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Keywords:

  • Coronatine insensitive1;
  • induced resistance;
  • Manduca sexta;
  • Tupiocoris notatus;
  • Epitrix hirtipennis;
  • caterpillar movement;
  • Nicotiana  attenuata

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Arabidopsis and tomato plants mutated in the F-box protein COI1 mediating jasmonate (JA) responses are more susceptible to herbivores in laboratory trials, but the exact mechanisms of COI1-mediated resistance are not known. We silenced COI1 by transformation with an inverted repeat construct (ir-coi1) in Nicotiana  attenuata, a plant the direct and indirect defenses of which against various herbivores have been well studied. ir-coi1 plants are male sterile and impaired in JA-elicited direct [nicotine, caffeoylputrescine and trypsin proteinase inhibitor (TPI) activity] and indirect (cis-α-bergamotene emission) defense responses; responses not elicited by JA treatment (ethylene production and flower TPI activity) were unaffected. Larvae of Manduca  sexta, a common herbivore of N. attenuata, gained three times more mass feeding on ir-coi1 than on wild-type (WT) plants in glasshouse experiments. By regularly moving caterpillars to unattacked leaves of the same plant, we demonstrate that larvae on WT plants can grow and consume leaves as fast as those on ir-coi1 plants, a result that underscores the role of COI1 in mediating locally induced resistance in attacked leaves, and the importance of herbivore movement in avoiding the induced defenses of a plant. When transplanted into native habitats in the Great Basin Desert, ir-coi1 plants suffer greatly from damage by the local herbivore community, which includes herbivores not commonly found on N. attenuata WT plants. Choice assays with field-grown plants confirmed the increased attractiveness of ir-coi1 plants for both common and unusual herbivores. We conclude that NaCOI1 is essential for induced resistance in N. attenuata, and that ir-coi1 plants highlight the benefits of herbivore movement for avoiding induced defenses.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Coronatine insensitive 1 (COI1), an F-box protein, was originally identified in a screen for Arabidopsis mutants insensitive to the bacterial phytotoxin coronatine (Feys et al., 1994). The structural similarity of coronatine to jasmonic acid (JA) suggested that the mutants would also be insensitive to JA, which was supported by bioassays of root growth inhibition by coronatine and methyl jasmonate (MeJA) (Feys et al., 1994; Li et al., 2004). Studies of COI1 in Arabidopsis and tomato highlighted its role in fertility, but in contrast to the Arabidopsis coi1 mutant, which is male sterile and impaired in anther dehiscence (Feys et al., 1994), the tomato coi1 mutant shows female sterility (Li et al., 2004). Numerous laboratory bioassays revealed that in most existing coi1 mutants, the defects in JA signaling are associated with increases in susceptibility to herbivores and pathogens.

When infested by Bradysia  impatiens larvae, Arabidopsis coi1 plants rarely survived, whereas wild-type (WT) plants did (Stintzi et al., 2001). Mewis et al. (2005) reported that both the specialist aphid Myzus  persica and the generalist aphid Brevicoryne  brassicae developed significantly greater populations on the coi1 mutant. In addition, Spodoptera  exigua larvae gained more mass on coi1 than on WT plants, although the caterpillars consumed less leaf material. Compared with WT plants, Arabidopsis coi1 plants are also more susceptible to the fungal pathogens Alternaria  brassicicola and Botrytis  cinerea, but not to Hyaloperonospora  parasitica (Thomma et al., 1998). Similarly, when soybean coi1 mutants were infested with B. cinerea or Erwinia  carotovora, all of the plants died, yet the death rate of WT plants was below 6% (Wang et al., 2005). The two-spotted spider mite Tetranychus  urticae, reared on lima bean, clearly preferred the tomato coi1 mutant over WT plants in choice assays, and laid more eggs on the mutant (Li et al., 2004). The altered attractiveness of tomato and Arabidopsis coi1 mutants has been shown to coincide with the suppressed induction of defense genes and/or decreased levels of induced secondary defense metabolites. In contrast to WT plants, eliciting tomato coi1 mutants with MeJA does not up-regulate trypsin proteinase inhibitor (TPI) activity or defense-related genes such as those encoding PI-II, threonine deaminase (TD) and polyphenol oxidase (Li et al., 2004). Similarly, in Arabidopsis coi1 mutants decreased levels of defensive metabolites such as glucosinolates (Mewis et al., 2005) as well as impaired wound-induced gene expression (Reymond et al., 2000) have been reported. However, to date no field experiments have been conducted with coi1 mutants to test how the function of COI1 affects the natural herbivore community of a plant. In addition, whether defects in constitutive or induced defense mechanisms account for the increased susceptibility of coi1 mutants to herbivores remains unknown. To resolve the question of whether COI1 plays a role in defending plants against their natural herbivores, a model plant, the defense physiology and ecology of which are understood, is required (Baldwin, 2001).

Nicotiana  attenuata is a native plant of the southwestern USA and grows in the immediate post-fire environment. Timing germination from a long-lived seed bank, it responds to pyrolysis products found in wood smoke (Preston and Baldwin, 1999). Because the plant has to ‘chase’ the ephemeral post-fire environment, its herbivore community, which has been characterized during more than 15 years of fieldwork, is forced to re-establish itself with each new plant population (Baldwin, 2001; Kessler and Baldwin, 2001; Kessler et al., 2004; Steppuhn et al., 2004). As a consequence, the composition of the herbivore community of a given plant population is highly unpredictable. Such unpredictability may have selected for plants that have access to a wide array of inducible defenses (Kessler and Baldwin, 2004), as such an array allows plants to tailor their defense responses to a particular herbivore community (Kessler and Baldwin, 2004; Voelckel and Baldwin, 2004). How the plant tailors its defense responses is best understood in the case of attack by the larvae of the specialist lepidopteran herbivore Manduca sexta, which regularly accounts for most of the leaf area lost to insect herbivores in native populations (Kessler and Baldwin, 2004).

