Nicotiana Attenuata Research Articles

Isolation and characterization of the threonine deaminase promoter in Nicotiana attenuata

The enzyme encoded by the threonine deaminase (TD) gene catalyzes the conversion of threonine to α-keto butyrate in the biosynthesis of isoleucine (Ile). In Nicotiana attenuata, TD transcripts accumulate constitutively in cotyledons and flowers and are elicited in leaves by wounding, herbivore attack, and methyl jasmonic acid (MeJA) treatment. To understand TD's unique pattern of expression, we isolated a genomic clone of the TD gene from N. attenuata and characterized its promoter. T2 transgenic plants, each harboring single copies of fusions of different parts of the 5′ non-coding region to the β-glucuronidase reporter gene, demonstrated that the promoter was constitutively expressed in seedlings and flowers, and elicited in leaves by wounding or by MeJA treatment. Promoter deletion analysis defined the promoter regions capable of directing minimal expression in cotyledons and anthers as −142 to −31bp, and in stigmas as −289 to −231bp. Regions from −142 to −31bp were found to be important for basal elicitation in leaves by both wounding and MeJA treatment. These promoter elements may prove valuable in biotechnological applications.

Using nutritional indices to study LOX3‐dependent insect resistance

Induced resistance to biotic attackers is thought to be mediated by responses elicited by jasmonic acid (JA), a subset of which are lipoxygenase 3 (LOX3) dependent. To understand the importance of LOX3-mediated insect resistance, we analysed the performance of Manduca sexta larvae on wild-type (WT) and on isogenic Nicotiana attenuata plants silenced in NaLOX3 expression and JA signalling, and we used Waldbauer nutritional indices to measure the pre- and post-ingestive effects. LOX3-mediated defenses reduced larval growth, consumption and frass production. These defenses reduced how efficiently late-instar larvae converted digested food to body mass (ECD). In contrast, LOX3-mediated defenses decreased approximate digestibility (AD) in early instar larvae without affecting the ECD and total food consumption. Larvae of all instars feeding on defended WT plants behaviourally compensate for their reduced body mass by consuming more food per unit of body mass gain. We suggest that larvae feeding on plants silenced in NaLOX3 expression (as-lox) initially increase their AD, which in turn enables them to consume more food in the later stages and consequently, to increase their ECD and efficiency of conversion of ingested food (ECI). We conclude that N. attenuata's oxylipin-mediated defenses are important for resisting attack from M. sexta larvae, and that Waldbauer nutritional assays reveal behavioural and physiological counter responses of insects to these plant defenses.

Caterpillar‐elicited methanol emission: a new signal in plant–herbivore interactions?

Plants release into the atmosphere large quantities of volatile organic compounds (VOCs), of which methanol (MeOH), a putative waste product, is the second most abundant. Using online proton-transfer-reaction mass spectrometry (PTR-MS), we demonstrate that when Manduca sexta larvae attack Nicotiana attenuata plants, the wound-induced release of MeOH dramatically increases. The sustained MeOH emission 24 h after herbivore feeding is already substantially greater than the release of the well-characterized green-leaf VOC E-2-hexenal. Herbivore attack and treatment of puncture wounds with larval oral secretions (OS) increased the transcript accumulation and activity of leaf pectin methylesterases (PMEs), and decreased the degree of pectin methylation, as determined by (1)H-NMR; therefore, we propose that the released MeOH originates from the activation of PMEs by herbivore attack. The herbivore- and OS-elicited MeOH results not from the activity of previously characterized elicitors in OS but from a pH shift at the wound site when larval OS (pH 8.5-9.5) are introduced into the wounds during feeding. Applying MeOH to plants in quantities that mimic the herbivory-elicited release decreases the activity of the potent plant defense proteins trypsin proteinase inhibitors (TPI), and increases the performance of the attacking larvae. The pH of lepidopteran larvae regurgitants is commonly very high, and the MeOH released during feeding that is elicited by the pH change at the wound site functions as a quantitative signal that influences the outcome of the plant-herbivore interaction.

