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1.  Lipoxygenase-mediated modification of insect elicitors 
Plant Signaling & Behavior  2010;5(12):1674-1676.
Plants can distinguish mechanical damage from larval folivory through the recognition of specific constituents of larval oral secretions (OS) which are deposited on the surface of leaf wounds during feeding. Fatty acid-amino acid conjugates (FACs) are major constituents of the OS of Lepidopteran larvae and they are strong elicitors of herbivore-induced defense responses in several plant species, including the wild tobacco Nicotiana attenuata. When OS from Manduca sexta larvae is deposited on N. attenuata wounded leaves, the major FAC N-linolenoyl-glutamic acid (18:3-Glu) is modified within seconds by a heat labile process. Some of the major modified forms are oxygenated products derived from 13-lipoxygenase activity and one of these derivatives, 13-oxo-13:2-Glu, is an active elicitor of enhanced JA biosynthesis and differential monoterpene emission in N. attenuata leaves.
doi:10.4161/psb.5.12.14036
PMCID: PMC3115133  PMID: 21150262
lipoxygenase; plant-insect interactions; fatty acid-amino acid conjugates; FAC; fatty acid-amides; insect elicitor; jasmonic acid; volatiles; herbivore-associated-elicitors; HAEs
2.  SuperSAGE analysis of the Nicotiana attenuata transcriptome after fatty acid-amino acid elicitation (FAC): identification of early mediators of insect responses 
BMC Plant Biology  2010;10:66.
Background
Plants trigger and tailor defense responses after perception of the oral secretions (OS) of attacking specialist lepidopteran larvae. Fatty acid-amino acid conjugates (FACs) in the OS of the Manduca sexta larvae are necessary and sufficient to elicit the herbivory-specific responses in Nicotiana attenuata, an annual wild tobacco species. How FACs are perceived and activate signal transduction mechanisms is unknown.
Results
We used SuperSAGE combined with 454 sequencing to quantify the early transcriptional changes elicited by the FAC N-linolenoyl-glutamic acid (18:3-Glu) and virus induced gene silencing (VIGS) to examine the function of candidate genes in the M. sexta-N. attenuata interaction. The analysis targeted mRNAs encoding regulatory components: rare transcripts with very rapid FAC-elicited kinetics (increases within 60 and declines within 120 min). From 12,744 unique Tag sequences identified (UniTags), 430 and 117 were significantly up- and down-regulated ≥ 2.5-fold, respectively, after 18:3-Glu elicitation compared to wounding. Based on gene ontology classification, more than 25% of the annotated UniTags corresponded to putative regulatory components, including 30 transcriptional regulators and 22 protein kinases. Quantitative PCR analysis was used to analyze the FAC-dependent regulation of a subset of 27 of these UniTags and for most of them a rapid and transient induction was confirmed. Six FAC-regulated genes were functionally characterized by VIGS and two, a putative lipid phosphate phosphatase (LPP) and a protein of unknown function, were identified as important mediators of the M. sexta-N. attenuata interaction.
Conclusions
The analysis of the early changes in the transcriptome of N. attenuata after FAC elicitation using SuperSAGE/454 has identified regulatory genes involved in insect-specific mediated responses in plants. Moreover, it has provided a foundation for the identification of additional novel regulators associated with this process.
doi:10.1186/1471-2229-10-66
PMCID: PMC3095340  PMID: 20398280
3.  New insights into the early biochemical activation of jasmonic acid biosynthesis in leaves 
Plant Signaling & Behavior  2010;5(3):287-289.
In plants, herbivore attack elicits the rapid accumulation of jasmonic acid (JA) which results from the activation of constitutively expressed biosynthetic enzymes. The molecular mechanisms controlling the activation of JA biosynthesis remain largely unknown however new research has elucidated some of the early regulatory components involved in this process. Nicotiana attenuata plants, a wild tobacco species, responds to fatty acid amino acid conjuguates (FAC) elicitors in the oral secretion of its natural herbivore, Manduca sexta, by triggering specific defense and tolerance responses against it; all of the defense responses known to date require the amplification of the wound-induced JA increase. We recently demonstrated that this FAC-elicited JA burst requires an increased flux of free linolenic acid (18:3) likely originating from the activation of a plastidial glycerolipase (GLA1) which is activated by an abundant FAC found in insect oral secretions, N-linolenoyl-glutamate (18:3-Glu). The lack of accumulation of free 18:3 after elicitation suggests a tight physical association between GLA1 and LOX3 in N. attenuata leaves. In addition, the salicylate-induced protein kinase (SIPK) and the nonexpressor of PR-1 (NPR1) participate in this activation mechanism that controls the supply of 18:3. In contrast, the wound-induced protein kinase (WIPK) does not but instead regulates the conversion of 13(S)-hydroperoxy-18:3 into 12-oxo-phytodienoic acid (OPDA). These results open new perspectives on the complex network of signals and regulatory components inducing the JA biosynthetic pathway.
PMCID: PMC2881280  PMID: 20037473
jasmonic acid; lipase; lipoxygenase; wounding; plant-insect interactions; FAC
4.  Root growth of Nicotiana attenuata is decreased immediately after simulated leaf herbivore attack 
Plant Signaling & Behavior  2008;3(4):236-237.
