Nicotine's Defensive Function in Nature

Plants produce metabolites that directly decrease herbivore performance, and as a consequence, herbivores are selected for resistance to these metabolites. To determine whether these metabolites actually function as defenses requires measuring the performance of plants that are altered only in the production of a certain metabolite. To date, the defensive value of most plant resistance traits has not been demonstrated in nature. We transformed native tobacco(Nicotiana attenuata) with a consensus fragment of its two putrescine N-methyl transferase (pmt) genes in either antisense or inverted-repeat (IRpmt) orientations. Only the latter reduced (by greater than 95%) constitutive and inducible nicotine. With D4-nicotinic acid (NA), we demonstrate that silencing pmt inhibits nicotine production, while the excess NA dimerizes to form anatabine. Larvae of the nicotine-adapted herbivore Manduca sexta (tobacco hornworm) grew faster and, like the beetle Diabrotica undecimpunctata, preferred IRpmt plants in choice tests. When planted in their native habitat, IRpmt plants were attacked more frequently and, compared to wild-type plants, lost 3-fold more leaf area from a variety of native herbivores, of which the beet armyworm, Spodoptera exigua, and Trimerotropis spp. grasshoppers caused the most damage. These results provide strong evidence that nicotine functions as an efficient defense in nature and highlights the value of transgenic techniques for ecological research.


Introduction
Plants produce many secondary metabolites, of which some are thought to function as direct defenses against pathogens and herbivores by reducing their performance, survival, and reproduction. Numerous plant allelochemicals with antiherbivore properties are classified according to their mode of action (e.g., toxins, antifeedants, antidigestive proteins, etc.) (Bennett and Wallsgrove 1994) and have been used in agriculture to control insect pests (Hedin 1991). The fact that a secondary metabolite reduces herbivore performance does not by itself demonstrate that the endogenously expressed metabolite functions defensively in the plant's natural environment (Bell 1987), because the evolutionary interaction between herbivores and their host plants may have reduced the defensive efficacy of the metabolite. Phytophagous insects have evolved various strategies to cope with allelochemicals (Karban and Agrawal 2002) and tend to tolerate, or even co-opt, plant defenses for their own defenses (Wink and Theile 2002).
Pharmacological studies demonstrating a resistance effect of metabolites applied to plants or artificial diets (Yamamoto et al. 1968;Bowers and Puttick 1988), and studies using heterologously expressed genes in agricultural systems (Carozzi and Koziel 1997;Hilder and Boulter 1999), represent a first step in evaluating the defensive function of a secondary metabolite. The interpretation of these studies is confounded by both the altered ecological context in which the resistance is measured and the altered chemical milieu, which is also known to influence the defensive function of a metabolite. Stronger evidence for resistance effects of allelochemicals arises from studies establishing correlations between plant resistance against herbivores and the genetically variable accumulation of secondary metabolites (Berenbaum et al. 1986;Shonle and Bergelson 2000) or from studies demonstrating the defensive role played by a suite of elicited metabolites (Orozco-Cardenas et al. 1993;Baldwin 1998;Halitschke and Baldwin 2003). Ideally, the benefits of a putative defense trait should be determined in plants differing only in a single gene that controls the expression of a resistance trait and are otherwise identical (Bergelson and Purrington 1996). To date, studies measuring resistance of ''near isogenic'' lines with altered metabolite accumulations  provide the strongest evidence for their resistance, but these lines, which are created by repetitive backcrossing, are likely to differ in many loci linked to the target locus, which may also affect resistance. Such problems of genetic linkage have been overcome through the use of genetic transformation to explore the fitness effects of herbicide resistance (Bergelson et al. 1996;Purrington and Bergelson 1997) and pathogen resistance (Tian et al. 2003) in field populations of Arabidopsis. In this study, we use transgenic silencing to alter a single putative resistance trait-the production of nicotine-and thereby establish its contribution to plant resistance in the field.