Feeding of M. sexta larvae or the application of larval oral secretions and regurgitant (OS) to puncture wounds elicits a rapid burst of JA, which in turn functions upstream of the induced accumulation of defense metabolites (Halitschke and Baldwin, 2004) such as nicotine (Baldwin, 1999), caffeoylputrescine and diterpene glycosides (Keinanen et al., 2001), and the increased activity of anti-digestive proteins (e.g. TPIs and TD; Van Dam et al., 2001; Zavala et al., 2004a; Chen et al., 2005; Kang et al., 2006). Silencing nicotine and TPI production in transformed plants has demonstrated the importance of these compounds in herbivore defense (Steppuhn et al., 2004; Zavala et al., 2004b; Kang et al., 2006). In addition, feeding of M. sexta larvae or the treatment of wounds with M. sexta OS also elicits quantitative and qualitative changes in the volatile organic compounds (VOCs; Halitschke et al., 2000) released from plants, which function as indirect defenses by attracting predators of M. sexta larvae (Kessler and Baldwin, 2001). The herbivore-induced release of terpenoids, a specific class of VOCs, has been shown to depend on JA signaling (Halitschke and Baldwin, 2003), whereas other emitted compounds such as green leaf volatiles are produced independently of JA signaling. The feeding of M. sexta larvae or treatment of puncture wounds with OS also elicits the release of ethylene (Kahl et al., 2000), a volatile hormone that interferes with the wound-induced production of nicotine (Winz and Baldwin, 2001). Kahl et al. (2000) showed that exogenous MeJA treatment failed to induce the release of ethylene, which suggests that the ethylene response is not mediated by JA.

We identified, isolated and characterized the N. attenuata COI1 gene and generated stable transformants that are RNAi-silenced in their expression of COI1 (ir-coi1). We examined the performance of M. sexta larvae on these plants in glasshouse experiments and the performance of the native herbivore community in field trials (performed in Utah, USA). In addition, we profiled the herbivore-induced secondary metabolites that are known to mediate herbivore resistance in N. attenuata. By regularly moving M. sexta larvae on WT and ir-coi1 plants, we analyzed the function of NaCOI1 in eliciting defense responses in the attacked leaf and demonstrated that herbivore movement is important for avoiding direct defenses.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Isolation and characterization of COI1 in N. attenuata

Comparing known COI1 sequences and selecting conserved regions, we designed PCR primers and used them to amplify a part of the putative COI1 gene in N. attenuata. The isolated 932-bp fragment was used as a probe to screen a cDNA library from N. attenuata shoot tissue induced by feeding M. sexta larvae (Hermsmeier et al., 2001). Sequencing the positive clones resulted in a full-length sequence of COI1 of N. attenuata (NaCOI1), as confirmed by alignment with known COI1 cDNA sequences from other plant species. To analyze the copy number of COI1 in the N. attenuata genome, we amplified a fragment of NaCOI1, and used it as the probe for a Southern hybridization, which revealed that one copy of COI1 is present in the genome of N. attenuata.

To analyze the expression pattern of NaCOI1 in response to herbivory, we performed a time-course experiment. Leaf tissue was harvested from individual plants at different times after wounding and applying M. sexta OS, a standardized treatment that has been shown to mimic the feeding of M. sexta (Halitschke et al., 2001). NaCOI1-specific qRT-PCR revealed that the accumulation of NaCOI1 transcript is suppressed after elicitation by wounding and M. sexta OS application (Fig. 1a; anova, treatment: F1,70 = 24.65; < 0.0001).

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Figure 1.  Accumulation of NaCOI1 transcripts is suppressed by herbivory. NaCOI1 was analyzed in (a) Nicotiana  attenuata wild-type (WT) plants (n = 5) at different times after elicitation by puncture wounds treated with the oral secretions of M. sexta (wounding + M. sexta OS) and in untreated (control) WT plants, (b) in the rosette leaves of WT and ir-coi1 (line 1 and 2) plants (n = 5) 60 min after elicitation by puncture wounds treated with water (wounding + water) or M. sexta oral secretions (wounding + M. sexta OS), and (c) in different tissues of untreated WT and ir-coi1 (line 1 and 2) plants (n > 3; ND, not determined). The abundance of NaCOI1 transcripts was analyzed by qRT-PCR and normalized to the abundance of transcripts of an unregulated gene (sulfite reductase). In this and all following figures, values are expressed as the mean (±SE).

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Silencing COI1 in N. attenuata

We silenced NaCOI1 in N. attenuata by Agrobacterium tumefaciens-mediated transformation (Krügel et al., 2002) using a pSOL3 transformation vector containing an inverted repeat construct of NaCOI1. Using Southern hybridization we identified two independently transformed lines with a single transgene insertion, which we selected for all further experiments (ir-coi1, lines 1 and 2).

The levels of NaCOI1 transcripts in ir-coi1 plants 60 min after wounding and applying water or M. sexta OS were analyzed by qRT-PCR (Fig. 1b). The levels of NaCOI1 transcripts in untreated (anova: F2,12 = 15.460, = 0.0005) and elicited (wound + water, F2,12 = 25.564, < 0.0001; Wounding + OS, F2,12 = 13.739, = 0.0008) leaves was significantly decreased in both ir-coi1 lines in comparison with the transcript levels in identically treated WT plants (Bonferroni’s post hoc test, < 0.05). The transcript analysis of different plant tissues revealed that NaCOI1 is expressed in leaves, stem, roots and flowers, and that the gene is silenced in all tissues of both ir-coi1 lines (Fig. 1c; anova, F2,45 = 42.199, < 0.0001; Bonferroni’s post hoc test, P < 0.05).

To test if ir-coi1 plants are altered in their response to exogenous MeJA treatment, as has been shown for other coi1 mutants (Feys et al., 1994; Li et al., 2004), we transferred germinating seeds to agar medium containing 20 or 50 μM MeJA. Compared with WT seedlings, both ir-coi1 lines developed significantly longer roots when the medium contained 20 μM (Fig. 2a; anova, F2,23 = 33.073, < 0.0001) or 50 μM MeJA (Fig. 2a; anova, F2,23 = 69.254, < 0.0001), confirming the results of previous studies.