Occurrence of tetraploidy in Nicotiana attenuata plants after Agrobacterium-mediated transformation is genotype specific but independent of polysomaty of explant tissue

Genotypes of Nicotiana attenuata collected from Utah and Arizona were transformed with 17 different vectors (14 unpublished vectors based on 3 new backbone vectors) using an Agrobacterium-mediated procedure to functionally analyze genes important for plant-insect interactions. None of the 51 T1-T3 transgenic Utah lines analyzed by the flow cytometry were tetraploid, as opposed to 18 of 33 transgenic Arizona lines (55%). Analysis of T0 regenerants transformed with the same vector carrying an inverted repeat (IR) N. attenuata pro-systemin construct confirmed the genotype dependency of tetraploidization: none of the 23 transgenic Utah lines were tetraploid but 31 (72%) of 43 transgenic Arizonas were tetraploid. We tested the hypothesis that the differences in polysomaty of the explant tissues accounted for genotype dependency of tetraploid formation by measuring polysomaty levels in different seedling tissues. Hypocotyls, cotyledons, and roots of Utah and Arizona genotypes contained similar percentages of 4C nuclei (61 and 60; 7 and 5; and 58 and 61%, respectively). Since we used hypocotyls as explant sources and the nonoccurrence of tetraploid Utah transformants does not correspond to the high percentage of 4C nuclei in Utah hypocotyls, we can rule out a direct relationship between tetraploid formation and polysomaty level. We hypothesize that the difference between the Utah and Arizona genotypes results from the failure of polyploid Utah callus to regenerate into fully competent plants. We propose that future work on post-transformation polyploidy concentrate on the processes that occur during callus formation and plant regeneration from callus.

Priming of plant defense responses in nature by airborne signaling between Artemisia tridentata and Nicotiana attenuata

Plants release volatile organic compounds (VOCs) in response to wounding and herbivore attack, some of which trigger responses in neighboring unattacked plants in the laboratory under conditions that are not likely to occur in the real world. Whether plants 'eavesdrop' on the volatile emissions of their neighbors in nature is not known. The best documented field study of between-species signaling via above-ground VOCs involves increases in fitness parameters of native tobacco (Nicotiana attenuata) transplanted adjacent to clipped sagebrush (Artemesia tridentata tridentata). Clipped sagebrush releases many biologically active VOCs, including methyl jasmonate (MeJA), methacrolein and a series of terpenoid and green leaf VOCs, of which MeJA, while active under laboratory conditions, is not released in sufficient quantities to directly elicit induced resistance in the field. Here we demonstrate, with laboratory and field-based experiments, that priming (rather than direct elicitation) of native N. attenuata's induced chemical defenses by a sagebrush-released VOC bouquet can account for earlier findings. With microarrays enriched in N. attenuata herbivore-regulated genes, we found transcriptional responses in tobacco growing adjacent to clipped sagebrush foliage, but failed to detect the direct elicitation of defensive chemicals or proteins. However, we observed an accelerated production of trypsin proteinase inhibitors when Manduca sexta caterpillars fed on plants previously exposed to clipped sagebrush. This readying of a defense response, termed priming, results in lower total herbivore damage to plants exposed to clipped sagebrush and in a higher mortality rate of young Manduca caterpillars. Our study demonstrates priming of plant defense responses as a mechanism of plant-plant signaling in nature, and provides an example for the analysis of between-plant signaling under ecologically realistic conditions. Although we describe priming as a potential mechanism for signaling between plants in nature, we critically discuss the ecological relevance of the particular interaction.