The pyridine alkaloid nicotine is one of the best-studied putative plant resistance traits. Because it can interact with the acetylcholine receptors in the nervous systems of animals, nicotine is extremely toxic to most herbivores and, consequently, was one of the first insecticides used to control pests in agriculture (Schmeltz 1971). Evidence for the resistance value of nicotine arises from the agricultural practice of using nicotine sprays and genotypes of cultivated tobacco differing in nicotine levels . Although nicotine is widely toxic, insects adapted to nicotine-producing plants have evolved resistance to this alkaloid (Glendinning 2002). The tobacco specialist Manduca sexta (tobacco hornworm) tolerates doses of nicotine that are fatal to unadapted herbivores but grows more slowly on high-nicotine diets (Appel and Martin 1992;Wink and Theile 2002). Other studies suggest that M. sexta might even be better defended by dietary nicotine against its parasitoid, Cotesia congregata, which suffers higher mortality when parasitizing larvae fed on high-rather than low-nicotine diets Thorpe and Barbosa 1986). Thus, the coevolutionary arms race between nicotine-producing plants and their adapted herbivores may have reduced the defensive value of nicotine.
In the native tobacco species Nicotiana attenuata and N. sylvestris, nicotine is the most abundant alkaloid. Elicitation of N. attenuata with jasmonic acid methyl ester (MeJA) in its native habitat increases nicotine content, which is correlated with enhanced plant fitness when plants are attacked (Baldwin 1998). However, herbivore attack and MeJA elicitation (as well as the plant's endogenous jasmonic acid cascade [Halitschke and Baldwin 2003]) regulate many resistance traits, including trypsin protease inhibitors (TPIs), diterpene glycosides, and volatile emissions involved in indirect defense. Hence, nicotine is only one of a suite of putative defense traits elicited by herbivore attack, and its specific role remains to be determined.
In laboratory trials, resistance benefits of nicotine production against M. sexta larvae were established using transgenic N. sylvestris plants silenced in their nicotine biosynthesis by antisense expression of putrescine N-methyl transferase (PMT). Plant consumption and the performance of M. sexta larvae were negatively correlated with constitutive nicotine levels in laboratory feeding trials (Voelckel et al. 2001); whether this result applies to plants in their natural habitat is unclear. To examine the resistance effect of nicotine, we transformed N. attenuata with inverted-repeat pmt (IRpmt) and antisense pmt constructs and found that only IRpmt plants had strongly reduced nicotine content. We characterized the defense and growth phenotypes of two independently transformed homozygous IRpmt lines and found that measured direct and indirect defenses did not differ from those of the wild-type (WT) plants, except for a dramatic reduction (greater than 95%) of MeJA-elicited and constitutive nicotine production and an increase in anatabine content. In pulsechase experiments with D 4 -nicotinic acid (NA) ethyl ester, we demonstrated that the increased anatabine likely results from a dimerization of the NA that would normally have been used in nicotine biosynthesis. In feeding trials, M. sexta larvae preferred and grew faster on IRpmt than WT leaves. We transplanted WT and IRpmt plants into N. attenuata's native habitat in southwestern Utah and elicited a subset with MeJA. Several naturally occurring herbivore species attacked and damaged unelicited IRpmt plants more than unelicited or elicited WT and elicited IRpmt plants. These results demonstrate that nicotine functions as an effective resistance trait under natural conditions.

Results/Discussion IRpmt Constructs Silence Nicotine Production
Nicotine accumulation was not reduced in most of the independent lines transformed with antisense pmt constructs (25 lines of pNATPMT1 and six lines of pCAMPMT1) compared to WT ( Figure 1A). None of the five lines with lower nicotine accumulation in the T 1 screen had nicotine levels lower than those of WT in the homozygous T 2 generation. In contrast, 29 of 34 independently transformed lines with the IRpmt construct pRESC5PMT had dramatically reduced constitutive and MeJA-induced nicotine accumulations ( Figure 1B). The suppression of nicotine accumulation was stable during plant development and when plants were grown in the glasshouse or in the field in Utah. Clearly, inverted-repeat constructs are more efficient at silencing the expression of endogenous genes, as has been previously described (Wesley et al. 2001).