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Figure 2.  Morphological changes in ir-coi1 plants. (a) Silencing NaCOI1 resulted in insensitivity to methyl jasmonate (MeJA) treatment. Wild-type (WT) and ir-coi1 (lines 1 and 2) seedlings were grown on Gamborg B5 medium containing MeJA in different concentrations (20 and 50 μM). Root growth of seedlings grown on MeJA-containing medium is shown as the percentage of roots of seedling grown on MeJA-free medium. Although the growth of WT roots was inhibited by MeJA, the growth of ir-coi1 roots was normal. (b) The flowers of ir-coi1 plants are impaired in anther dehiscence. ir-coi1 plants develop flowers that do not differ from WT plants in number and size, but are male sterile because of defects in dehiscence. In contrast to the flowers of WT plants, the flowers of both ir-coi1 lines have closed anthers when the corollas open. Although the anthers of line 1 will occasionally dehisce and release pollen, the anthers of line 2 remain unable to release pollen until the flower wilts.

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The vegetative growth phenotype of ir-coi1 plants does not differ from that of WT plants, except for slightly accelerated elongation of the shoot (data not shown). Like Arabidopsis coi1 mutants, the flowers of tobacco ir-coi1 plants are male sterile because of impaired anther dehiscence (Fig. 2b). The anthers of both ir-coi1 lines are closed when the flower opens, whereas the anthers of WT flowers are completely dehisced at the time of flower opening. Whereas the anthers of line 2 remain closed until the flower wilts, the anthers of line 1 would occasionally open and result in self-pollination. Drying cut anthers promotes their dehiscence, enabling manual pollination and the production of homozygous lines. Manual pollination of ir-coi1 plants resulted in viable seed production, demonstrating that pollen viability was not affected by silencing NaCOI1 (data not shown). This was confirmed by staining pollen grains with fluoresceine diacetate, which revealed that pollen from both WT and ir-coi1 flowers is viable.

JA-mediated herbivore-induced responses are COI1 dependent

The induction of TPI activity and the accumulation of nicotine in N. attenuata plants in response to herbivory is mediated by JA signaling (Halitschke and Baldwin, 2003), and both defense responses negatively affect herbivore performance (Glawe et al., 2003; Steppuhn et al., 2004). We analyzed the responses of WT and ir-coi1 plants to feeding M. sexta larvae, and to wounding and treatment with M. sexta OS (Fig. 3). Both treatments elicited similar levels of caffeoylputrescine and TPI activity. As the standardized treatment reduces variability in the amount and intensity of damage, we used this type of elicitation in most experiments.

image

Figure 3.  Jasmonic acid-mediated defense responses are not induced in ir-coi1 plants. Wild-type (WT) and ir-coi1 plants were elicited with puncture wounds that were treated either with water (wound + water) or the oral secretions of Manduca  sexta (wound + M. sexta OS). After 3 days, the leaf tissue was harvested and analyzed for: (a) accumulation of nicotine; (b) activity of trypsin proteinase inhibitors (TPIs); (c) accumulation of caffeoylputrescine; (d) accumulation of diterpene glycosides (DTGs).

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Although not elicited in response to wounding and M. sexta OS, nicotine levels (Fig. 3a; anova, genotype, F2,66 = 70.630, < 0.0001) in both ir-coi1 lines are lower than in WT plants (Bonferroni's post hoc test, P < 0.05). Moreover, TPI activity, which is strongly induced by wounding, and by wounding with the additional application of M. sexta OS in WT plants, is barely detectable in the ir-coi1 lines (Fig. 3b; anova, genotype, F2,58 = 35.074, < 0.0001, Bonferroni's post hoc test, P < 0.05). Similarly, caffeoylputrescine, which is also known to be induced by JA (Keinanen et al., 2001), was induced in WT plants after wounding and M. sexta OS treatment, but its accumulation was completely suppressed in both ir-coi1 lines (Fig. 3c; anova, genotype, F2,39 = 17.863, < 0.0001, Bonferroni's post hoc test, P < 0.05). A series of diterpene glycosides (DTGs) is induced by MeJA in the leaves of N. attenuata (Jassbi et al., 2006). In WT plants, the levels of DTGs increased in response to wounding and the application of OS, but the levels were significantly decreased in ir-coi1 plants (Fig. 3d; anova, genotype, F2,39 = 118.416, < 0.0001; Bonferroni's post hoc test, P < 0.05). The reduced levels of all analyzed metabolites were comparable in the two different ir-coi1 lines (Bonferroni’s post hoc test, line 1 versus line 2, P > 0.05).

The release of terpenoid VOCs, such as cis-α-bergamotene, is known to be induced by herbivory and to depend on JA signaling (Halitschke and Baldwin, 2003). The analysis of the herbivore-induced VOC bouquet of field-grown plants revealed that ir-coi1 (line 2) plants released significantly less cis-α-bergamotene (0.031 ± 0.026 ng h−1 plant−1) than did WT plants (0.221 ± 0.036 ng h−1 plant−1) after elicitation with puncture wounds and the application of M. sexta OS (Student’s t-test, = 4.306, = 0.0026).

The accumulation of rutin and chlorogenic acid, two secondary metabolites that are reportedly not inducible by MeJA treatment (Keinanen et al., 2001), was not suppressed in the ir-coi1 lines. Although rutin concentrations decreased slightly after wounding and the application of water or M. sexta OS in all three genotypes (anova, treatment, F2,39 = 9.785, = 0.0004), overall rutin levels were not significantly different in ir-coi1 plants (genotype, F2,39 = 3.261, = 0.0490; Bonferroni’s post hoc test, P > 0.05). Chlorogenic acid levels increased slightly after elicitation treatments (anova, treatment, F2,39 = 3.235, = 0.0501). Both ir-coi1 lines accumulated chlorogenic acid levels that were higher than those of WT plants (anova genotype, F2,39 = 12.197, < 0.0001; Bonferroni’s post hoc test, < 0.05). No differences for each substance were detected between the two ir-coi1 lines (Bonferroni’s post hoc test, P > 0.05).