Image-based non-destructive methods were used to quantify root growth reactions happening within hours following simulated leaf herbivore attack.1 The induction of wound reactions in leaves of seedlings of Nicotiana attenuata led to transiently decreased root growth rates: Upon application of the oral secretions and regurgitants of the specialist herbivore Manduca sexta, a transient decrease in root growth was observed that was more pronounced than if a mere mechanical wounding was imposed. Root growth reduction was more severe than leaf growth reduction and the timing of the transient growth reduction coincided with endogenous bursts of jasmonate (JA) and ethylene emissions reported in literature. The reaction of root growth was superimposed by a strong diel variation of root growth, which was caused by the fluctuating temperature to which the plants were exposed. Apart from the observed root growth reaction, other defense-related traits such as increased nicotine concentration, trichome length and density were activated within 72 h after wounding. Further experiments indicated that the response was elicited by fatty acid-amino acid conjugates that are contained in the oral secretions and that JA signalling is crucial for root-shoot communication here.
PMCID: PMC2634187  PMID: 19704639
image analysis; plant-insect interactions; Manduca sexta
5.  Challenges in the Investigation of the Metabolic Changes in Nicotiana Attenuata during Insect Herbivory Using an Improved HPLC-TOF-MS Method 
RP-67
This study investigates the metabolic changes in leaf extracts of Nicotiana attenuata after simulated insect herbivory by mechanically wounding and applying oral secretions of Manduca sexta (W+OS) using HPLC-ESI-TOF-MS. Responses of wild-type (WT) plants are compared with genetically modified plants in which the hydroxyproline-rich glycopeptide systemin precursor (ppHypSys) is either over-expressed (OVsys) or down-regulated (IRsys). The challenges inherent to this type of study and strategies for quality control are discussed. Treated and untreated leaves (control samples) from WT, IRsys, and OVsys plants were harvested 1, 14, 86, and 120 h after treatment. The flash-frozen plant tissue was extracted with a MeOH/acetic acid buffer 40:60 (v/v). Individual extracts from five biological replicates per time-point, treatment and genotype were analyzed by HPLC-MS with a 45min binary acetonitrile-water gradient using an ESI-Qq-TOF for MS and MS/MS information for structure elucidation. Statistical analysis was performed using the Bruker ProfileAnalysis as well as XCMS and R software. Preliminary results obtained from a first data set of the same sample origin were used to design the experiment presented here. The number of biological replicates was increased from three to five to access the biological variation. The chromatographic separation was optimized in order to minimize suppression effects due to coelutions. A pooled sample generated from all individual samples was spiked with internal standards and served as quality control sample to monitor instrumental variation. The samples were analyzed in two different laboratories independently. The application of accurate and high resolution MS/MS data to support structure elucidation will be demonstrated.
PMCID: PMC2918224
6.  BAK1 regulates the accumulation of jasmonic acid and the levels of trypsin proteinase inhibitors in Nicotiana attenuata's responses to herbivory 
Journal of Experimental Botany  2010;62(2):641-652.
BAK1 is a co-receptor of brassinosteroid (BR) receptor BRI1, and plays a well-characterized role in BR signalling. BAK1 also physically interacts with the flagellin receptor FLS2 and regulates pathogen resistance. The role of BAK1 in mediating Nicotiana attenuata's resistance responses to its specialist herbivore, Manduca sexta, was examined here. A virus-induced gene-silencing system was used to generate empty vector (EV) and NaBAK1-silenced plants. The wounding- and herbivory-induced responses were examined on EV and NaBAK1-silenced plants by wounding plants or simulating herbivory by treating wounds with larval oral secretions (OS). After wounding or OS elicitation, NaBAK1-silenced plants showed attenuated jasmonic acid (JA) and JA-isoleucine bursts, phytohormone responses important in mediating plant defences against herbivores. However, these decreased JA and JA-Ile levels did not result from compromised MAPK activity or elevated SA levels. After simulated herbivory, NaBAK1-silenced plants had EV levels of defensive secondary metabolites, namely, trypsin proteinase inhibitors (TPIs), and similar levels of resistance to Manduca sexta larvae. Additional experiments demonstrated that decreased JA levels in NaBAK1-VIGS plants, rather than the enzymatic activity of JAR proteins or Ile levels, were responsible for the reduced JA-Ile levels observed in these plants. Methyl jasmonate application elicited higher levels of TPI activity in NaBAK1-silenced plants than in EV plants, suggesting that silencing NaBAK1 enhances the accumulation of TPIs induced by a given level of JA. Thus NaBAK1 is involved in modulating herbivory-induced JA accumulation and how JA levels are transduced into TPI levels in N. attenuata.
doi:10.1093/jxb/erq298
PMCID: PMC3003809  PMID: 20937731
BAK1; defence; herbivory; jasmonic acid (JA); jasmonic acid-isoleucine (JA-Ile); Nicotiana attenuata; SERK; trypsin proteinase inhibitor
7.  Diverting the Flux of the JA Pathway in Nicotiana attenuata Compromises the Plant's Defense Metabolism and Fitness in Nature and Glasshouse 
PLoS ONE  2011;6(10):e25925.
