliana LecRK-I.9 has been identified as a putative mediator of cell wall–plasma membrane adhesion during infection with Phytophthora species (Bouwmeester et al., 2011). LecRK-I.9 binds to a P. infestans effector protein through an RGD (Arg-Gly-Asp) motif that is also present as a cell attachment motif in extracellular matrix proteins that mediate cell adhesion to the cell walls (Bouwmeester et al., 2011). In this study, we show that LecRKs also participate in mechanisms regulating the induction of defense responses against folivorous insects.

The Induction of LecRK1 Expression Is Tightly Regulated by OS/FACs and JAs

Elicitation of leaves from wild-type N. attenuata plants with OS from the generalist Spodoptera exigua induces the accumulation of SA to levels higher than those induced by OS from the specialist M. sexta, whereas the latter induces the accumulation of higher levels of JA and ET (Diezel et al., 2009). The same study showed that the lower levels of M. sexta OS-elicited SA are brought about by a suppression of SA accumulation mediated by enhanced ET levels; however, pathways independent of ET also operate (Diezel et al., 2009). As a final outcome, the induction of defense responses is attenuated after elicitation of leaves with S. exigua OS compared with M. sexta OS (Diezel et al., 2009).

In ir-lecRK1 plants, elicitation of leaves by M. sexta OS and larval folivory induced the accumulation of SA to levels twofold higher than those in wild-type plants, suggesting that LecRK1 is one signal transduction component operating during M. sexta herbivory to suppress SA accumulation. Interestingly, the levels of ET produced by ir-lecRK1 plants were similar to those produced by wild-type plants, indicating that the LecRK1-mediated signaling pathway works in a pathway independent of the ET-mediated suppression of SA accumulation.

Analysis of LecRK1 mRNA expression in plants deficient in JA biosynthesis or perception suggested that jasmonates inhibit the induction of this gene (Figure 1C). Moreover, plants with reduced expression of SIPK and WIPK showed that these two regulatory components have a positive effect on the expression of LecRK1, consistent with their central role in the activation of defense responses against M. sexta herbivory (Wu et al., 2007; Figure 9). These results revealed that the induction of LecRK1 expression is under tight control; it is induced by OS/FAC elicitation but the levels of induction are checked by JAs (in a COI1-dependent manner), which are also produced after OS/FAC elicitation. Thus, in this case, jasmonate levels would tune LecRK1 expression and thereby the accumulation of SA levels during insect herbivory (Figure 9).

Figure 9.
Proposed Model for the Role of LecRK1 in the N. attenuata Defense Response against M. sexta.

The Induced Accumulation of Central Defense Metabolites Is Affected by Reducing Expression of LecRK1 in N. attenuata Plants

When N. attenuata plants are reduced either in the accumulation of HGL-DTGs or nicotine or in the expression of TPI or TD, M. sexta larvae feeding on these plants can gain from 50 to 300% more mass than larvae feeding on wild-type plants (Steppuhn et al., 2004; Zavala et al., 2004; Kang et al., 2006; Jassbi et al., 2008). Consistent with these previous studies, an increased performance of M. sexta larvae on ir-lecRK1 plants was accompanied by reductions in the induced accumulation of these defense metabolites or proteins (Figures 5 to to77).

In agreement with the role of TD in the deamination of Thr in the insect gut (Chen et al., 2005), the levels of Thr in the midgut tissue and content of M. sexta larvae fed on ir-lecRK1 were 34 and 70% higher, respectively, than the level of Thr in the midgut tissue and content of larvae fed on wild-type plants (Figures 7B and and7C).7C). Also in agreement with the role of TD in the supplying of Ile for JA-Ile biosynthesis in leaves after M. sexta OS elicitation (Kang et al., 2006), the accumulation of JA-Ile was reduced on average by 30% at 1 h after this treatment (Figure 3D). TD presents 77 and 78% amino acid sequence similarity to tomato TD1 and TD2, respectively (Chen et al., 2007). In tomato, TD1 is involved in Ile biosynthesis in leaves, while TD2 participates in the deamination of Thr in the gut of M. sexta larvae (Chen et al., 2007). Our results suggest that TD participates in both processes in N. attenuata.