The quantity of ethylene released from wound plus OS-elicited ir-coi1 leaves did not differ from that of elicited WT leaves (Student’s t-test, = 0.919, = 0.3851).

High proteinase inhibitor activity has been reported in the flowers of undamaged plants (Ausloos et al., 1995; Atkinson et al., 1993; Damle et al., 2005). Consistent with these findings, the activity of TPIs in the undamaged flowers of WT N. attenuata plants was much higher than in WT leaves (Fig. 3b). Nevertheless, no difference was detected between WT and ir-coi1 flowers (Student’s t-test, = 0.968, = 0.9848).

Exogenous JA does not recover defects of ir-coi1 plants

To test whether defense metabolites in ir-coi1 plants can be elicited by exogenous JA, we wounded WT and ir-coi1 plants and treated the wounds with JA. Levels of caffeoylputrescine (Fig. 4a; anova, F2,11 = 3.269, P = 0.0042) and TPI activity (Fig. 4b; anova, F2,12 = 12.977, P = 0.001, data log-transformed), increased slightly in elicited ir-coi1 leaves but were significantly lower than in elicited WT leaves (Fisher’s PLSD, P = 0.0003). Levels of both metabolites did not exceed control levels of WT plants in response to exogenous JA. In contrast, when as-lox plants deficient in JA biosynthesis (Halitschke and Baldwin, 2003) were treated with JA, levels of both secondary metabolites increased. Although levels of caffeoylputrescine could be restored to the levels found in WT plants, TPI activity was only partly recovered, a result that confirms previous analyses (Halitschke and Baldwin, 2003).

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Figure 4.  Treatment with jasmonic acid (JA) does not elicit defense responses in ir-coi1 plants. Rosette-stage wild-type (WT), as-lox and ir-coi1 (line 1) plants were elicited by treating puncture wounds with water (wound + water) or 0.625 μmol JA (wound + JA). After 3 days, leaves were harvested and analyzed for: (a) caffeoylputrescine and (b) trypsin proteinase inhibitor (TPI) activity. Although the induced levels of compounds could be at least partly recovered in as-lox plants, levels remained low in ir-coi1 plants. Lowercase letters refer to comparisons between control plants (wound + water), whereas capital letters indicate differences between JA-treated plants. Different letters indicate significant differences at P < 0.05.

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COI1 mediates resistance to M. sexta

To test the degree to which the deficiencies in defense metabolites caused by silencing NaCOI1 affect the performance of herbivores, we placed M. sexta neonate larvae on rosette leaves of WT and ir-coi1 (line 1) N. attenuata plants and allowed them to feed freely for 12 days. Larvae feeding on ir-coi1 plants gained significantly more mass after 6 days compared with larvae feeding on WT plants (Fig 5A; anova, F2,24 = 8.547, P = 0.016; Fisher’s PLSD, P = 0.0048). This difference continued to increase until the end of the experiment (after 12 days; Fisher’s PLSD, P < 0.0001).

image

Figure 5. Manduca  sexta performance is impaired by COI-mediated induced resistance. (a) Weight gain of M. sexta larvae on wild-type (WT) and ir-coi1 (line 1) plants. Freshly hatched larvae were placed on the fully developed leaves of rosette-stage plants and larval mass was recorded over 12 days. M. sexta gained significantly more weight on ir-coi1 than on WT plants. The upper row of stars refers to larvae feeding on ir-coi1 line 1 plants, and the lower row refers to ir-coi1 line 2. To analyze the impact of locally induced defenses on caterpillar growth, (b) larval mass and (c) consumed leaf area were analyzed in a clip-cage assay. Freshly hatched M. sexta larvae were placed on fully developed leaves of elongated WT (solid line) and ir-coi (dashed line) plants and enclosed in clip cages. The clip cages with caterpillars were moved to another randomly assigned leaf of the same plant after 3- (first and second feeding interval) or 1-day feeding intervals (third and fourth interval). Before cages and caterpillars were moved, caterpillar mass and consumed leaf area were recorded. As the feeding intervals were shortened, the difference in larval mass and consumed leaf area between WT and ir-coi1 waned and caterpillars performed similarly on WT and ir-coi1 plants. Significant differences between caterpillars feeding on WT plants: *P < 0.05, **P < 0.001 and ***P < 0.0001.

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With a clip-cage assay, we determined if the performance of M. sexta was affected by locally or by systemically induced responses, and if both responses were similarly mediated by NaCOI1. Enclosed in clip cages, freshly hatched M. sexta larvae were placed on fully developed leaves of WT and ir-coi1 plants. The caterpillars were weighed and moved to another leaf of the same plant after 3, 6, 7 and 8 days. When feeding on the same leaf for 3 days, caterpillars on ir-coi1 plants gained more weight (Fig. 5b; Student’s t-test, t = 3.021, P = 0.0061) and consumed significantly more leaf area (Fig. 5c; Student’s t-test, t = 2.245, P = 0.0347) than caterpillars feeding on WT plants. The differences between larvae vanished as the time between movements was shortened. Between day 7 and day 8 – when the caterpillars fed for only one day on each leaf – the weight gained (Student’s t-test, t = 1.204, P = 0.2413) and the leaf area consumed (Student’s t-test, t = 0.796, P = 0.4347) by caterpillars feeding on both genotypes were the same.