A plant's inducible defenses against herbivores as well as certain developmental processes are known to be controlled by the jasmonic acid (JA) pathway. We have previously shown that ectopically expressing Arabidopsis thaliana JA O-methyltransferase in Nicotiana attenuata (35S-jmt) strongly reduces the herbivory-elicited jasmonate bursts by acting as metabolic sink that redirects free JA towards methylation; here we examine the consequences of this metabolic sink on N. attenuata's secondary metabolism and performance in nature. In the glasshouse, 35S-jmt plants produced fewer seed capsules due to shorter floral styles, which could be restored to wild type (WT) levels after hand-pollination, and were more susceptible to Manduca sexta larvae attack. When transplanted into the Great Basin Desert in Utah, 35S-jmt plants grew as well as WT empty vector, but were highly attacked by native herbivores of different feeding guilds: leaf chewers, miners, and single cell feeders. This greater susceptibility was strongly associated with reduced emissions of volatile organic compounds (hexenylesters, monoterpenes and sesquiterpenes) and profound alterations in the production of direct defenses (trypsin proteinase inhibitors [TPI], nicotine, diterpene glycosides [DTGs] and phenylpropanoid-polyamine conjugates) as revealed by a combination of targeted and metabolomics analyses of field collected samples. Complementation experiments with JA-Ile, whose formation is outcompeted in 35S-jmt plants by the methylation reaction, restored the local TPI activation to WT levels and partially complemented nicotine and DTG levels in elicited but not systemic leaves. These findings demonstrate that MeJA, the major JA metabolite in 35S-jmt plants, is not an active signal in defense activation and highlights the value of creating JA sinks to disrupt JA signaling, without interrupting the complete octadecanoid pathway, in order to investigate the regulation of plants' defense metabolism in nature.
doi:10.1371/journal.pone.0025925
PMCID: PMC3189938  PMID: 22022469
8.  S-Nitrosoglutathione reductase (GSNOR) mediates the biosynthesis of jasmonic acid and ethylene induced by feeding of the insect herbivore Manduca sexta and is important for jasmonate-elicited responses in Nicotiana attenuata 
Journal of Experimental Botany  2011;62(13):4605-4616.
S-nitrosoglutathione reductase (GSNOR) reduces the nitric oxide (NO) adduct S-nitrosoglutathione (GSNO), an essential reservoir for NO bioactivity. In plants, GSNOR has been found to be important in resistance to bacterial and fungal pathogens, but whether it is also involved in plant–herbivore interactions was not known. Using a virus-induced gene silencing (VIGS) system, the activity of GSNOR in a wild tobacco species, Nicotiana attenuata, was knocked down and the function of GSNOR in defence against the insect herbivore Manduca sexta was examined. Silencing GSNOR decreased the herbivory-induced accumulation of jasmonic acid (JA) and ethylene, two important phytohormones regulating plant defence levels, without compromising the activity of two mitogen-activated protein kinases (MAPKs), salicylic acid-induced protein kinase (SIPK) and wound-induced protein kinase (WIPK). Decreased activity of trypsin proteinase inhibitors (TPIs) were detected in GSNOR-silenced plants after simulated M. sexta feeding and bioassays indicated that GSNOR-silenced plants have elevated susceptibility to M. sexta attack. Furthermore, GSNOR is required for methyl jasmonate (MeJA)-induced accumulation of defence-related secondary metabolites (TPI, caffeoylputrescine, and diterpene glycosides) but is not needed for the transcriptional regulation of JAZ3 (jasmonate ZIM-domain 3) and TD (threonine deaminase), indicating that GSNOR mediates certain but not all jasmonate-inducible responses. This work highlights the important role of GSNOR in plant resistance to herbivory and jasmonate signalling and suggests the potential involvement of NO in plant–herbivore interactions. Our data also suggest that GSNOR could be a target of genetic modification for improving crop resistance to herbivores.
doi:10.1093/jxb/err171
PMCID: PMC3170554  PMID: 21622839
Defence; ethylene; insect herbivore; jasmonic acid; jasmonate signalling; Manduca sexta; Nicotiana attenuata; S-nitrosoglutathione reductase (GSNOR); secondary metabolites; trypsin proteinase inhibitor
9.  Calcium-dependent protein kinases, CDPK4 and CDPK5, affect early steps of jasmonic acid biosynthesis in Nicotiana attenuata 
Plant Signaling & Behavior  2012;8(1):e22784.
Calcium-dependent protein kinases (CDPKs) modulate plant development and growth and are important regulators of biotic and abiotic stress responses. Recently it was found that simultaneously silencing Nicotiana attenuata NaCDPK4 and NaCDPK5 (IRcdpk4/5 plants) results in accumulation of exceptionally high JA levels after wounding or simulated herbivory treatments, which in turn induced high levels of defense metabolites that slowed the growth of Manduca sexta, a specialist insect herbivore. To investigate the mechanism by which NaCDPK4 and NaCDPK5 regulate JA accumulation, we analyzed the transcript levels of all important enzymes involved in JA biosynthesis, but these genes showed no differences between wild-type and IRcdpk4/5 plants. Moreover, the dynamics of JA were similar between these plants, excluding the possibility of decreased degradation rates in IRcdpk4/5 plants. To gain insight into the mechanism by which NaCDPK4 and NaCDPK5 regulate JA biosynthesis, free fatty acids, including C18:3, and (9S,13S)-12-oxo-phytodienoic acid (OPDA), two important precursors of JA were quantified at different times before and after wounding and simulated herbivore feeding treatments. We show that after these treatments, IRcdpk4/5 plants have decreased levels of C18:3, but have enhanced OPDA and JA levels, suggesting that NaCDPK4 and NaCDPK5 have a role in the early steps of JA biosynthesis. The possible role of NaCDPK4 and NaCDPK5 regulating AOS and AOC enzymatic activity is discussed.
doi:10.4161/psb.22784
PMCID: PMC3745584  PMID: 23221744
CDPK; jasmonic acid; defense; herbivore; insect; wounding
10.  An Ecological Analysis of the Herbivory-Elicited JA Burst and Its Metabolism: Plant Memory Processes and Predictions of the Moving Target Model 
PLoS ONE  2009;4(3):e4697.