Genomic and Transcriptional Characterization
Two homozygous T 2 IRpmt lines (108 and 145) with reduced nicotine levels were further characterized. Southern blot analysis using a probe hybridizing to the selective marker in the IRpmt construct demonstrated that both lines contained a single insertion ( Figure S1). Transformation with a pRESC transformation vector allowed the transferred DNA (T-DNA) and flanking DNA at the insertion site to be recovered from the plant genomic DNA. These experiments demonstrated that the T-DNA integrated into the N. attenuata genome at a single site in each line, since all sequenced clones from a line (108, n = 4; 145, n = 5) contained the same flanking sequence (see Figure S1 and Protocol S1).
Transcripts of the pmt genes in the two lines were significantly reduced to approximately 10% of the constitutive and MeJA-induced WT mRNA levels (Figure 2A), demonstrating that the targeted genes were successfully silenced. Metabolic Consequences of pmt Silencing in N. attenuata Consistent with the observed silencing of pmt transcripts, the constitutive and induced nicotine levels in transformed plants of both lines were dramatically reduced to 3%-4% of the levels found in WT plants ( Figure 2B). All 29 IRpmt lines with reduced nicotine levels accumulated the alkaloid anatabine, which was not detected in WT plants. Constitutive and MeJA-induced total (nicotine, anabasine, and anatabine) alkaloid contents of the two IRpmt lines were about one-half and one-third of the WT levels, respectively, of which anatabine comprised 30% and 23% ( Figure 2C). Levels of anabasine representing 20% of the constitutive and 8% of the MeJA-elicited total alkaloid contents in WT plants were unchanged in IRpmt plants ( Figure S2). Elevated anatabine levels were also found in recently published studies with antisense pmt transformation of N. tabacum; elevated anatabine levels did not affect transcript levels of other genes encoding enzymes involved in alkaloid metabolism (Chintapakorn and Hamill 2003).
Anatabine consists of a pyridine and a piperideine ring. Both are likely derived from NA, which is also the precursor of the pyridine ring of nicotine (Leete and Slattery 1976). Disrupting nicotine biosynthesis at the formation of the pyrrolidine ring by silencing PMT activity might cause an oversupply of the NA used in the biosynthesis of anatabine. Feeding the roots of hydroponically grown MeJA-elicited WT plants with NA ethyl ester resulted in formation of anatabine at levels of about a third of the total alkaloids (nicotine and anatabine) ( Figure 3); in the IRpmt lines, anatabine constitutes 98% of the total alkaloids. Feeding plants with D 4 -NA ethyl ester results in the formation not only of D 4 -nicotine and D 4anatabine but also of D 8 -anatabine, demonstrating that the last integrates two D 4 -NA units. When these experiments are conducted with WT plants, about half of the anatabine is labeled, suggesting that the unlabeled half was formed from endogenous unlabeled NA. In addition, about one-fourth of the WT nicotine was D 4 -nicotine. In IRpmt plants, in contrast, only traces of D 4 -nicotine were found, but one-third of the anatabine was either D 4 -or D 8 -labeled. In summary, exogenously supplied NA is taken up by the roots of N. attenuata plants and used in alkaloid biosynthesis, and an  oversupply of NA results in the formation of anatabine. These results support the hypothesis that the silencing of pmt disrupts nicotine biosynthesis, causing an oversupply of NA and the subsequent formation of anatabine.