COI1-deficient plants are highly susceptible to herbivores in the field

To determine how NaCOI1-mediated responses affect the resistance of N. attenuata to herbivory under natural conditions, we transplanted WT and ir-coi1 plants into a field plot in their natural habitat and monitored damage by herbivorous insects. The most abundant herbivores on the tobacco plants were mirid bugs (Tupiocoris  notatus), which inflicted significantly more damage on ir-coi1 than on WT plants (Fig. 6a; Student’s t-test, t = 4.057, P = 0.0004). Similarly, we detected more flea beetle (Epitrix  hirtipennis) damage on ir-coi1 plants (Fig. 6b; Student’s t-test, t = 2.939, P = 0.0070). In addition to these common herbivores of N. attenuata, leaf hoppers (Empoasca ssp.) – herbivores that do not feed on WT tobacco plants – damaged ir-coi1 plants (Fig. 6c; Student’s t-test, t = 3.596, P = 0.0014).

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Figure 6.  Herbivory on field-grown ir-coi1 (line 2) plants. The characteristic feeding damage to field-grown plants caused by (a) mirids (Tupiocoris  notatus), (b) flea beetles (Epitrix  hirtipennis) and (c) leaf hoppers (Empoasca ssp.) was estimated as the percentage of leaf area removed 16 days after plants were transplanted to the field plot. Significant difference: *P < 0.05.

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Choice assays on field-grown plants confirmed the increased attractiveness of ir-coi1 plants. Flea beetles clearly preferred ir-coi1 to WT leaves (paired t-test, t = 2.875, P = 0.0183) and inflicted more damage on them (Fig. 7a; paired t-test, t = 3.038, P = 0.0040,). Additionally, Diabrotica  undecimpunctata, a leaf beetle that usually does not rely on N. attenuata as a host plant, damaged ir-coi1 leaves more than leaves of WT plants (Fig. 7b; paired t-test, t = 6.741, P = 0.0011).

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Figure 7.  Choice assays confirm the increased attractiveness of ir-coi1 plants. Leaf material from field-grown wild-type (WT) and ir-coi1 (line 2) plants was placed in plastic boxes together with herbivorous insects. The damage inflicted by (a) flea beetle (Epitrix  hirtipennis) and (b) Diabrotica  undecimpunctata was recorded. ir-coi1 plants received significantly more damage from both species. Significant differences: *P < 0.05; ***P < 0.0001.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

A comparison of the phenotypes of the different COI1-silenced plants provides a number of new insights into the role that this F-box protein plays in defense against herbivores, as well as in morphological responses that are not clearly defense related. Consistent with findings from tomato and Arabidopsis coi1 (Feys et al., 1994; Xie et al., 1998; Li et al., 2004), the root growth of ir-coi1 N. attenuata seedlings is relatively insensitive to MeJA treatments. The dramatic inhibition of root growth by exogenous JA treatments in WT plants highlights the presence of JA signaling in roots. For most species, the wound- and elicitor-induced dynamics of JA in the roots are unknown. Given the importance of JA signaling in the responses elicited by beneficial rhizobacteria in the roots in the mediation of induced systemic resistance (Pozo et al., 2004), a detailed analysis of JA signaling and biosynthesis in the roots of model systems is sorely needed to understand the function of jasmonates on a holistic level.

In contrast to the similar root responses among the available COI1-deficient plants, flower phenotypes are diverse. The sterility of the tomato coi1 plants is mainly caused by the defective maternal control of seed maturation (Li et al., 2004), whereas in N. attenuata, silencing COI1 results in defects in anther dehiscence that resemble the Arabidopsis coi1 flower phenotype (Feys et al., 1994). The stamens of tobacco ir-coi1 flowers are shorter than those of WT plants (Fig. 2b), a difference that was also observed in Arabidopsis coi1 flowers (Feys et al., 1994). In tomato coi1 plants, trichome shape and number, and the constitutive levels of proteinase inhibitors differed from those in WT flowers (Li et al., 2004). These phenotypes have not been reported in either Arabidopsis coi1 or N. attenuata ir-coi1 lines. Taken together the results suggest that COI1 is essential for the development of fertile flowers and viable seeds, but it remains to be determined if and how these differences are taxonomically related. Although N. attenuata is closely related to tomato, silencing COI1 in N. attenuata results in a floral phenotype that more closely resembles the Arabidopsis phenotype than the tomato phenotype. Both N. attenuata and Arabidopsis produce relatively small fruits in comparison with tomato (Li et al., 2004), and the differences in floral phenotypes may result from how COI1 mediates source-sink relationships in apoplastically connected sinks such as fruits and pollen.

In addition to its role in fertility, COI1 plays a central role in herbivore resistance: many studies have demonstrated the increased susceptibility of coi1 mutants to herbivores and pathogens (Thomma et al., 1998; Stintzi et al., 2001; Li et al., 2004; Mewis et al., 2005; Wang et al., 2005). The results of our field experiments confirm the findings of the previous studies. Silencing COI1 in N. attenuata, a model plant in which the ecology and mechanisms of induced resistance have been extensively studied, broadens our understanding of this gene. The defensive function of several secondary metabolites has been demonstrated by silencing the genes that produce them (Steppuhn et al., 2004; Zavala et al., 2004a). In addition, more than a decade of fieldwork in the natural habitat of N. attenuata has identified the community of herbivores that attack this species (Baldwin and Ohnmeiss, 1993; Kessler and Baldwin, 2002; Kessler et al., 2004; Steppuhn et al., 2004). The knowledge of secondary metabolites that mediate induced resistance in N. attenuata allows the identification of defects in ir-coi1 plants that increase the attractiveness of these plants to natural herbivores. In contrast to previous results (Steppuhn et al., 2004; Halitschke and Baldwin, 2003), in this experiment nicotine is not induced in WT plants in response to wounding and M. sexta OS, a result that could be ascribed to the early sampling time. That ir-coi1 plants contain less nicotine than WT plants suggests a role for NaCOI1 in the accumulation of basal nicotine levels. Induced levels of caffeoylputrescine, DTGs and TPIs in WT plants, and lower levels in ir-coi1 plants, were detected. Treatment with JA increases but does not fully recover TPI levels or the caffeoylputrescine levels found in JA-elicited WT plants. Similar results have been reported for tomato coi1, which does not up-regulate the PI-II gene (Li et al., 2004) and does not accumulate caffeoylputrescine in response to MeJA treatment (Chen et al., 2006). These findings demonstrate that NaCOI1 is required to elicit these direct defenses, and are consistent with the hypothesis that COI1 is involved in JA perception. The release of cis-α-bergamotene, which is known to be mediated by JA (Halitschke and Baldwin, 2003), functions as an indirect defense by attracting predatory insects to their prey (Kessler and Baldwin, 2001), and is also impaired in ir-coi1 plants. Similarly, in tomato, terpenoid production in trichomes has been shown to be COI1 dependent (Li et al., 2004). Taken together, this suggests that the ability to release volatile terpenoids as an indirect defense depends on COI1. We conclude that the inability of ir-coi1 plants to accumulate these defense compounds accounts for the increased susceptibility to herbivores such as M. sexta, T. notatus and E. hirtipennis.