Background
Rapid herbivore-induced jasmonic acid (JA) accumulation is known to mediate many induced defense responses in vascular plants, but little is known about how JA bursts are metabolized and modified in response to repeated elicitations, are propagated throughout elicited leaves, or how they directly influence herbivores.
Methodology/Principal Findings
We found the JA burst in a native population of Nicotiana attenuata to be highly robust despite environmental variation and we examined the JA bursts produced by repeated elicitations with Manduca sexta oral secretions (OS) at whole- and within-leaf spatial scales. Surprisingly, a 2nd OS-elicitation suppressed an expected JA burst at both spatial scales, but subsequent elicitations caused more rapid JA accumulation in elicited tissue. The baseline of induced JA/JA-Ile increased with number of elicitations in discrete intervals. Large veins constrained the spatial spread of JA bursts, leading to heterogeneity within elicited leaves. 1st-instar M. sexta larvae were repelled by elicitations and changed feeding sites. JA conjugated with isoleucine (JA-Ile) translates elicitations into defense production (e.g., TPIs), but conjugation efficiency varied among sectors and depended on NaWRKY3/6 transcription factors. Elicited TPI activity correlated strongly with the heterogeneity of JA/JA-Ile accumulations after a single elicitation, but not repeated elicitations.
Conclusions/Significance
Ecologically informed scaling of leaf elicitation reveals the contribution of repeated herbivory events to the formation of plant memory of herbivory and the causes and importance of heterogeneity in induced defense responses. Leaf vasculature, in addition to transmitting long-distance damage cues, creates heterogeneity in JA bursts within attacked leaves that may be difficult for an attacking herbivore to predict. Such unpredictability is a central tenet of the Moving Target Model of defense, which posits that variability in itself is defensive.
doi:10.1371/journal.pone.0004697
PMCID: PMC2650097  PMID: 19277115
11.  Host-plant-mediated effects of Nadefensin on herbivore and pathogen resistance in Nicotiana attenuata 
BMC Plant Biology  2008;8:109.
Background
The adage from Shakespeare, "troubles, not as single spies, but in battalions come," holds true for Nicotiana attenuata, which is commonly attacked by both pathogens (Pseudomonas spp.) and herbivores (Manduca sexta) in its native habitats. Defense responses targeted against the pathogens can directly or indirectly influence the responses against the herbivores. Nadefensin is an effective induced defense gene against the bacterial pathogen Pseudomonas syringae pv tomato (PST DC3000), which is also elicited by attack from M. sexta larvae, but whether this defense protein influences M. sexta's growth and whether M. sexta-induced Nadefensin directly or indirectly influences PST DC3000 resistance are unknown.
Results
M. sexta larvae consumed less on WT and on Nadefensin-silenced N. attenuata plants that had previously been infected with PST DC3000 than on uninfected plants. WT plants infected with PST DC3000 showed enhanced resistance to PST DC3000 and decreased leaf consumption by M. sexta larvae, but larval mass gain was unaffected. PST DC3000-infected Nadefensin-silenced plants were less resistant to subsequent PST DC3000 challenge, and on these plants, M. sexta larvae consumed less and gained less mass. WT and Nadefensin-silenced plants previously damaged by M. sexta larvae were better able to resist subsequent PST DC3000 challenges than were undamaged plants.
Conclusion
These results demonstrate that Na-defensin directly mediates defense against PST DC3000 and indirectly against M. sexta in N. attenuata. In plants that were previously infected with PST DC3000, the altered leaf chemistry in PST DC3000-resistant WT plants and PST DC3000-susceptible Nadefensin-silenced plants differentially reduced M. sexta's leaf consumption and mass gain. In plants that were previously damaged by M. sexta, the combined effect of the altered host plant chemistry and a broad spectrum of anti-herbivore induced metabolomic responses was more effective than Nadefensin alone in resisting PST DC3000.
doi:10.1186/1471-2229-8-109
PMCID: PMC2613890  PMID: 18950524
12.  Trichomes as dangerous lollipops 
Plant Signaling & Behavior  2011;6(12):1893-1896.
When attacked by herbivores, plants produce toxic secondary metabolites that function as direct defenses, as well as indirect defenses that attract and reward predators of the offending herbivores. These indirect defenses include both nutritive rewards such as extra floral nectar, as well as informational rewards, such as the production and release of volatile compounds that betray the location of feeding herbivores to predators. Herbivory of Nicotiana attenuata by the tobacco hornworm (Manduca larvae) alters the volatile profiles of both the plant and larval headspace. Herbivory-elicited specific changes in the volatile profiles are detected by arthropod predators of Manduca larvae. The known predators that perceive volatile cues induced by Manduca herbivory of N. attenuata are insects that target Manduca at early developmental stages, when the larvae are still small; large, late-instar larvae may have outgrown these predation risks. However, here we offer evidence that branched chain aliphatic acids derived from the digestion of plant O-acyl sugars from trichomes may betray Manduca larvae to lizard predators during late developmental stages as well.
doi:10.4161/psb.6.12.18028
PMCID: PMC3337173  PMID: 22095147
O-acyl sugars; body odor; frass odor; lizards; Manduca quinquemaculata; Manduca sexta; Nicotiana attenuata
13.  Tobacco Rattle Virus Vector: A Rapid and Transient Means of Silencing Manduca sexta Genes by Plant Mediated RNA Interference 
PLoS ONE  2012;7(2):e31347.