After 11 d of continuous M. sexta larval feeding, the levels of JA-Ile were similar between wild-type and ir-lecRK1 plants (Figure 7E), indicating that the reduced levels of TD expression in the latter did not affect the accumulation of JA-Ile after several days of larval feeding. This effect could be brought about, for example, by changes in the metabolism of JA-Ile in ir-lecRK1 plants. It has been shown that not only the biosynthesis but also the metabolism of JA-Ile is tightly regulated in plants, as exemplified by the misregulated accumulation of JA-Ile in plants with reduced expression of COI1 (Paschold et al., 2008; VanDoorn et al., 2011). Importantly, together with the twofold increased levels of SA observed after 11 d of M. sexta larvae continuous feeding (Figure 7F), the results suggest that the reduced induction of nicotine, HGL-DTGs, TPI, and TD levels was the result not of reduced JA-Ile accumulation, but instead of a suppressive effect of SA.

The Suppression of SA Accumulation in N. attenuata during M. sexta Herbivory Is Linked to the Unfettered Induction of Defense Responses

In tomato, it has been shown that the SA produced upon pathogen infection inhibits the accumulation of JA-induced PIs (Doares et al., 1995) and that the positive regulation of PI gene expression by JA and ET is suppressed by exogenous application of SA (O’Donnell et al., 1996). Similarly, in N. sylvestris and N. attenuata plants, the JA-induced accumulation of nicotine is suppressed by the exogenous application of methyl-salicylate, salicylhydroxamic acid, and acetosalicylic acid (Baldwin et al., 1996, 1997). Consistent with this, N. attenuata plants with increased accumulation of endogenous SA levels (two- to threefold) produce lower levels of nicotine, caffeoylputrescine, and rutin during herbivory (Rayapuram and Baldwin, 2007).

Although the levels of OS- and M. sexta–elicited SA were only moderately increased (twofold) in ir-lecRK1 plants (Figures 3E, ,7F,7F, and and8A;8A; see Supplemental Figure 13A online), the expression of nahG in these plants suppressed the increased SA accumulation (Figure 8A) and recovered the defense response against M. sexta herbivory (Figure 8B). Moreover, total HGL-DTG levels and TPI activity were fully recovered in ir-lecRK1xov-nahG plants (Figures 8C and and8D),8D), suggesting that the accumulation of these compounds was fully inhibited by SA in ir-lecRK1 plants. By contrast, nicotine levels were only partially recovered in lecRK1xov-nahG plants compared with wild-type plants (Figure 8E). This partial recovery was probably due to the fact that nicotine levels were significantly reduced by 15% in ov-nahG plants compared with the wild type, indicating that the ectopic expression of nahG has a slight negative effect on nicotine accumulation (Figure 8E). However, M. sexta larvae performed similarly on wild-type and ov-nahG plants (Figure 8A), indicating that defense responses were not impaired in the latter. Consistent with this, the levels of HGL-DTGs, TPI activity, and TD expression were not significantly different between wild-type and ov-nahG plants (Figures 8C, 8D, and and8F8F).

Similar to induced nicotine accumulation, TD expression was also partially recovered in ir-lecRK1xov-nahG plants compared with the wild type, and since TD expression levels were statistically similar between wild-type and ov-nahG plants, this partial recovery was probably the result of a partial involvement of the OS-elicited SA burst in the suppression of TD expression in ir-lecRK1 plants (Figure 8F).

In Arabidopsis, van Wees and Glazebrook (2003) showed that the ectopic expression of nahG induces the accumulation of catechol, which renders the plants more susceptible to P. syringae infection. However, Heck et al. (2003) demonstrated that these changes in defense resistance were not an effect of catechol and hypothesized that nahG may use multiple substrates in addition to SA. In tobacco, catechol has also no effect on the plant’s resistance response to the tobacco mosaic virus (Friedrich et al., 1995). Moreover, the same study showed that nahG has higher specificity toward SA as a substrate compared with other aromatic molecules (Friedrich, et al., 1995). In Arabidopsis, the ectopic expression of nahG also alters JA and ET accumulation after inoculation with P. syringae (Heck et al., 2003). In our case and in contrast with Arabidopsis, N. attenuata ov-nahG plants were not affected in the accumulation of jasmonates and ET after M. sexta OS elicitation. Thus, the pleiotropic effect caused by the ectopic expression of nahG varies between plant species, and it remains unclear which other mechanisms in addition to the increased SA metabolism affect defense responses in plants expressing nahG. Moreover, the lack of whole-plant phenotype in N. attenuata ov-nahG plants supports the conclusion that pleiotropic effects of the transgene are unlikely to be major determinants of the changes in defense responses we observed in ir-lecRK1xov-nahG plants. To summarize, our data strongly suggest a causal link between the suppression of SA accumulation and LecRK1 function and thereby to the unfettered JA-mediated induction of defense responses against M. sexta herbivory. However, since the expression of nahG can have pleiotropic effects on plant metabolism, further experimentation will be required to conclusively rule out the effect of other metabolites on the phenotype observed in ir-lecRK1 plants.