Interestingly, ir-coi1 plants planted into the natural habitat of N. attenuata in Utah attract herbivorous insects that usually do not feed on WT plants, such as the leaf beetle D. undecimpunctata and the generalist leaf hopper Empoasca ssp. Similar results were obtained in field experiments with as-lox plants silenced in JA biosynthesis (Kessler et al., 2004), suggesting that JA signaling is involved in the resistance to leaf hoppers. In as-lox plants, the JA burst and JA-mediated defenses are not elicited, but it remains unclear if the lack of jasmonates or the lack of induced defense responses downstream of JA attracts these unusual herbivores. Additional field experiments are required to identify the factors that mediate the attraction of leafhoppers and allow them to utilize N. attenuata plants impaired in JA biosynthesis or JA responses as hosts. In summary, defenses that are known to be elicited by JA are clearly reduced in ir-coi1 plants, demonstrating that JA-mediated defense responses require COI1 in N. attenuata. Ethylene is known to be elicited by herbivory, but we did not detect differences in ethylene production between WT and ir-coi1 plants. Similarly levels of rutin and of chlorogenic acid did not decrease, which is consistent with the fact that these traits are not elicited by JA treatment (Kahl et al., 2000; Keinanen et al., 2001).

This work demonstrates that COI1 mediates aspects of the signaling that increases defense metabolites in tissues in the vicinity of the attack site. Depending on the speed and extent of the spatial spread of the zone of induced metabolites around the attacking herbivore, induced resistance would be expected to elicit movement in caterpillars as they attempt to feed on unelicited tissues (Edwards and Wratten, 1983). Previous work with M. sexta has shown that larvae usually do not move from the leaf on which they hatched for the first two instars of development (Kessler and Baldwin, 2002); hence the leaf on which they hatch provides the early instars with food. One reason they are stationary may be that movement at this developmental stage is costly. Movement increases the risk of being predated or parasitized, incurs metabolic costs and prolongs the time of starvation (Schultz, 1983), which might be more deleterious for larval fitness than having to cope with increased levels of TPI or similar defenses. When M. sexta larvae are allowed to feed freely, they grow larger on ir-coi1 than on WT plants. The fact that differences in weight gain are already detectable 6 days after the larvae hatched emphasizes the importance of these elicited defenses in slowing caterpillar development during early instars.

Interestingly, when larvae are manually moved to unattacked leaves after 1 day of feeding, larvae attain growth rates on WT plants that are comparable with those attained on COI1-silenced plants, and they consume similar quantities of leaf material. This result confirms that induced defenses require time to become effective against the herbivore that elicits them. In addition, it indicates that, in contrast to local responses, systemically induced defenses play a minor role in diminishing the larval performance of M. sexta, and that the induction of local defenses depends on NaCOI1. Moreover, these data suggest that caterpillars of later instars might be able to escape the ‘induced resistance’ of a plant by ‘induced movement’. As a result, caterpillars are expected to move less on ir-coi1 plants than on WT plants, resulting in lower metabolic costs and predation risks. As induced movement responses are likely to be sculpted by numerous ecological parameters, tests of this hypothesis are best conducted under field conditions with native herbivores. We anticipate that mutant plants impaired in systemic signaling will help elucidate the adaptive value of herbivore movement. The ability to dodge the defenses that a plant launches against damaging insects may increase longevity and fitness of the herbivores.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Plant growth and M. sexta rearing

We used seeds of the fourteenth generation of an inbred line of N. attenuata Torr. Ex Watts (synonymous with Nicotiana  torreyana: Solanaceae) as the WT genotype in all experiments. The original seeds were collected in 1988 in a natural population at the DI ranch in southwestern Utah, USA. Seed germination and plant growth were conducted as described by Krügel et al. (2002). In addition to WT and ir-coi1 plants, we used as-lox plants that are silenced in LOX3, a key enzyme of JA biosynthesis (Halitschke and Baldwin, 2003). The tissue-specific expression of NaCOI1 was analyzed in hydroponically grown plants (Van Dam et al., 2001).

In the field, seeds were pre-treated and sowed identically. The plates were then kept at 25°C/16-h light (200 μm s−1 m−2) and 20°C/8-h dark. After 10 days, seedlings were transferred to Jiffy 703 pots (1 ¾ in × 1 ¾ in; AlwaysGrows, http://www.alwaysgrows.com), which had been soaked in borax solution (0.4 mg/45 ml water). The seedlings were fertilized with iron solution (stock solution: 2.78 g FeSO4 7H2O and 3.93 g Titriplex in 1-liter distilled H2O, diluted 100-fold for fertilization) after 7 days. After 3–4 weeks, plants were transferred to the field plot. Plants were placed in a watered field plot at the Lytle Preserve research station (Santa Clara, UT, USA). Releases of the transformed plants were conducted under APHIS notification numbers 06-003-08n.