Silencing of a Germin-Like Gene in Nicotiana attenuata Improves Performance of Native Herbivores

Germins and germin-like proteins (GLPs) are known to function in pathogen resistance, but their involvement in defense against insect herbivores is poorly understood. In the native tobacco Nicotiana attenuata, attack from the specialist herbivore Manduca sexta or elicitation by adding larval oral secretions (OS) to wounds up-regulates transcripts of a GLP. To understand the function of this gene, which occurs as a single copy, we cloned the full-length NaGLP and silenced its expression in N. attenuata by expressing a 250-bp fragment in an antisense orientation with an Agrobacterium-based transformation system and by virus-induced gene silencing (VIGS). Homozygous lines harboring a single insert and VIGS plants had significantly reduced constitutive (measured in roots) and elicited NaGLP transcript levels (in leaves). Silencing NaGLP improved M. sexta larval performance and Tupiocoris notatus preference, two native herbivores of N. attenuata. Silencing NaGLP also attenuated the OS-induced hydrogen peroxide (H(2)O(2)), diterpene glycosides, and trypsin proteinase inhibitor responses, which may explain the observed susceptibility of antisense or VIGS plants to herbivore attack and increased nicotine contents, but did not influence the OS-elicited jasmonate and salicylate bursts, or the release of the volatile organic compounds (limonene, cis-alpha-bergamotene, and germacrene-A) that function as an indirect defense. This suggests that NaGLP is involved in H(2)O(2) production and might also be related to ethylene production and/or perception, which in turn influences the defense responses of N. attenuata via H(2)O(2) and ethylene-signaling pathways.

Using ‘mute’ plants to translate volatile signals

When attacked by herbivores, plants release volatile organic compounds (VOCs) that attract natural enemies of the herbivores and function as indirect defenses. Whether or not neighboring plants 'eavesdrop' on these VOCs remains controversial because most studies use unrealistic experimental conditions and VOC exposures. In order to manipulate exposures of wild-type (WT) Nicotiana attenuata'receiver' plants, we elicited transformed 'emitter' plants, whose production of herbivore-induced C6 green leaf volatiles (GLVs) or terpenoid volatiles was genetically silenced, and placed them up-wind of WT 'receiver' plants in open-flow experimental chambers. We compared the transcriptional and secondary metabolite defense responses of WT receiver plants exposed to VOCs from these transgenic emitter plants with those of plants exposed to VOCs from WT emitter plants. No differences in the constitutive accumulation of defense metabolites and the signal molecule jasmonic acid (JA) were found. Additional elicitation of receiver plants revealed that exposure to WT, GLV-deficient and terpenoid-deficient volatile blends did not prime induced defenses, JA accumulation, or the expression of lipoxygenase 3 (NaLOX3), a gene involved in JA biosynthesis. However, exposure to wound- and herbivore-induced VOCs significantly altered the transcriptional patterns in receiver plants. We identified GLV-dependent genes by complementing the GLV-deficient volatile blend with a mixture of synthetic GLVs. Blends deficient in GLVs or cis-alpha-bergamotene regulated numerous genes in receiver plants that did not respond to the complete VOC blends of WT emitters, indicating a suppressive effect of GLVs and terpenoids. Whether these transcriptional responses translate into changes in plant fitness in nature remains to be determined.

Induced defense in Nicotiana attenuata (Solanaceae) fruit and flowers

Plants protect themselves against herbivory using a continuum of strategies, ranging from constitutive defenses to intermittent induced responses. Induced defenses may not provide immediate and maximum protection, but could be advantageous when continuous defense is either energetically or ecologically costly. As such, induced defenses in flowers could help defend relatively valuable tissue while keeping reproductive structures accessible and attractive to pollinators. Thus far, no one has demonstrated the efficacy of induced defenses against floral herbivores (florivores) in the field. Here we show that mechanical leaf damage in wild tobacco, Nicotiana attenuata (Solanaceae), reduced both flower and fruit herbivory in the field and that exogenous application of methyl jasmonate, a potent elicitor of induced responses, reduced both leaf and floral damage in natural populations. This result is consistent with a survey of damage in the field, which showed a negative relationship between leaf damage and flower and fruit damage. Although optimal defense theory predicts that induced defenses should be rare in reproductive tissues, owing to their high fitness value, our results suggest otherwise. Induced defenses in leaves and reproductive tissues may allow plants to respond effectively to the concomitant pressures of defending against herbivory and attracting pollinators.