Interestingly, N. attenuata ov-nahG plants accumulate similar basal levels of SA as wild-type plants in unelicited leaves. A similar result was obtained when nahG was ectopically expressed in a poplar hybrid (Populus tremula × Populus alba; Morse et al., 2007). In this previous study, the authors hypothesized that poplar has a mechanism to maintain constitutive SA levels to a specific threshold at the expense of shikimate and phenylpropanoid metabolites and the compartmentalization of SA (Morse et al., 2007). Thus, one possible explanation for the unchanged basal levels of SA in leaves of N. attenuata ov-nahG is that, similar to poplar, N. attenuata maintains basal SA levels in unelicited leaves by changing the flux of the SA biosynthetic pathway and SA compartmentalization. However, this hypothesis requires further experimentation.

Additional Changes in the Metabolome and Transcriptome Affected by Reduced Expression of LecRK1

Even though the work was primarily focused on the effect of LecRK1 on known defense responses in N. attenuata, the accumulation of 148 ions was affected in ir-lecRK1 plants compared with the wild type (see Supplemental Data Set 2 online). The identity of most of the corresponding metabolites could not be unambiguously determined and remains the focus of future work. Some of these metabolites may also be critical for the interaction between M. sexta and N. attenuata, and their accumulation may also be affected by increased OS-elicited SA levels (in a manner similar to the defense metabolites analyzed in detail in this study). Consistent with the number of ions detected as differentially regulated in ir-lecRK1 versus wild-type plants, a previous study identified 173 metabolites as differentially regulated in N. attenuata wild-type plants at 1 h and 5 d after M. sexta elicitation (Gaquerel et al., 2010). Thus, from these numbers it can be preliminarily concluded that LecRK1 has a prominent effect on the accumulation of metabolites induced by M. sexta OS elicitation in leaves of N. attenuata plants. However, this effect is specific to some degree since the induction of defense metabolites derived from the phenyl-propanoid pathway were not affected in ir-lecRK1 plants (see Supplemental Figure 10 online).

Gene expression analysis of leaf tissue from ir-lecRK1 and wild-type plants at 1 h after M. sexta OS elicitation showed that the reduced expression of LecRK1 affects the accumulation of 77 transcripts (using the conditions described in Results) and, based on gene ontology annotation (see Supplemental Data Set 4 online), the functions of these genes were distributed among diverse cellular processes (e.g., defense, metabolism, transcriptional regulation, transport, and oxidative reduction processes). What roles these genes play in the response of N. attenuata plants to M. sexta herbivory is at present unknown and the focus of future work.

The unraveling of the complexity of the changes occurring in the metabolome and transcriptome of ir-lecRK1 plants during M. sexta herbivory will provide further insights into additional mechanisms mediated by LecRK1 and affecting the response of N. attenuata to M. sexta herbivory.

METHODS

Plant Growth and Treatments

Seeds of the 31st generation of an inbred genotype of Nicotiana attenuata, originally collected from southwestern Utah in 1988, were used for all experiments. N. attenuata as-tdm2 plants have been previously described (Kang et al., 2006). Seeds from wild-type and genetically transformed plants were germinated as previously described (Krügel et al., 2002). Plants were grown in the glasshouse under high-pressure sodium lamps (200 to 300 μmol s−1 m−2 light) with a day/night cycle of 16 h (26 to 28°C)/8 h (22 to 24°C) and 45 to 55% humidity. Crosses between N. attenuata lines were performed by removing anthers from flowers of homozygous ir-lecRK1-378 plants before pollen maturation and by brushing the stigma with pollen from homozygous plants ectopically expressing the nahG gene (ov-nahG) or the wild type. For wounding and elicitation treatments, leaves were wounded by rolling a fabric-pattern wheel three times on each side of the midvein, and the wounds were supplemented either with 20 μL of water (wounding treatment), 20 μL of 18:3-Glu (0.03 nmol/μL; FAC elicitation), or 20 μL of Manduca sexta, Spodoptera littoralis, or Schistocera gregaria OS diluted 1:5 (v/v) in water (OS elicitation). The tissue expression profile of LecRK1 was evaluated by collecting different plant tissues from wild-type N. attenuata plants; rosette leaves and roots were collected from 30-d-old plants (rosette stage; Figure 2C), whereas stems, systemic leaves, sepals, pistils, corolla, inflorescence, and stamens were collected from 50-d-old (early flowering) plants.