Eggs of M. sexta were ordered from North Carolina State University (Raleigh, NC, USA) and kept in a growth chamber (Snijders Scientific, http://www.snijders-tilburg.nl) at 26°C 16-h light, 24°C 8-h dark until the larvae hatched. For M. sexta performance assays, neonates were directly placed on fully developed leaves of rosette-stage plants. M. sexta OS were collected from 3rd to 4th instar larvae and diluted 1:2 (v/v) with deionized water before being added to puncture wounds.

Isolation of the COI1 gene in N. attenuata

An exon-specific fragment of the genomic COI1 gene of N. attenuata was amplified using primers derived from conserved cDNA regions of LeCOI1 (Li et al., 2004), AtCOI1 (Xie et al., 1998) and OsCOI1 (Hu et al., 2006). The isolated fragment was cloned into a pGEM®-T EASY vector (Promega, http://www.promega.com), purified, sequenced (Nucleo-Trap; Macherey-Nagel, https://http://www.macherey-nagel.com) and used as a probe to screen a cDNA library from N. attenuata shoot tissue induced by the feeding of M. sexta (Hermsmeier et al., 2001). Sequencing of several positive cDNA clones resulted in a full-length cDNA sequence of NaCOI1 (accession number EF025087). Alignment of the isolated NaCOI1 cDNA sequence with other known COI1 sequences revealed 89% similarity with LeCOI1 and 82% similarity with AtCOI1.

Generation of transgenic plants

A 288-bp fragment of the cDNA sequence of NaCOI1 was inserted into the pSOL3 transformation vector as an inverted repeat construct (Bubner et al., 2006). This vector was transformed into N. attenuata WT plants using A. tumefaciens-mediated transformation (Krügel et al., 2002). The presence of the hygromycin resistance gene (hptII) in the transformation vector allowed transformed plants to be identified by selecting hygromycin-resistant individuals (Krügel et al., 2002). The number of insertions was determined by Southern hybridization of genomic DNA using a PCR fragment of the hptII gene as probe. Two single-insertion lines (ir-coi1 lines 1 and 2) were identified. These were screened for homozygocity and used in all subsequent experiments.

One of the striking morphological phenotypes of both ir-coi1 lines was the inability of their anthers to dehisce and the resulting male sterility. Cutting the anther-carrying filaments and drying in a desiccator for 24 h caused the anthers to open and release fertile pollen grains. The dried anthers were used to pollinate stigmas of open flowers, which resulted in capsules containing viable seeds. This method was used to generate homozygous progeny of both ir-coi1 lines.

Fluoresceine diacetate staining was used to determine viability of pollen grains. For the fluorescein diacetate stock solution, 60 mg was dissolved in 30 ml acetone. The stock solution was added to 10 ml of a 10% (w/v) sucrose solution until it turned milky. One drop of this solution was placed on a glass slide. Open anthers were dipped into the solution and released pollen grains were incubated for 3 min. Stained pollen grains were observed under the fluorescence microscope.

To determine the MeJA sensitivity of the plants, seedlings were grown on MeJA-containing agar. As MeJA inhibits germination of the seeds, they had to be pre-germinated on GB5 plant agar (Krügel et al., 2002). After 3 days, the seed and the surrounding agar were excised and transferred to freshly prepared agar plates containing 20 and 50 μM MeJA. Root length was measured after 10 days.

Nucleic acid analysis

DNA was extracted from the leaf tissue of fully developed plants using the cetyltrimethylammonium bromide (CTAB) method (Rogers and Bendich, 1985) with the following modification: after the second chloroform/isoamyl alcohol extraction, a two-thirds volume of ice-cold isopropanol was added to the supernatant and the sample was incubated for 30 min. After being centrifuged briefly, the supernatant was discarded and the pellet was dissolved in high-salt Tris-EDTA. The solution was then incubated with 100 ng μl−1 RNAse (30 min at 37°C) followed by another extraction with chloroform/isoamyl alcohol.

DNA samples (10 μg) were digested with different restriction enzymes, size fractionated in a 0.8% (w/v) agarose gel and Southern blotted (Brown, 1997) onto a nylon membrane (GeneScreen Plus; Perkin Elmer, http://www.perkinelmer.com). Fragments of NaCOI1 (forward primer, 5′-GTTGAGAATGATGGAGAATGGG-3′; reverse primer, 5′-GGCACCTTTGCAGTAAGAAAC-3′) and of hptII (forward primer, 5′-CGTCTGTCGAGAAGTTTCTG-3′; reverse primer, 5′-CCGGATCGGACGATTGCG-3′), respectively, were amplified by PCR and used as probes for Southern hybridization. The probes were labeled with α-32P (Rediprime® II DNA labeling system; Amersham Biosciences, http://www.amersham.com).

To analyze NaCOI1 expression, we extracted total RNA with TRI reagent following the protocol from TIGR (http://www.tigr.org/tdb/potato/images/SGED_SOP_3.1.1.pdf). cDNA was synthesized from 150 ng RNA using MultiScribe® reverse transcriptase (Applied Biosystems, http://www.appliedbiosystems.com). Quantitative real-time PCR (ABI PRISM®7000; Applied Biosystems) was conducted using the qPCR® core reagent kit (Eurogentec, http://www.eurogentec.be), a gene-specific TaqMan primer pair (forward primer, 5′-CAGGGCATCTTCAGCTGGTC-3′; reverse primer, 5′-CGGGATGCTCAGCAACGA-3′) and a double fluorescent dye-labeled probe (5′-CTCTTGGCGATGGCTCGGCCATT-3′). The relative gene expression was calculated using a 10-fold dilution series of cDNAs containing NaCOI1 transcripts. Sulfite reductase, which is not regulated under our experimental conditions (B. Bubner and I.T. Baldwin, unpublished data), served as the endogenous control gene (Bubner and Baldwin, 2004).