Differential Elicitation of Two Processing Proteases Controls the Processing Pattern of the Trypsin Proteinase Inhibitor Precursor in Nicotiana attenuata

Trypsin proteinase inhibitors (TPIs) of Nicotiana attenuata are major antiherbivore defenses that increase dramatically in leaves after attack or methyl jasmonate (MeJA) elicitation. To understand the elicitation process, we characterized the proteolytic fragmentation and release of TPIs from a multidomain precursor by proteases in MeJA-elicited and unelicited plants. A set of approximately 6-kD TPI peptides was purified from leaves, and their posttranslational modifications were characterized. In MeJA-elicited plants, the diversity of TPI structures was greater than the precursor gene predicted. This elicited structural heterogeneity resulted from differential fragmentation of the linker peptide (LP) that separates the seven-domain TPI functional domains. Using an in vitro fluorescence resonance energy transfer assay and synthetic substrates derived from the LP sequence, we characterized proteases involved in both the processing of the TPI precursor and its vacuolar targeting sequence. Although both a vacuolar processing enzyme and a subtilisin-like protease were found to participate in a two-step processing of LP, only the activity of the subtilisin-like protease was significantly increased by MeJA elicitation. We propose that MeJA elicitation increases TPI precursor production and saturates the proteolytic machinery, changing the processing pattern of TPIs. To test this hypothesis, we elicited a TPI-deficient N. attenuata genotype that had been transformed with a functional NaTPI gene under control of a constitutive promoter and characterized the resulting TPIs. We found no alterations in the processing pattern predicted from the sequence: a result consistent with the saturation hypothesis.

Piriformospora indica and Sebacina vermifera increase growth performance at the expense of herbivore resistance in Nicotiana attenuata

A Sebacinales species was recovered from a clone library made from a pooled rhizosphere sample of Nicotiana attenuata plants from 14 native populations. Axenic cultures of the related species, Piriformospora indica and Sebacina vermifera, were used to examine their effects on plant performance. Inoculation of N. attenuata seeds with either fungus species stimulated seed germination and increased growth and stalk elongation. S. vermifera inoculated plants flowered earlier, produced more flowers and matured more seed capsules than did non-inoculated plants. Jasmonate treatment during rosette-stage growth, which slows growth and elicits herbivore resistance traits, erased differences in vegetative, but not reproductive performance resulting from S. vermifera inoculation. Total nitrogen and phosphorous contents did not differ between inoculated and control plants, suggesting that the performance benefits of fungal inoculation did not result from improvements in nutritional status. Since the expression of trypsin proteinase inhibitors (TPI), defensive proteins which confer resistance to attack from Manduca sexta larvae, incur significant growth and fitness costs for the plant, we examined the effect of S. vermifera inoculation on herbivore resistance and TPI activity. After 10 days of feeding on S. vermifera-inoculated plants, larval mass was 46% higher and TPI activity was 48% lower than that on non-inoculated plants. These results suggest that Sebacina spp. may interfere with defense signaling and allow plants to increase growth rates at the expense of herbivore resistance mediated by TPIs.

Specificity in Ecological Interactions. Attack from the Same Lepidopteran Herbivore Results in Species-Specific Transcriptional Responses in Two Solanaceous Host Plants