Background
RNAi can be achieved in insect herbivores by feeding them host plants stably transformed to express double stranded RNA (dsRNA) of selected midgut-expressed genes. However, the development of stably transformed plants is a slow and laborious process and here we developed a rapid, reliable and transient method. We used viral vectors to produce dsRNA in the host plant Nicotiana attenuata to transiently silence midgut genes of the plant's lepidopteran specialist herbivore, Manduca sexta. To compare the efficacy of longer, undiced dsRNA for insect gene silencing, we silenced N. attenuata's dicer genes (NaDCL1- 4) in all combinations in a plant stably transformed to express dsRNA targeting an insect gene.
Methodology/Principal Findings
Stable transgenic N. attenuata plants harboring a 312 bp fragment of MsCYP6B46 in an inverted repeat orientation (ir-CYP6B46) were generated to produce CYP6B46 dsRNA. After consuming these plants, transcripts of CYP6B46 were significantly reduced in M. sexta larval midguts. The same 312 bp cDNA was cloned in an antisense orientation into a TRV vector and Agro-infiltrated into N. attenuata plants. When larvae ingested these plants, similar reductions in CYP6B46 transcripts were observed without reducing transcripts of the most closely related MsCYP6B45. We used this transient method to rapidly silence the expression of two additional midgut-expressed MsCYPs. CYP6B46 transcripts were further reduced in midguts, when the larvae fed on ir-CYP6B46 plants transiently silenced for two combinations of NaDCLs (DCL1/3/4 and DCL2/3/4) and contained higher concentrations of longer, undiced CYP6B46 dsRNA.
Conclusions
Both stable and transient expression of CYP6B46 dsRNA in host plants provides a specific and robust means of silencing this gene in M. sexta larvae, but the transient system is better suited for high throughput analyses. Transiently silencing NaDCLs in ir-CYP6B46 plants increased the silencing of MsCYP6B46, suggested that insect's RNAi machinery is more efficient with longer lengths of ingested dsRNA.
doi:10.1371/journal.pone.0031347
PMCID: PMC3270032  PMID: 22312445
14.  Dietary plant phenolic improves survival of bacterial infection in Manduca sexta caterpillars 
Plant phenolics are generally thought to play significant roles in plant defense against herbivores and pathogens. Many plant taxa, including Solanaceae, are rich in phenolic compounds and some insect herbivores have been shown to acquire phenolics from their hosts to use them as protection against their natural enemies. Here we demonstrate that larvae of an insect specialist on Solanaceae, the tobacco hornworm, Manduca sexta L. (Lepidoptera: Sphingidae), acquire the plant phenolic chlorogenic acid (CA), and other caffeic acid derivatives as they feed on one of their hosts, Nicotiana attenuata L. (Solanaceae), and on artificial diet supplemented with CA. We test the hypothesis that larvae fed on CA-supplemented diet would have better resistance against bacterial infection than larvae fed on a standard CA-free diet by injecting bacteria into the hemocoel of fourth instars. Larvae fed CA-supplemented diet show significantly higher survival of infection with Enterococcus faecalis (Andrewes & Horder) Schleifer & Kilpper-Bälz, but not of infection with the more virulent Pseudomonas aeruginosa (Schroeter) Migula. Larvae fed on CA-supplemented diet possess a constitutively higher number of circulating hemocytes than larvae fed on the standard diet, but we found no other evidence of increased immune system activity, nor were larvae fed on CA-supplemented diet better able to suppress bacterial proliferation early in the infection. Thus, our data suggest an additional defensive function of CA to the direct toxic inhibition of pathogen proliferation in the gut.
doi:10.1111/eea.12032
PMCID: PMC3570171  PMID: 23420018
chemical defense; acquired plant metabolite; immune defense; Lepidoptera; Solanaceae; Sphingidae; Nicotiana attenuata; tobacco hornworm; Enterococcus faecalis; Pseudomonas aeruginosa; chlorogenic acid
15.  Silencing an N-Acyltransferase-Like Involved in Lignin Biosynthesis in Nicotiana attenuata Dramatically Alters Herbivory-Induced Phenolamide Metabolism 
PLoS ONE  2013;8(5):e62336.
In a transcriptomic screen of Manduca sexta-induced N-acyltransferases in leaves of Nicotiana attenuata, we identified an N-acyltransferase gene sharing a high similarity with the tobacco lignin-biosynthetic hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyl transferase (HCT) gene whose expression is controlled by MYB8, a transcription factor that regulates the production of phenylpropanoid polyamine conjugates (phenolamides, PAs). To evaluate the involvement of this HCT-like gene in lignin production as well as the resulting crosstalk with PA metabolism during insect herbivory, we transiently silenced (by VIGs) the expression of this gene and performed non-targeted (UHPLC-ESI/TOF-MS) metabolomics analyses. In agreement with a conserved function of N. attenuata HCT-like in lignin biogenesis, HCT-silenced plants developed weak, soft stems with greatly reduced lignin contents. Metabolic profiling demonstrated large shifts (up to 12% deregulation in total extracted ions in insect-attacked leaves) due to a large diversion of activated coumaric acid units into the production of developmentally and herbivory-induced coumaroyl-containing PAs (N′,N′′-dicoumaroylspermidine, N′,N′′-coumaroylputrescine, etc) and to minor increases in the most abundant free phenolics (chlorogenic and cryptochlorogenic acids), all without altering the production of well characterized herbivory-responsive caffeoyl- and feruloyl-based putrescine and spermidine PAs. These data are consistent with a strong metabolic tension, exacerbated during herbivory, over the allocation of coumaroyl-CoA units among lignin and unusual coumaroyl-containing PAs, and rule out a role for HCT-LIKE in tuning the herbivory-induced accumulation of other PAs. Additionally, these results are consistent with a role for lignification as an induced anti-herbivore defense.