Insect Rearing, Feeding Experiments, and OS Collection

Larvae of the tobacco hornworm (M. sexta, Lepidoptera, Sphingidae) were obtained from in-house colonies, originally generated from M. sexta eggs purchased from Carolina Biological Supply. One freshly hatched larva of M. sexta was placed on the leaf of early rosette stage (Figure 2C) N. attenuata plants (n = 30). Larval mass was determined using a microbalance after 4, 7, and 11 d of the start of the experiment. OS from M. sexta and S. littoralis reared on N. attenuata wild-type plants were collected as described by Roda et al. (2004), and OS from S. gregaria were collected as described by Schäfer et al. (2011). For the estimation of the leaf area consumed after 11 d of M. sexta larval feeding, 20 leaves in which larvae has fed were excised (one leaf per plant per genotype was used) and scanned (CanoScanLide20 scanner; Cannon). The area consumed was calculated by subtracting the total leaf area (estimated by reconstruction of the leaf contour) to the remaining leaf area.

VIGS

VIGS based on the tobacco rattle virus (Ratcliff et al., 2001) was used to transiently silenced Na-LecRK1 as previously described (Gilardoni et al., 2010). An 86-bp fragment corresponding to the 3′-untranslated region of LecRK1 (see accession number below) was amplified by PCR with the primers LecRK1-33 and LecRK1-30 (see Supplemental Table 1 online). The PCR product was digested with BamHI and SalI and inserted into plasmid pTV00 in antisense orientation to generate the VIGS-lecRK1 vector. Plants transformed with the empty vector (EV) were used as control. Efficiency of gene silencing was evaluated by real-time qPCR (see below) using the primers 5358 Fw and 5358 Rv (see Supplemental Table 1 online) 1 h after wounding and 18:3-Glu elicitation.

Generation of Stable Silenced Lines

The same 86-bp fragment used for generating the VIGS-lecRK1 vector was subcloned using SacI and XhoI (New England Biolabs) restriction sites into the pSOL8 transformation vector (Bubner et al., 2006) as an inverted-repeat construct. This construct was used to transform N. attenuata wild-type plants using Agrobacterium tumefaciens–mediated transformation and plant regeneration as previously described (Krügel et al., 2002). T1 transformed plants were analyzed for T-DNA insertion number by DNA gel blot hybridization (see below). Segregation analysis for hygromycin resistance in T2 seedlings was performed on agar plates supplemented with hygromycin (0.025 mg mL−1). Two lines, A-08-378-5-4-1 (ir-lecRK1-378) and A-08-380-6-3-8 (ir-lecRK1-380) each had a single T-DNA insertion in the genome, and they were used for all experiments. For DNA gel blot analysis, genomic DNA from wild-type and ir-lecRK1 plants was isolated by the cetyltrimethylammonium bromide method. DNA samples (10 μg) were digested with EcoRV (New England Biolabs) overnight at 37°C according to commercial instructions and separated on a 1% (w/v) agarose gel using standard conditions. DNA was blotted onto Gene Screen Plus Hybridization Transfer membranes (Perkin-Elmer Life and Analytical Sciences) using the capillary transfer method. A gene-specific probe for the hygromycin resistance gene hptII was generated by PCR using the primers HYG1-18 and HYG3-20 (see Supplemental Table 1 online). The probe was labeled with [α-32P]dCTP (Perkin-Elmer) using the Rediprime II kit (Amersham Pharmacia) according to commercial instruction. Efficiency of gene silencing was evaluated by qPCR (see below) after 1 h of wounding and 18:3-Glu treatment using the primers 5358 Fw and 5358 Rv (see Supplemental Table 1 online).