Model systems have proven enormously useful in elucidating the biochemical function of plant genes. However their ecological function, having been sculpted by evolutionary forces specific to a species, may be less conserved across taxa. Responses to wounding and herbivore attack differ among plant families and are known to be mediated by oxylipin, ethylene, and systemin-signaling networks. We analyzed transcriptional responses of two native Solanaceous species to the attack of an herbivore whose elicitors are known not to be influenced by diet. With The Institute for Genomic Research 10k-cDNA potato (Solanum tuberosum) microarray, we compared the transcriptional responses of Nicotiana attenuata with those of black nightshade (Solanum nigrum) when both were attacked by the Solanaceous generalist herbivore, Manduca sexta. Based on an NADH dehydrogenase subunit F phylogeny, S. nigrum is more closely related to potato than N. attenuata but responded significantly less to M. sexta attack. Apart from transcriptional differences anticipated from their differences in secondary metabolism, both species showed distinct transcriptional patterns (with only 10% overlap in significantly regulated genes), which point to fundamental differences in the signaling cascades and downstream genes mediating herbivore resistance. The lackluster transcriptional response of S. nigrum could not be attributed to its inability to respond to elicitation, because methyl jasmonate elicitation of S. nigrum resulted in a strong transcriptional response. Given that attack from the same herbivore elicits profoundly different responses in two Solanaceaous taxa, we conclude that blueprints for commonly regulated responses to plant-herbivore interactions appear unlikely.

Plant–Plant Signaling: Application of trans- or cis-Methyl Jasmonate Equivalent to Sagebrush Releases Does Not Elicit Direct Defenses in Native Tobacco

Nicotiana attenuata plants growing in close proximity to damaged sagebrush (Artemisia tridentata ssp. tridentata) suffer less herbivory than plants near undamaged sagebrush. Sagebrush constitutively releases methyl jasmonate (MeJA), a compound that when applied directly to N. attenuata, elicits herbivore resistance and the direct defense traits [protease inhibitors (PIs), nicotine]. Damage increases the release of volatile MeJA, primarily in the cis epimer, suggesting that cis-MeJA may mediate this apparent interplant signaling. We characterized sagebrush's MeJA plume before and after damage in nature and in the laboratory, and compared the activity of trans- and cis-MeJA in inducing PIs, nicotine, and Manduca sexta resistance in N. attenuata. We used both lanolin applications and aqueous sprays that mimic natural exposures, and we determined the amount of volatilized MeJA required to elicit a nicotine response in open-grown plants. Wounding rapidly and transiently increased cis-MeJA emissions from damaged parts (but not systemically), and the released plume did not rapidly dissipate in nature. cis-MeJA was not consistently more active than trans-MeJA, and the order of exposure (trans- then cis-) did not influence activity. We conclude that volatile MeJA, either trans- or cis-, when applied at levels consistent with those released by sagebrush does not elicit direct defenses in N. attenuata.

Microarray analysis of salicylic acid‐ and jasmonic acid‐signalling in responses of Nicotiana attenuata to attack by insects from multiple feeding guilds

ABSTRACTResistance to herbivore attack is elicited by damage‐ and herbivore‐specific signals. To understand the role of salicylic acid (SA) and jasmonic acid (JA) signalling in the herbivore‐induced transcriptional responses of the native tobacco Nicotiana attenuata, a comparison was made of the responses in undamaged systemic leaves after attack by five herbivore species (Manduca sexta, Spodoptera littoralis, Trichoplusia ni, Tupiocoris notatus and Myzus nicotianae) from three feeding guilds and to elicitation by SA, BTH and methyl‐JA with a 789‐gene oligo‐microarray and measured endogenous SA and JA levels. Systemic responses to M. sexta attack were similar to the responses in locally attacked tissues; however, the up‐regulation of genes (such as proteinase inhibitor, PI) was more attenuated and resembled the responses to attack from the other Lepidopteran, S. littoralis, and to methyl‐JA elicitation. Responses to attack from the puncture‐feeding T. notatus repressed some JA‐elicited genes (PI) elicited by the leaf‐chewing Lepidopterans and most closely resembled SA elicitation. Attack from the phloem‐feeding M. nicotianae aphids elicited few responses. Treatment with the SA‐mimic BTH induced both SA‐ and JA‐elicited genes and most closely resembled the aphid response. SA concentrations were uniformly increased by herbivore attack and correlated with the down‐regulation of photosynthetic genes in all treatments.

Silencing of hydroperoxide lyase and allene oxide synthase reveals substrate and defense signaling crosstalk in Nicotiana attenuata.