doi:10.1371/journal.pone.0062336
PMCID: PMC3660383  PMID: 23704878
16.  Identification and profiling of miRNAs during herbivory reveals jasmonate-dependent and -independent patterns of accumulation in Nicotiana attenuata 
BMC Plant Biology  2012;12:209.
Background
Plant microRNAs (miRNAs) play key roles in the transcriptional responses to environmental stresses. However, the role of miRNAs in responses to insect herbivory has not been thoroughly explored. To identify herbivory-responsive miRNAs, we identified conserved miRNAs in the ecological model plant Nicotiana attenuata whose interactions with herbivores have been well-characterized in both laboratory and field studies.
Results
We identified 59 miRNAs from 36 families, and two endogenous trans-acting small interfering RNAs (tasiRNA) targeted by miRNAs. We characterized the response of the precursor and mature miRNAs to simulated attack from the specialist herbivore Manduca sexta by quantitative PCR analysis and used ir-aoc RNAi transformants, deficient in jasmonate biosynthesis, to identify jasmonate-dependent and -independent miRNA regulation. Expression analysis revealed that groups of miRNAs and tasiRNAs were specifically regulated by either mechanical wounding or wounding plus oral secretions from M. sexta larvae, and these small RNAs were accumulated in jasmonate-dependent or -independent manners. Moreover, cDNA microarray analysis indicated that the expression patterns of the corresponding target genes were correlated with the accumulation of miRNAs and tasiRNAs.
Conclusions
We show that a group of miRNAs and tasiRNAs orchestrates the expression of target genes involved in N. attenuata’s responses to herbivore attack.
doi:10.1186/1471-2229-12-209
PMCID: PMC3502350  PMID: 23134682
Anti-herbivore defense; Jasmonate; Manduca sexta; miRNA; Nicotiana attenuata; tasiRNA
17.  Host Plant Odors Represent Immiscible Information Entities - Blend Composition and Concentration Matter in Hawkmoths 
PLoS ONE  2013;8(10):e77135.
Host plant choice is of vital importance for egg laying herbivorous insects that do not exhibit brood care. Several aspects, including palatability, nutritional quality and predation risk, have been found to modulate host preference. Olfactory cues are thought to enable host location. However, experimental data on odor features that allow choosing among alternative hosts while still in flight are not available. It has previously been shown that M. sexta females prefer Datura wrightii compared to Nicotiana attenuata. The bouquet of the latter is more intense and contains compounds typically emitted by plants after feeding-damage to attract the herbivore’s enemies. In this wind tunnel study, we offered female gravid hawkmoths (Manduca sexta) odors from these two ecologically relevant, attractive, non-flowering host species. M. sexta females preferred surrogate leaves scented with vegetative odors form both host species to unscented control leaves. Given a choice between species, females preferred the odor bouquet emitted by D. wrightii to that of N. attenuata. Harmonizing, i.e. adjusting, volatile intensity to similar levels did not abolish but significantly weakened this preference. Superimposing, i.e. mixing, the highly attractive headspaces of both species, however, abolished discrimination between scented and non-scented surrogate leaves. Beyond ascertaining the role of blend composition in host plant choice, our results raise the following hypotheses. (i) The odor of a host species is perceived as a discrete odor ‘Gestalt’, and its core properties are lost upon mixing two attractive scents (ii). Stimulus intensity is a secondary feature affecting olfactory-based host choice (iii). Constitutively smelling like a plant that is attracting herbivore enemies may be part of a plant’s strategy to avoid herbivory where alternative hosts are available to the herbivore.
doi:10.1371/journal.pone.0077135
PMCID: PMC3792911  PMID: 24116211
18.  Digestive Duet: Midgut Digestive Proteinases of Manduca sexta Ingesting Nicotiana attenuata with Manipulated Trypsin Proteinase Inhibitor Expression 
PLoS ONE  2008;3(4):e2008.
Background
The defensive effect of endogenous trypsin proteinase inhibitors (NaTPIs) on the herbivore Manduca sexta was demonstrated by genetically altering NaTPI production in M. sexta's host plant, Nicotiana attenuata. To understand how this defense works, we studied the effects of NaTPI on M. sexta gut proteinase activity levels in different larval instars of caterpillars feeding freely on untransformed and transformed plants.
Methodology/ Principal Findings
Second and third instars larvae that fed on NaTPI-producing (WT) genotypes were lighter and had less gut proteinase activity compared to those that fed on genotypes with either little or no NaTPI activity. Unexpectedly, NaTPI activity in vitro assays not only inhibited the trypsin sensitive fraction of gut proteinase activity but also halved the NaTPI-insensitive fraction in third-instar larvae. Unable to degrade NaTPI, larvae apparently lacked the means to adapt to NaTPI in their diet. However, caterpillars recovered at least part of their gut proteinase activity when they were transferred from NaTPI-producing host plants to NaTPI-free host plants. In addition extracts of basal leaves inhibited more gut proteinase activity than did extracts of middle stem leaves with the same protein content.