For the generation of N. attenuata plants ectopically expressing the bacterial salicylate hydroxylase gene (nahG) from Pseudomonas putida, a 690-bp fragment corresponding to this gene was amplified with primers NahG1-33 and NahG1-34 (see Supplemental Table 1 online) using P. putida genomic DNA as template and subcloned using XhoI and BstEII (New England Biolabs) restriction sites into the pSOL1 vector (Bubner et al., 2006) to generate pSOL1-nahG1, and this vector was used to transform N. attenuata wild-type plants using Agrobacterium-mediated transformation as previously described (Krügel et al., 2002). Segregation analysis for hygromycin resistance in T2 seedlings was performed on agar plates supplemented with hygromycin (0.025 mg mL−1), and homozygous T2 transformed plants were analyzed for expression of the nahG gene by RT-PCR using the primers NahG1-Fw and NahG1-Rv (see Supplemental Table 1 online). For DNA gel blot analysis, genomic DNA was isolated as described above. DNA samples (10 μg) were digested with EcoRV and DraI (New England Biolabs) overnight at 37°C according to commercial instructions and separated on a 1% (w/v) agarose gel using standard conditions. DNA was blotted onto nylon membranes as described above. Membranes were hybridized with a nahG-specific radiolabeled probe that was generated by PCR using the primer pairs NahG1 Fw and NahG1 Rv (see Supplemental Table 1 online) and [α-32P]dCTP (Perkin-Elmer) using the Rediprime II kit (Amersham Pharmacia) according to commercial instruction. Pseudomonas syringe DC3000 (Pst DC3000) grown at OD = 0.001 or water (control) was syringe infiltrated into leaves of wild-type and ov-nahG plants, and infiltrated leaf tissue was harvested 2 d after the treatment for analysis of SA levels (see below).

RACE and Sequence Analysis

For cloning of the full-length LecRK1 cDNA sequence, 5 μg of total RNA were isolated from leaves of N. attenuata plants. The 3′RACE and 5′RACE Systems for Rapid Amplification of cDNA Ends (Invitrogen) were used following the manufacturer’s instructions and the primers listed in Supplemental Table 1 online. The PCR products were cloned into the pGEM-T easy vector (Promega) and sequenced using universal primers. Sequence alignments were performed using BLAST (http://blast.ncbi.nlm.nih.gov) and ExPASy (http://expasy.org). Structural domain prediction was performed using SMART (http://smart.embl-heidelberg.de; Letunic et al., 2009) and Pfam (http://pfam.sanger.ac.uk) databases. Prediction of trasmembrane domains was performed using TMHMM (http://www.cbs.dtu.dk/services/TMHMM) and SOSUI (http://bp.nuap.nagoya-u.ac.jp/sosui) Web servers. Prediction of signal peptides was performed using SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP).

Real-Time qPCR

For all treatments, N. attenuata rosette leaves were harvested and immediately frozen in liquid nitrogen. Total RNA was extracted using the TRIzol reagent (Invitrogen), and 5 μg of total RNA was reverse transcribed using oligo(dT)18 and SuperScript reverse transcriptase II (Invitrogen). Real-time qPCR was performed with a Mx3005P Multiplex qPCR system (Stratagene) and the qPCR Core kit for SYBR Green I (Eurogentec). Relative quantification of mRNA levels was performed by the comparative Δ cycle threshold (CT) method using the eukaryotic elongation factor 1A (NaEF1A) mRNA as an internal standard (Gilardoni et al., 2010). The sequences of the primers used for qPCR are listed in Supplemental Table 1 online. All the reactions were performed using the following qPCR conditions: initial denaturation step of 95°C for 30 s, followed by 40 cycles each of 95°C for 30 s and 60°C for 1 min, with a final extension step of 95°C for 30 s and 60°C for 1 min. All samples were obtained from three independent biological replicates (n = 3) for each time point, plant genotype, and treatment.

Phytohormone Extraction and Quantification

For analysis of JA, JA-Ile, and SA, 0.1 g of frozen leaf tissue was homogenized to a fine powder with a Geno/Grinder 2000 (BTC and OPS Diagnostics) in the presence of liquid nitrogen. One milliliter of ethylacetate spiked with 200 ng [2H2]JA and [2H4]SA and 40 ng JA-[13C6]Ile was added to the samples, and after vortexing, the samples were centrifuged for 15 min at 12,000g (4°C). The upper organic phase was transferred into a fresh tube, and the leaf material was reextracted with 0.5 mL ethylacetate without internal standards. The organic phases were pooled and evaporated to dryness under reduced pressure. The dry residue was reconstituted in 0.4 mL of 70/30 (v/v) methanol/water for analysis with an LC-ESI-MS/MS instrument (Varian 1200 Triple-Quadrupole-LC-MS system; Varian). Ten microliters of the sample were injected in a ProntoSIL column (C18-ace-EPS, 50 × 2 mm, 5 μm, 120 Å; Bischoff) connected to a precolumn (C18, 4 × 2 mm; Phenomenex). As mobile phases, 0.05% formic acid in water (solvent A) and methanol (solvent B) were used in a gradient mode with the following conditions: time/concentration (min/%) for B: 0.0/15; 2.5/15; 4.5/98; 10.5/98; 12.0/15; 15.0/15; time/flow (min/mL min−1): 0.0/0.4; 1.5/0.2; 1.5/0.2; 10.5/0.4; 15.0/0.4. Compounds were detected in the ESI negative mode and multiple reaction monitoring according to the parameters previously published (Bonaventure et al., 2011b).