IRpmt plants did not differ from WT plants in any other measured secondary metabolite or growth parameter. Constitutive or MeJA-induced levels of caffeoylputrescine, chlorogenic aid, rutin ( Figure S2), TPI activity, or the release of cis-a-bergamotene ( Figure S3) in IRpmt-transformed plants did not differ from those of WT plants. Rosette-stage and elongation-stage growth in individual pots in both the glasshouse and the field ( Figure S4) did not differ between WT and IRpmt lines, and transformed lines were not visually or morphologically distinguishable from WT plants. Hence, the IRpmt plants represent an ideal construct with which to examine the ecological consequences of nicotine production.
Effects of Nicotine Silencing on N. attenuata Herbivores M. sexta larvae reared on IRpmt plants in the glasshouse gained significantly more mass and changed instars faster than larvae reared on WT plants (n = 17-20; ANOVA: p , 0.01, p WT-PMT108 , 0.02, p WT-PMT145 , 0.01). The differences were comparable to those observed for M. sexta larvae reared on nicotine-enriched artificial diets (Parr and Thurston 1972;Appel and Martin 1992) or on nicotine-enhanced WT (Baldwin 1988) or antisense-pmt-transformed N. sylvestris plants (Voelckel et al. 2001). Two-thirds of freshly eclosed M. sexta larvae, given the choice between leaf material from WT or IRpmt (108) plants, preferred to initiate feeding on the latter (n = 43; Chi 2 = 6.7, p , 0.01). Such behavior suggests that nicotine plays an important role in determining feeding sites of M. sexta larvae, as has been suggested in a study with cultivated tobacco (Kester et al. 2002). While the relative toxic effects of anatabine and nicotine remain unstudied, these results are likely to underestimate the influence of nicotine on M. sexta choice and performance, because IRpmt plants had enhanced levels of anatabine.
Since secondary metabolism is known to be sensitive to environmental parameters that differ between glasshouse and field conditions (e.g., UV-B influence; Caldwell et al. 1983), nicotine, anatabine, and TPI levels of WT and IRpmt plants grown in the field plantation were analyzed: they were found not to differ from plants grown under laboratory conditions ( Figure 4A). A M. sexta feeding choice test evaluating the larvae's choice between field-grown WT and IRpmt plants (n = 57; Chi 2 = 7.74, p , 0.01) verified the results described above for the same experiment conducted with glasshousegrown plants. Thus, the phenotype of glasshouse-grown IRpmt plants was not altered by growth under field conditions. In addition, choice tests with field-collected D. undecimpunctata, which was observed colonizing only IRpmt plants in the field plantation, revealed that 77% of these beetles preferred the nicotine-deficient IRpmt leaf material over WT (n = 35; Chi 2 = 10.31, p , 0.001). Another beetle species observed occasionally on WT plants, Trichobarus mucorea, does not distinguish between WT and IRpmt leaf material in choice tests (n = 19; Chi 2 = 0.05, p = 0.8).
In the field plantation, IRpmt plants lost significantly more leaf area to herbivores than did WT plants ( Figure 4B), demonstrating that nicotine indeed functions as a direct resistance trait of N. attenuata in its native habitat. Over a period of 16 d, IRpmt plants exposed to naturally occurring herbivores lost 16% of their total leaf area to herbivores, an amount that is more than double the amount of damage incurred by WT plants. In order to meet compliance requirements described in the Code of Federal Regulations (7CFR340.3c) for the introduction of organisms altered through genetic engineering, flowers were removed as they matured, and therefore we could not directly measure the fitness consequences of this greater herbivore load. However, in other experiments with N. attenuata plants grown in natural populations, leaf area damage is negatively correlated with capsule number (Baldwin 1998;Kessler and Baldwin 2004), suggesting that the strongly enhanced herbivore damage of the nicotine-deficient IRpmt plants translates into a fitness loss.
IRpmt plants were attacked by a variety of insect herbivores. About half of the total herbivore damage resulted from S. exigua feeding ( Figure 4C). One-third of the total herbivore damage was damage from grasshoppers of the genus Trimerotropis, which followed the same general pattern of distribution as S. exigua damage, but the differences between unelicited IRpmt and WT plants were not significant. The damage caused by Epetrix hirtipennis was variable but significantly higher for unelicited IRpmt compared to WT plants (ANOVA: F = 2.81, df = 3, p = 0.04, p PMT-WT , 0.05).