The fatty acid hydroperoxide (HP) substrates required for the biosynthesis of jasmonic acid (JA) and green leaf volatiles (GLVs) are supplied by separate lipoxygenases (LOX). We silenced the expression of two genes downstream of the LOX: allene oxide synthase (AOS) and HP lyase (HPL) by antisense expression of endogenous genes (NaAOS, NaHPL) in Nicotiana attenuata, in which the biosynthesis of JA is amplified by herbivore-specific elicitors. We report that these elicitors also amplify wound-induced GLV releases, but suppress the wound-induced increase of NaHPL transcripts, suggesting that substrate flux controls GLV biosynthesis. As expected, silencing of NaHPL and NaAOS reduced GLV release and JA accumulation, respectively. Surprisingly, HPL- and AOS-silenced plants had enhanced JA and GLV responses, suggesting substrate 'crosstalk' between these two oxylipin cascades. Plants with depleted GLVs (as-hpl) were less attractive than wild type (WT) or empty vector control plants in choice-tests with native lepidopteran herbivores. In feeding trials, Manduca sexta larvae developed slower on as-hpl plants. The reduced larval consumption and performance, which was not caused by increases in defense responses in as-hpl plants, could be restored to WT levels by the addition of synthetic GLVs, demonstrating that GLVs function as feeding stimulants. Gene expression profiling by cDNA microarray analysis and characterization of several induced defenses in herbivore-elicited as-hpl and as-aos plants revealed differential involvement of JA and GLVs in defense signaling. Elicitation of volatile terpenoids (an indirect defense) requires JA signaling, where as trypsin protease inhibitor elicitation (a direct defense) requires both functional JA and GLV cascades.

Generalist and specialist lepidopteran larvae elicit different transcriptional responses in Nicotiana attenuata, which correlate with larval FAC profiles

AbstractUnlike generalist herbivores, specialists are believed to share a history of evolutionary interactions with their host plants. We determine whether a specialist lepidopteran species alters plant metabolism differently from two generalist species despite similarities in feeding mode and larval elicitors. With a cDNA microarray enriched in defence‐related genes, we compared the transcriptional responses elicited in the native tobacco Nicotiana attenuata by the oligophagous larvae of Manduca sexta and the polyphagous larvae of Heliothis virescens and Spodoptera exigua, which are all members of N. attenuata's natural herbivore community. We found the differences in plant responses to be correlated with the profile of larval elicitors [fatty acid–amino acid conjugates (FAC)] and discuss how variation in FAC composition may shape the interaction between generalist or specialist lepidopteran larvae and plants.

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.

Silencing the jasmonate cascade: induced plant defenses and insect populations.

We transformed the native tobacco, Nicotiana attenuata, to silence its lipoxygenase, hydroperoxide lyase, and allene oxide synthase genes in order to inhibit oxylipin signaling, known to mediate the plant's direct and indirect defenses. When planted into native habitats, lipoxygenase-deficient plants were more vulnerable to N. attenuata's adapted herbivores but also attracted novel herbivore species, which fed and reproduced successfully. In addition to highlighting the value of genetically silencing plants to study ecological interactions in nature, these results show that lipoxygenase-dependent signaling determines host selection for opportunistic herbivores and that induced defenses influence herbivore community composition.

Nitrogen supply influences herbivore-induced direct and indirect defenses and transcriptional responses in Nicotiana attenuata.