For the analysis of ET, the youngest fully expanded leaf of rosette stage N. attenuata plants was either elicited with OS or 18:3-Glu or wounded, and it was immediately excised, weighed, and transferred into a 250-mL glass vessel. Five leaves per treatment and genotype were used (n = 5). After a 5-h incubation period (glass vessels were kept in the glasshouse under the same conditions as the plants), the headspace of the vessels was flushed into a laser photoacoustic spectrometer (INVIVO) for determination of ET levels as previously described (Körner et al., 2009). ET emissions are expressed as nL h−1 g−1 fresh weight. Untreated leaves were used to determine basal ET levels.

Metabolic Profiling of Leaves by Ultraperformance LC-ToF-MS

Leaf tissue from rosette stage ir-lecRK1-378 and wild-type plants was harvested at 0.5, 3, and 6 d after M. sexta OS elicitation, and three independent biological replicates per plant genotype per time point were used (total samples = 18). One hundred milligrams of leaf tissue was ground with a Geno/Grinder 2000 (BTC and OPS Diagnostics) in the presence of liquid nitrogen 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 12,000g for 20 min at 4°C, the supernatant was transferred into a fresh 1.5-mL microcentrifuge tube, and the samples were centrifuged again using the same conditions. One hundred microliters of the supernatant was transferred into HPLC vials.

Four microliters of the leaf extract was injected into a C18 Acclaim column (2.2-μm particle size, 150 × 2.1-mm inner diameter; Dionex) and separated using an RSLC system (Dionex). Solvent A was deionized water containing 0.1% (v/v) acetonitrile (Baker, HPLC grade) and 0.05% (v/v) formic acid. Solvent B was acetonitrile and 0.05% (v/v) formic acid. The gradient condition was applied as follows: 0 to 0.5 min 10% B, 0.5 to 6.5 min linear gradient 80% B, 6.5 to 10 min 80% B, and reequilibration at 10% B for 3 min. The flow rate was 300 μL min−1. Eluted compounds were detected with a MicroToF mass spectrometer (Bruker Daltonics) equipped with an electrospray ionization source in positive ion mode. Instrument settings were as follows: capillary voltage, 4500 V; capillary exit, 130 V; dry gas temperature, 200°C; dry gas flow, 8 liters min−1. Mass calibration was performed using sodium formate clusters (10 mM solution of NaOH in 50/50% [v/v] isopropanol/water containing 0.2% formic acid). Data sets were evaluated from 125 to 550 s in the mass range m/z 90 to 1400. The raw data files were converted to netCDF format using the export function of the Data Analysis version 4.0 software (Bruker Daltonics) and processed using the XCMS package (Tautenhahn et al., 2008) and the R-package CAMERA (http://www.bioconductor.org/biocLite.R) as previously described (Gaquerel et al., 2010). Peak detection was performed using the centWave method (Tautenhahn et al., 2008) and the parameter settings ppm = 20, snthresh = 10, peak width = 5 to 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).

The Metaboanalyst software (Xia et al., 2009; Xia and Wishart, 2011) was used to perform multivariate analysis (PCA and PLSDA). The data were filtered using the coefficient of variation, and it was normalized using Pareto scaling (Xia et al., 2009; Gaquerel et al., 2010). PLSDA (Eriksson et al., 2006; Xie et al., 2008) was validated using a permutation test as previously described (Westerhuis et al., 2008). An important output of the PLSDA analysis is that it estimates and ranks the influence of individual features (ions) on the model by assigning to each variable a VIP value, and VIP values bigger than or equal to 1.0 are considered statistically significant for group discrimination (Xie et al., 2008; Mazzara et al., 2011). For elemental molecular formula calculation, the SmartFormula algorithm (Data-Analysis 4.0 software; Bruker Daltonics) was used following maximum elemental composition CaHbNcOdNaeKf and the restrictions 1 ≤ b/a ≤ 3; e=0 or 1; f=0 or 1; a, b, c, and d not limited. Ring plus double bond values from −0.5 to 40, the nitrogen rule, and ions of even electron configuration were also considered (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 TPI Activity