MeJA elicitation significantly reduced the damage of IRpmt plants to levels found on WT plants, suggesting that MeJA treatment elicits defense traits that are as efficient as the constitutive levels of nicotine in protecting plants. MeJA elicitation of N. attenuata plants is known to induce a diverse suite of transcriptional responses and secondary metabolites including TPIs, phenolics, flavonoids, phenolic putrescine conjugates, diterpene sugar esters, and volatile organic compounds (Halitschke and Baldwin 2003;Roda and Baldwin 2003), some of which apparently function as resistance traits. Which component of this complex suite of elicited metabolites is as effective as nicotine remains to be determined. It should be noted that the overall amounts of leaf area lost to herbivores was relatively low during the field experiments. Only 5% of the canopy area was lost from control and MeJAelicited WT plants. In previous experiments (Baldwin 1998), fitness differences were observed between control and MeJAelicited WT plants in populations that had lost approximately 40% of their canopy area to herbivores.
Altogether, these results provide direct evidence for the defensive value of nicotine. In a field trial, we established that a native tobacco, which produces large amounts of nicotine, is better defended against its natural herbivores than are nicotine-deficient transformants of the same genetic background. This is likely mediated by the reduction of herbivore performance and by the fact that these phytophagous insects prefer low-nicotine diets. In contrast to studies demonstrating genetic correlations between the production of secondary metabolites and herbivore resistance (Berenbaum et al. 1986;Shonle and Bergelson 2000), the resistance effects established in this study can be directly attributed to the altered traits. The fact that the silencing of one enzyme in the nicotine biosynthetic pathway redirects metabolite flux, resulting in the accumulation of an apparently less toxic alkaloid, anatabine, underscores the importance of characterizing single-gene transformants for secondary effects.

Conclusion
Plant secondary metabolites are widely accepted as essential components of a plant's direct defenses against its natural enemies, but unambiguous proof has been lacking, mainly because of the difficulty of altering the expression of single traits in plants and testing the consequences of these manipulations under natural conditions. Transformation technology has provided biologists with the ability to manipulate and study the ecological consequences of singlegene manipulations. To date, the technology has largely been used for the heterologous expression of resistance genes (e.g., Bacillus thuringiensis d-endotoxin) in agricultural systems (see Tian et al. [2003] for an elegant exception), and therefore has provided little evidence for the defensive value of endogenously expressed traits against a plant's native herbivore community. The scientific value of transgenically silencing endogenous genes in native plants to understand the ecological function of particular genes has been undermined by the polarized attitudes towards the use of genetically modified organisms in agriculture. Transgenic down-regulation of nicotine demonstrates that N. attenuata is under relentless herbivore pressure. Disabling this resistance trait, even in a year of low herbivore abundance, results in a large increase in opportunistic herbivory and supports the conclusion that secondary metabolites play an important role in explaining why the earth is largely green (Hairston et al. 1960).

Materials and Methods
Plant material and transformation. N. attenuata Torr. ex Watson (synonymous with N. torreyana Nelson and Macbr.; Solanaceae) grown from field-collected seeds (Baldwin 1998) and inbred 11 or 14 generations were used for transformation and all experiments. Seed germination and the Agrobacterium tumifaciens (strain LBA 4404)mediated transformation procedure are described in Krü gel et al. (2002). In order to silence the expression of the two N. attenuata pmt genes, plants were transformed with pCAMPMT1 and pNATPMT1 vectors, which contain a gene fragment of pmt1 (which has 95% identity to pmt2) in an antisense orientation, and pRESC5PMT, which contains the pmt gene fragment twice in an inverted orientation separated by intron 3 of the Flaveria trinervia gene pyruvate orthophosphate dikinase (pdk) (for vector construction and plasmids see Figure S5 and Protocol S1). T 1 plants were screened for hygromycin resistance (hygromycin phosphotransferase II gene of the vector pCAM-BIA-1301) and constitutive and induced nicotine accumulation; homozygosity was determined by resistance screening of the T 2 plants. Two independently transformed homozygous IRpmt lines (108 and 145) were further characterized by Southern blot analysis and by the rescuing of the transformation vector from genomic DNA into Escherichia coli to identify copy number and insertion site of the T-DNA (see Figure S1 and Protocol S1).