Conclusions/ Significance
Although larvae can minimize the effects of high NaTPI levels by feeding on leaves with high protein and low NaTPI activity, the host plant's endogenous NaTPIs remain an effective defense against M. sexta, inhibiting gut proteinase and affecting larval performance.
doi:10.1371/journal.pone.0002008
PMCID: PMC2292244  PMID: 18431489
19.  Methyl jasmonate-elicited herbivore resistance: does MeJA function as a signal without being hydrolyzed to JA? 
Planta  2008;227(5):1161-1168.
Treatment with methyl jasmonate (MeJA) elicits herbivore resistance in many plant species and over-expression of JA carboxyl methyltransferase (JMT) constitutively increases JA-induced responses in Arabidopsis. When wild-type (WT) Nicotiana attenuata plants are treated with MeJA, a rapid transient endogenous JA burst is elicited, which in turn increases levels of nicotine and trypsin proteinase inhibitors (TPIs) and resistance to larvae of the specialist herbivore, Manduca sexta. All of these responses are impaired in plants silenced in lipoxygenase 3 expression (asLOX3) but are restored to WT levels by MeJA treatment. Whether these MeJA-induced responses are directly elicited by MeJA or by its cleavage product, JA, is unknown. Using virus-induced gene silencing (VIGS), we silenced MeJA-esterase (NaMJE) expression and found this gene responsible for most of the MeJA-cleaving activity in N. attenuata protein extracts. Silencing NaMJE in asLOX3, but not in WT plants, significantly reduced MeJA-induced nicotine levels and resistance to M. sexta, but not TPI levels. MeJA-induced transcript levels of threonine deaminase (NaTD) and phenylalanine ammonia lyase (NaPAL1) were also decreased in VIGS MJE (asLOX3) plants. Finally the performance of M. sexta larvae that fed on plants treated with JA or MeJA demonstrated that silencing NaMJE inhibited MeJA-induced but not JA-induced resistance in asLOX3 plants. From these results, we conclude that the resistance elicited by MeJA treatment is directly elicited not by MeJA but by its de-methylated product, JA.
Electronic supplementary material
The online version of this article (doi:10.1007/s00425-008-0690-8) contains supplementary material, which is available to authorized users.
doi:10.1007/s00425-008-0690-8
PMCID: PMC2756367  PMID: 18214527
MeJA esterase (NaMJE); Methyl jasmonate (MeJA); Jasmonate (JA); Nicotiana attenuata; Manduca sexta
20.  Pectin methylesterase NaPME1 contributes to the emission of methanol during insect herbivory and to the elicitation of defence responses in Nicotiana attenuata 
Journal of Experimental Botany  2009;60(9):2631-2640.
Pectin methylesterases (PMEs) catalyse the demethylation of pectin within plant cell walls, releasing methanol (MeOH) in the process. Thus far, PMEs have been found to be involved in diverse processes such as plant growth and development and defence responses against pathogens. Herbivore attack increases PME expression and activity and MeOH emissions in several plant species. To gain further insights into the role of PMEs in defence responses against herbivores, the expression of a Manduca sexta oral secretion (OS)-inducible PME in Nicotiana attenuata (NaPME1) was silenced by RNA interference (RNAi)-mediated gene silencing. Silenced lines (ir-pme) showed 50% reduced PME activity in leaves and 70% reduced MeOH emissions after OS elicitation compared with the wild type (WT), demonstrating that the herbivore-induced MeOH emissions originate from the demethylation of pectin by PME. In the initial phase of the OS-induced jasmonic acid (JA) burst (first 30 min), ir-pme lines produced WT levels of this hormone and of jasmonyl-isoleucine (JA-Ile); however, these levels were significantly reduced in the later phase (60–120 min) of the burst. Similarly, suppressed levels of the salicylic acid (SA) burst induced by OS elicitation were observed in ir-pme lines even though wounded ir-pme leaves contained slightly increased amounts of SA. This genotype also presented reduced levels of OS-induced trypsin proteinase inhibitor activity in leaves and consistently increased M. sexta larvae performance compared with WT plants. These latter responses could not be recovered by application of exogenous MeOH. Together, these results indicated that PME contributes, probably indirectly by affecting cell wall properties, to the induction of anti-herbivore responses.
doi:10.1093/jxb/erp106
PMCID: PMC2692009  PMID: 19380422
Defence; herbivory; jasmonic acid; Manduca; methanol; Nicotiana; pectin methylesterase; proteinase inhibitor
21.  Nicotine's Defensive Function in Nature 
PLoS Biology  2004;2(8):e217.
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.
Transgenic plants confirm the role that nicotine has in protecting tobacco plants from predators in nature and demonstrates the power of transgenic tools in studying ecological interactions in the field
doi:10.1371/journal.pbio.0020217
PMCID: PMC509292  PMID: 15314646
22.  Fitness benefits of trypsin proteinase inhibitor expression in Nicotiana attenuata are greater than their costs when plants are attacked. 
BMC Ecology  2004;4:11.