Leaf tissue from 40-d-old ir-lecRK1-378, ir-lecRK-380, and wild-type plants was harvested 2 and 3 d after wounding and M. sexta OS elicitation. Unelicited tissue was used as control for basal TPI activity. One hundred milligrams of leaf tissue was ground using a Geno/Grinder 2000 (BTC and OPS Diagnostics) in 2-mL microcentrifuge tubes and extracted with 0.3 mL of ice-cold extraction buffer (0.1 M Tris-Cl, pH 7.6, 5% [w/w] polyvinylpolypyrrolidone [Sigma-Aldrich], 2 mg/mL phenylthiourea [Sigma-Aldrich], 5 mg/mL diethyldithiocarbamate [Sigma-Aldrich], and 0.05 M Na2EDTA). The samples were thoroughly vortexed and centrifuged at 4°C for 20 min at 12,000g. The supernatant was transferred into a fresh tube and kept on ice for protein quantification and TPI analysis. Protein concentration was determined with the protein assay kit (Bio-Rad) using BSA (Sigma-Aldrich) as a standard. TPI activity was analyzed by a radial diffusion assay as previously described (van Dam et al., 2001).

Analysis of HGL-DTGs, Nicotine, and Phenyl-Propanoid Derivatives

For quantification of total HGL-DTGs and nicotine levels, leaf samples were harvested after the treatments and times indicated in the figure captions and homogenized to a fine powder with a Geno/Grinder 2000 (BTC and OPS Diagnostics). Samples were extracted with 40% (v/v) methanol/water containing 0.5% (v/v) acetic acid and analyzed by HPLC as previously described (Keinänen et al., 2001; Jassbi et al., 2008). An external calibration curve was generated with a dilution series of nicotine and glycyrrhizinic acid, and samples were normalized by gram fresh weight. Individual HGL-DTG species were quantified by reverse-phase LC-MS/MS as previously described (Heiling et al., 2010). Derivatives of the phenyl-propanoid pathway were analyzed as previously described (Kaur et al., 2010).

Amino Acid Determination in M. sexta Midgut and Leaf Tissue

M. sexta larvae reared on N. attenuata plants for 11 d were placed on ice, and their midguts were dissected with forceps under a stereomicroscope. The midgut tissue and content were separated and immediately transferred into screw cap 10-mL glass tubes placed on ice. Midgut tissue and content were homogenized separately in the presence of 1 mL 1/1 (v/v) chloroform/methanol containing 0.01% (v/v) formic acid. After centrifugation for 15 min at 700g (4°C), the supernatant was transferred into a fresh 10-mL glass tube, and the aqueous phase/tissue was reextracted with 1 mL of chloroform. After centrifugation for 15 min at 700g (4°C), the supernatants were pooled and transferred into 4-mL HPLC vials. Midgut and midgut content were diluted 1/40 (v/v) in water before analysis. Leaves were extracted using the same protocol but diluted 1/1 (v/v) in water before analysis. Chromatography was performed on an Agilent 1200 HPLC system (Agilent Technologies) using a Zorbax Eclipse XDB-C18 column (50 × 4.6 mm, 1.8 μm; Agilent Technologies). Formic acid (0.05%) in water and acetonitrile were employed as mobile phases A and B, respectively. The elution profile was 0 to 1 min, 100% A; 1 to 3 min, 0 to 100% B in A; 3 to 4 min 100% B; and 4.1 to 7 min 100% A. The mobile phase flow rate was 0.8 mL min−1. The column temperature was maintained at 25°C. An API 3200 tandem mass spectrometer (Applied Biosystems) equipped with a Turbospray ion source was operated in positive ionization mode. The instrument parameters were optimized by infusion experiments with pure Thr standard (Sigma-Aldrich). The ionspray voltage was maintained at 5500 eV. The turbo gas temperature was set at 700°C. Nebulizing gas was set at 70 p.s.i., curtain gas at 35 p.s.i., heating gas at 70 p.s.i., and collision gas at 2 p.s.i. Multiple reaction monitoring was used to monitor analyte parent ion → product ion as described by Jander et al. (2004). External calibration curves for amino acids were created by linear regression in the range from 0.2 to 5 μM.