PMT mRNA accumulation and secondary metabolites. Transformed lines (108 and 145) and WT plants were grown in 1-l hydroponic vessels in a climate chamber as described in Hermsmeier et al. (2001), and 4-wk-old rosette-stage plants were treated (elicited) on the first two fully expanded (source) leaves with 150 lg of MeJA per plant applied in 20 ll of lanolin paste, or left untreated. Approximately 200 mg of young roots was harvested and frozen in liquid nitrogen 10 h after elicitation, and RNA was extracted with Tri Reagent (Sigma, Taufkirchen, Germany) according to the manufacturer's instructions (n = 3/line/treatment). PMT transcript accumulation was analyzed by real-time PCR (ABI PRISM 7000; Applied Biosystems, Darmstadt, Germany). cDNA was generated from 20 ng of RNA with MultiScribe reverse transcriptase (Applied Biosystems), and amplified using the qPCR core reagent kit (Eurogentec, Searing, Belgium) and a probe and primers that were gene-specific (for sequences see Figure S6). For analysis of secondary metabolites, leaves growing one node above the sink-source transition leaf and young root tissue were harvested 4 d after elicitation (n = 8-10/line/ treatment). Samples were analyzed by HPLC for alkaloids, caffeoylputrescine, chlorogenic acid, and rutin (Keinä nen et al. 2001;Halitschke and Baldwin 2003). A peak occurring in IRpmt alkaloid extracts but not in extracts of WT N. attenuata was collected and identified by nuclear magnetic resonance imaging as anatabine (for spectra and method, see Protocol S1).
To determine whether a NA oversupply was responsible for the formation of anatabine in the transformed lines, we supplied 4-wk-old plants with either unlabeled or D 4 -NA ethyl ester (1 mM) in their hydroponic solution 24 h after MeJA elicitation (n = 4/line/treatment). After 4 d, the treated leaf was harvested and extracted as above, but analyzed by LC/MS/MS to detect incorporation of the deuterium into nicotine and anatabine (for instrument conditions, see Protocol S1).
To examine the release of cis-a-bergamotene in the transformed lines compared to WT, volatiles from hydroponically grown plants (n = 3-5/line/treatment) enclosed in open-top volatile collection chambers were collected for an 8 h period starting 24 h after MeJA elicitation of the first two source leaves, and analyzed by GC/MS (Halitschke et al. 2000). TPI activity in the MeJA-treated leaf 3 d after elicitation was analyzed in plants (n = 5/line/treatment) by radial diffusion activity assay .
M. sexta performance and feeding choice. In the glasshouse, 2-wkold seedlings were planted individually into 2-l pots with potting soil (C 410;Stender,Schermbeck,Germany)  were placed on the first-stem leaf of 8-wk-old WT and IRpmt (108 and 145) plants and allowed to feed for 14 d. Larval mass was recorded at 8, 10, 12, and 14 d.
The first feeding choice of M. sexta was determined by placing newly eclosed larvae in the center of a 3-cm-diameter cup containing, on opposite sides, 1.5-cm 2 WT and IRpmt (108) leaf pieces and recording the leaf on which larvae started feeding (n = 44).