To analyze the expression of NaCOI1 in WT plants, we conducted a kinetic experiment. Fully developed leaves were either left untreated (control) or elicited by wounding and the application of M. sexta OS. Leaf tissue was harvested from five independent plants for each time point and treatment. To analyze NaCOI1 expression in transgenic plants, leaves were treated identically, but tissue was only harvested 60 min after elicitation (n = 5). The expression of NaCOI1 in roots, stem, rosette leaves, stem leaves, flowers and anthers was analyzed in untreated hydroponically grown plants (n > 3).

Analysis of herbivory

To analyze M. sexta performance, freshly hatched larvae were placed on the second fully developed leaf of rosette-stage plants. After feeding for 3 days, larvae were weighed every second day for 9 days. To restrict the area of feeding, the caterpillars were enclosed on leaves using clip cages. The time during which the caterpillars were allowed to feed on a single leaf decreased over the time of the experiment (first and second intervals, 3 days; third and fourth intervals, 1 day). Each caterpillar was weighed before being placed on another leaf. After the larva was moved to another leaf, the leaf area it had consumed during the feeding interval was determined using graph paper. The clip-cage assays had to be determined after 9 days because of the size and consumption of caterpillars.

In the field, we measured the abundance of naturally occurring herbivores and the particular feeding damage they cause on WT and ir-coi1 (line 2) plants 16 days after they were transplanted to the field site. In addition, choice assays were conducted in plastic boxes with detached leaves of field-grown WT and ir-coi1 (line 2) plants that were matched in size and shape. The locations of the leaves from the different genotypes were alternated inside the box to avoid position effects. All insects used in choice assays were collected at the field site. For the E.  hirtipennis choice assay, three beetles were enclosed in each box. After 48 h, the damage (percentage of leaf area removed) and the choice of the insects were recorded. The D. undecimpunctata choice assay was conducted under identical conditions, except that only two beetles were enclosed in each box and the damage was recorded after 48 h.

Analysis of direct defense traits and VOC emission

Leaf tissue (100–150 mg) was analyzed for nicotine, rutin, caffeoylputrescine, chlorogenic acid, DTGs and TPI activity 3 days after elicitation by treating puncture wounds with water or OS. In complementation experiments, leaves were elicited by puncture wounding and applying water or 0.625 μmol JA. The accumulation of secondary metabolites was analyzed by high-performance liquid chromatography as previously described (Halitschke and Baldwin, 2003). TPI activity was analyzed by the radial diffusion assay described by (Van Dam et al., 2001).

Terpenoid VOCs were trapped from individual leaves of field-grown plants 24 h after elicitation by manual wounding and applying M. sexta OS as described above. VOCs released from the treated leaf were trapped for 8 h using charcoal traps (Orbo®32; Supelco, http://www.sigmaaldrich.com/Brands/Supelco_Home.html). The traps were spiked with 400 ng tetraline as an internal standard and eluted with 500 μl dichlormethane. Samples were analyzed by gas chromatography-mass spectrometry as described by Halitschke et al. (2000). cis-α-Bergamotene was quantified with a trans-caryophyllene calibration curve.

To analyze ethylene release, fully developed stem leaves of WT and ir-coi1 (line 1) plants were wounded and treated with M. sexta OS. The leaves were cut and placed in 250-ml glass cuvettes. After 5 h, the cuvettes were connected to a photoacoustic laser spectrometer to measure accumulated ethylene levels. The air coming out of the cuvettes was pumped through a cooling trap (130–150 ml min−1) to remove CO2 and H2O. To remove ambient hydrocarbons the air was cleaned by organic oxidation at 540°C through a platinum catalyst (Sylatech, http://www.sylatech.de) and further directed to the sampling devices. A line-tunable infrared laser was used as a light source and the detection device was a resonant photoacoustic cell (INVIVO, http://www.invivo-gmbh.de). The two critical laser lines for ethylene (10p14 and 10p16) were measured. Ethylene was detected by two acoustic cells. To calibrate and continuously adjust the laser line, one cell was filled with a known ethylene concentration (516 ppb) withdrawn from a calibration gas source. The sampling cell was calibrated with the gas (516 ppb) before each measurement.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

We thank Anke Sieler, Sven Kumme, Holger Merker, Antje Dudda, Ashley McDonough and Anne-Kathrin Adler for invaluable help in the greenhouse, and in the laboratory, Thomas Hahn for sequencing, Susan Kutschbach and Antje Wissgott for generating transformed plants, Caroline von Dahl for help with ethylene measurements and Danny Keßler for assistance during the field experiments and for maintaining herbivore colonies. We thank Andre Keßler for kindly providing insect images. In addition, we thank Merijn Kant and Emily Wheeler for commenting on and editing the manuscript, as well as two three anonymous reviewers for helpful comments.

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  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Figure S1. (a) Southern hybridization shows that only one copy of NaCOI1 is present in Nicotiana attenuata. (b) The transformation vector that was used to generate transgenic plants silenced in NaCOI1. (c) Southern hybridization of genomic DNA from ir-coi1 lines 1 and 2 with a hptII-specific probe identified those lines containing only one transgene. Figure S2. (a) Fluorescein diacetate-stained pollen grains from wildtype (WT) and ir-coi1 plants. (b) Ethylene released from elicited leaves of WT and ir-coi1 (line 1) plants. (c) Activity of trypsin proteinase inhibitor in flowers of WT and ir-coi1 plants. Figure S3. The accumulation of (a) rutin and (b) chlorogenic acid 3 days after elicitation. Figure S4. Flea beetles (Epitrix hirtipennis) clearly preferred ir-coi1 to WT leaves in choice assays. Figure S5. Caffeoylputrescine levels and TPI activity in response to Manduca sexta feeding.

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TPJ_3119_sm_fig1.pdf1624KSupporting info item
TPJ_3119_sm_fig2.pdf1214KSupporting info item
TPJ_3119_sm_fig3.pdf1014KSupporting info item
TPJ_3119_sm_fig4.pdf1084KSupporting info item
TPJ_3119_sm_fig5.pdf1068KSupporting info item

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