Although nitrogen (N) availability is known to alter constitutive resistance against herbivores, its influence on herbivore-induced responses, including signaling pathways, transcriptional signatures, and the subsequently elicited chemical defenses is poorly understood. We used the native tobacco, Nicotiana attenuata, which germinates in the postfire environment and copes with large changes in soil N during postfire succession, to compare a suite of Manduca sexta- and elicitor-induced responses in plants grown under high- and low-N (LN) supply rates. LN supply decreased relative growth rates and biomass by 35% at 40 d compared to high-N plants; furthermore, it also attenuated (by 39 and 60%) the elicitor-induced jasmonate and salicylate bursts, two N-intensive direct defenses (nicotine and trypsin proteinase inhibitors, albeit by different mechanisms), and carbon-containing nonvolatile defenses (rutin, chlorogenic acid, and diterpene glycosides), but did not affect the induced release of volatiles (cis-alpha-bergamotene and germacrene A), which function as indirect defenses. M. sexta and methyl jasmonate-induced transcriptional responses measured with a microarray enriched in herbivore-induced genes were also substantially reduced in plants grown under LN supply rates. In M. sexta-attacked LN plants, only 36 (45%) up-regulated and 46 (58%) down-regulated genes showed the same regulation as those in attacked high-N plants. However, transcriptional responses frequently directly countered the observed metabolic changes. Changes in a leaf's sensitivity to elicitation, an attacked leaf's waning ability to export oxylipin wound signals, and/or resource limitations in LN plants can account for the observed results, underscoring the conclusion that defense activation is a resource-intensive response.

Herbivore-induced plant vaccination. Part I. The orchestration of plant defenses in nature and their fitness consequences in the wild tobacco Nicotiana attenuata.

A plant's responses to attack from particular pathogens and herbivores may result in resistance to subsequent attack from the same species, but may also affect different species. Such a cross-resistance, called immunization or vaccination, can benefit the plant, if the fitness consequences of attack from the initial attacker are less than those from subsequent attackers. Here, we report an example of naturally occurring vaccination of the native tobacco plant, Nicotiana attenuata, against Manduca hornworms by prior attack from the mirid bug, Tupiocoris notatus (Dicyphus minimus), which results from the elicitation of two categories of induced plant responses. First, attack from both herbivore species causes the plants in nature to release predator-attracting volatile organic compounds (VOCs), and the attracted generalist predator, Geocoris pallens, preferentially attacks the less mobile hornworm larvae. Second, attack from both mirids and hornworms increases the accumulation of secondary metabolites and proteinase inhibitors (PIs) in the leaf tissue, which is correlated with the slow growth of Manduca larvae. Mirid damage does not significantly reduce the fitness of the plant in nature, whereas attack from the hornworm reduces lifetime seed production. Consequently, plants that are attacked by mirids realize a significant fitness advantage in environments with both herbivores. The combination of growth-slowing direct defenses and predator-attracting indirect defenses results in greater hornworm mortality on mirid-attacked plants and provides the mechanism of the vaccination phenomenon.

Herbivore-induced plant vaccination. Part II. Array-studies reveal the transience of herbivore-specific transcriptional imprints and a distinct imprint from stress combinations.

Summary Microarray technology has given plant biologists the ability to simultaneously monitor changes in the expression of hundreds of genes, and yet, to date, this technology has not been applied to ecological phenomena. In native tobacco (Nicotiana attenuata), prior attack of sap-feeding mirids (Tupiocoris notatus) results in vaccination of the plant against subsequent attacks by chewing hornworms (Manduca sexta). This vaccination is mediated by a combination of direct and indirect defenses and tolerance responses, which act in concert with the attack preferences of a generalist predator. Here, we use microarrays enriched in herbivore-elicited genes with a principal components analysis (PCA) to characterize transcriptional 'imprints' of single, sequential, or simultaneous attacks by these two main herbivores of N. attenuata. The PCA identified distinctly different imprints left by individual attack from the two species after 24 h, but not after 5 days. Moreover, imprints of sequential or simultaneous attacks differed significantly from those of single attack, suggesting the existence of a distinct gene expression program responsive to the combination of biological stressors. A dissection of the transcriptional imprints revealed responses in direct and indirect defense genes that were well correlated with observed increases in defense metabolites. Attack from both herbivores elicits a switch from growth- to defense-related transcriptional processes, and herbivore-specific changes occur largely in primary metabolism and signaling cascades. PCA of these polygenic transcriptional imprints characterizes the ephemeral changes in the transcriptome that occur during the maturation of ecologically relevant phenotypic responses.