Resistance of WT and IRpmt plants to herbivores in the natural habitat. In a field plantation (15 m 3 18 m; GPS: lat 3780894599N, long 11480191299) in N. attenuata's natural habitat in southwest Utah, transformed IRpmt (108) and WT plants were exposed to naturally occurring herbivores dispersing from adjacent populations. To allow for spatial heterogeneity, plants were transplanted in a paired design (with 0.3 m and 1.5 m between plants of a pair and between pairs, respectively) in which plants were matched for equal rosette diameters. Plants were grown in soil (Potting Mix; Miracle-Gro, Marysville, Ohio, United States) for 5 wk after germination (Krü gel et al. 2002), and were transplanted into the field plot (10 columns by 15 lines) in their 3.8-l pots. Seven days after transplantation, 30 WT and IRpmt plants were elicited with 150 lg of MeJA per plant applied in 20 ll of lanolin paste to the two youngest rosette leaves. Starting 4 d after transplantation, each plant was examined for damage and insects (including predators and eggs) every other day for 14 d. Damage amount was estimated as a percentage of the total leaf area, and the characteristic damage caused by caterpillars, beetles, grasshoppers, myrids, and leafhoppers was noted separately. The most abundant herbivores observed in the field plantation during the release were S. exigua, Trimerotropis spp., E. hirtipennis, and D. undecimpunctata. M. sexta and M. quinquemaculata occurred in the season only rarely, and no eggs were laid in the plantation during the 14 d. As plants began to elongate and produce flowers, they were examined daily, and all flowers were removed before opening and anthesis to meet the performance standards in the Code of Federal Regulations (7CFR340.3c). Consequently, direct fitness measures were unobtainable in this experiment.
For analysis of alkaloids and TPIs under field conditions, leaf samples of WT and IRpmt plants in the plot (n = 6) were taken 7 wk after transplantation and frozen (dry ice). To determine if the herbivore phenotype of IRpmt plants observed in glasshouse-grown plants was retained in plants grown under natural light conditions, the M. sexta choice experiment was repeated. The first feeding choices of freshly eclosed M. sexta larvae (North Carolina State University) and of adults of field-collected D. undecimpunctata and Trichobarus mucorea (Chrysomelidae and Curculionidae) found on N. attenuata were determined as described above. Figure S1. Copy Number of T-DNA in the Two Studied IRpmt Lines (A) Southern blot analysis of two independently transformed N. attenuata IRpmt lines (108 and 145) and WT plants. Genomic DNA (15 lg) from individual plants of the three genotypes and the plasmid used for transformation pRESC5PMT (4 ng) were digested with EcoRV and blotted onto nylon membranes (Winz and Baldwin 2001). The blot was hybridized with a PCR fragment of the hygromycin phosphotransferase II gene from pCAMBIA-1301, which is specific for the selective marker on the T-DNA and signifies one insertion in each of the two lines. (B) Ethidium bromide staining of the DNA revealed an overload of the DNA of the IRpmt lines and therefore loading of the WT was controlled by stripping and rehybridization with a PMT probe, which clearly revealed the endogenous pmt1 and pmt 2 genes described (Winz and Baldwin 2001) (unpublished data). Found at DOI: 10.1371/journal.pbio.0020217.sg001 (6.3 MB TIF). Figure S2. Secondary Metabolite Levels in the Studied IRpmt Lines Inverted-repeat silencing of pmt did not change the levels of (A) anabasine, (B) caffeoylputrescine, (C) chlorogenic acid, and (D) rutin (mean 6 standard error [SE]) in two independently transformed N. attenuata lines (108 and 145) compared to WT plants. Plants were harvested 4 d after receiving one of four treatments: untreated control (Con), wounding (W), wounding and regurgitate application (WþR), and application of 150 lg of MeJA per plant applied in a lanolin paste. Plants were treated at the first two fully expanded (source) leaves and wounding was performed by generating three rows of puncture wounds on each leaf side using a pattern wheel. Subsequently, 10 ll per leaf of either water or M. sexta regurgitate diluted 1:1 (v:v) was dispersed over the puncture wounds (n = 8-10).