Starch Quantification

Leaf samples were harvested at different times after M. sexta OS elicitation and homogenized to a fine powder with a Geno/Grinder 2000. Samples were extracted with 1 mL 80% (v/v) ethanol/water and centrifuged at 12,000g for 15 min at 4°C. The pellet was washed with 1 mL 80% (v/v) ethanol/water and centrifuged at 12,000g for 15 min. Five hundred microliters of water were added to the pellet, and the samples were vortexed for 5 min. Perchloric acid (650 μL; Carl-Roth) was added, and the samples were completely homogenized by pipetting several times up and down with a micropipette. The homogenates were incubated on ice for 20 min and then centrifuged at 12,000g for 15 min at 4°C. The starch-containing supernatant was transferred to a new 1.5-mL microcentrifuge tube, and 10 μL were diluted with 90 μL of water. A total of 400 μL of anthrone reagent (100 mg anthrone [Sigma-Aldrich] in 100 mL 95% [v/v] H2SO4/water [Carl-Roth]) was added, and the reactions were kept for 8 min in a boiling water bath (Viles and Silverman, 1949). After samples cooled down, absorbance was measured at 630 nm with the Ultrospec 3000 spectrophotometer (Pharmacia Biotech). Standard curves were generated with d-glucose (Sigma-Aldrich) from 0.05 to 1 mg/mL.

Microarray Analysis

Leaf tissue from rosette stage ir-lecRK1-378 and wild-type plants (three independent biological replicates per genotype were used) was harvested at 1 h after M. sexta OS elicitation. Total RNA was extracted based on the method of Kistner and Matamoros (2005) and its quality checked by spectrophotometry (NanoDrop). Genomic DNA was removed by DNase treatment following commercial instructions (Turbo DNase; Ambion), RNA was cleaned up with RNeasy MinElute columns (Qiagen), and the RNA quality was checked with the RNA 6000 Nano kit (Agilent) using an Agilent 2100 bioanalyzer. Total RNA was used to generate labeled cRNA with the Quick Amp labeling kit (Agilent) following commercial specifications, and the yield of cRNA was determined spectrophotometrically (NanoDrop). Labeled cRNA was hybridized using the gene expression hybridization kit (Agilent) following commercial instructions onto 44K custom-designed 60-mer N. attenuata Agilent microarrays (Kallenbach et al., 2011) containing 43,533 sequences (see accession numbers). Microarrays were hybridized overnight at 65°C, and slides were washed with the Gene Expression Wash Buffer kit (Agilent) as outlined in the One-Color Microarray-Based Gene Expression Analysis manual (Agilent). Three biological replicates were used per treatment with a total of six arrays (see accession numbers). Arrays were scanned with an Agilent G2565BA scanner, and image data were acquired with the Agilent Scan Control software (version A.7.0.1 for the B scanner). Data were extracted using the Agilent Feature Extraction software (version 9.5) and analyzed with the Significance Analysis of Microarrays (SAM) software (Tusher et al., 2001). The q-values for each gene corresponded to a computed FDR of 4%. Changes in gene expression were considered to be significant when the log2(FC; treatment versus control) was >1.3 or smaller than −1.3 and q-values lower than 0.05 (according to the FDR value calculated by SAM).

Statistical Analysis

Statistics were calculated using SPSS software version 17.0.

Accession Numbers

Sequence data from this article can be found under the following accession numbers: Na-LecRK1 (JF919621; GenBank database), Agilent Chip platform (GPL13527; National Center for Biotechnology Information Gene Expression Omnibus [NCBI GEO] database), and microarray data (GSE29905; NCBI GEO database).

Supplemental Data

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

Acknowledgments

We thank E. Gaquerel for his help with the analysis of the metabolic profiling data and M. Reichelt for his assistance with amino acid analysis. P.A.G. is a fellow of the Deutscher Akademischer Austausch Dienst. This work was funded by the Deutsche Forschungsgemeinschaft (Project BO3260/3-1) and the Max Planck Society.

AUTHOR CONTRIBUTIONS

P.A.G. carried out the experiments, analyzed the data, and wrote the article. C.H. characterized and provided the ov-nahG plants. I.T.B. participated in the design and coordination of the study and wrote the article. G.B. conceived of the study, participated in its design and coordination, and wrote the article.

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