Background
The commonly invoked cost-benefit paradigm, central to most of functional biology, explains why one phenotype cannot be optimally fit in all environments; yet it is rarely tested. Trypsin proteinase inhibitors (TPIs) expression in Nicotiana attenuata is known to decrease plant fitness when plants compete with unattacked conspecifics that do not produce TPIs and also to decrease the performance of attacking herbivores.
Results
In order to determine whether the putative benefits of TPI production outweigh its cost, we transformed N. attenuata to silence endogenous TPI production or restore it in a natural mutant that was unable to produce TPIs. We compared the lifetime seed production of N. attenuata genotypes of the same genetic background with low or no TPI to that of genotypes with high TPI levels on which M. sexta larvae were allowed to feed freely. Unattacked low TPI-producing genotypes produced more seed capsules than did plants with high TPI levels. Caterpillar attack reduced seed capsule production in all genotypes and reversed the pattern of seed capsule production among genotypes. M. sexta larvae attacking genotypes with high TPI activity consumed more TPI, less protein, and move later to the young leaves. Larval masses were negatively correlated (R2 = 0.56) with seed capsule production per plant.
Conclusions
Our results demonstrate that the fitness benefits of TPI production outweigh their costs in greenhouse conditions, when plants are attacked and that despite the ongoing evolutionary interactions between plant and herbivore, TPI-mediated decreases in M. sexta performance translates into a fitness benefit for the plant.
doi:10.1186/1472-6785-4-11
PMCID: PMC514560  PMID: 15304198
23.  Herbivore induction of jasmonic acid and chemical defences reduce photosynthesis in Nicotiana attenuata  
Journal of Experimental Botany  2013;64(2):685-694.
Herbivory initiates a shift in plant metabolism from growth to defence that may reduce fitness in the absence of further herbivory. However, the defence-induced changes in carbon assimilation that precede this reallocation in resources remain largely undetermined. This study characterized the response of photosynthesis to herbivore induction of jasmonic acid (JA)-related defences in Nicotiana attenuata to increase understanding of these mechanisms. It was hypothesized that JA-induced defences would immediately reduce the component processes of photosynthesis upon attack and was predicted that wild-type plants would suffer greater reductions in photosynthesis than plants lacking JA-induced defences. Gas exchange, chlorophyll fluorescence, and thermal spatial patterns were measured together with the production of defence-related metabolites after attack and through recovery. Herbivore damage immediately reduced electron transport and gas exchange in wild-type plants, and gas exchange remained suppressed for several days after attack. The sustained reductions in gas exchange occurred concurrently with increased defence metabolites in wild-type plants, whereas plants lacking JA-induced defences suffered minimal suppression in photosynthesis and no increase in defence metabolite production. This suppression in photosynthesis occurred only after sustained defence signalling and defence chemical mobilization, whereas a short bout of feeding damage only transiently altered components of photosynthesis. It was identified that lipoxygenase signalling interacted with photosynthetic electron transport and that the resulting JA-related metabolites reduced photosynthesis. These data represent a metabolic cost to mounting a chemical defence against herbivory and link defence-signalling networks to the differential effects of herbivory on photosynthesis in remaining leaf tissues in a time-dependent manner.
doi:10.1093/jxb/ers364
PMCID: PMC3542056  PMID: 23264519
chlorophyll fluorescence; defence; lipoxygenase; nicotine; plant–insect interaction
24.  Intraspecific Combinations of Flower and Leaf Volatiles Act Together in Attracting Hawkmoth Pollinators 
PLoS ONE  2013;8(9):e72805.
Insects pinpoint mates, food and oviposition sites by olfactory cues. Recognizing and localizing a suitable target by olfaction is demanding. Odor sources emit characteristic blends of compounds that have to be identified against an environmentally derived olfactory background. This background, however, does not necessarily disturb the localization of a source. Rather, the contrary. Sex pheromones become more attractive to male moths when being presented against a relevant plant background. Here we asked whether such olfactory coaction also characterizes foraging cues. The tobacco hornworm Manduca sexta feeds on nectar from wild tobacco Nicotiana attenuata and sacred datura Datura wrightii flowers. We tested how leaf-derived volatile blends as a background affect the moths' approach to flower blends. We found coaction when a flower blend was presented against a conspecific leaf volatile background but not when the blend was presented against volatiles emitted by the other host plant or by a non-host plant. Hence, our results reveal a species-specific coaction between flower blend and leaf volatile background. The ability to integrate information from different odor sources on one plant might provide the moth with a fine-grained analysis of food site quality.
doi:10.1371/journal.pone.0072805
PMCID: PMC3771915  PMID: 24069159
25.  The Nicotiana attenuata LECTIN RECEPTOR KINASE 1 is involved in the perception of insect feeding 
Plant Signaling & Behavior  2011;6(12):2060-2063.
The Nicotiana attenuata LECTIN RECEPTOR KINASE 1 (LecRK1) has been recently identified as a component of the mechanism used by plants to suppress the Manduca sexta-triggered accumulation of salicylic acid (SA). The suppression of the SA burst by LecRK1 allows for the unfettered induction of jasmonic acid (JA)-mediated defense responses against M. sexta herbivory. LecRK1 contains a multi-domain extracellular region composed of a G-type Lectin domain and a PAN-AP domain separated by a variable sequence with low similarity to an EGF domain. The LecRK1 intracellular region is composed of a single domain structure with predicted Ser/Thr protein kinase activity. The multi-domain structure of the extracellular region of LecRK1 adds a level of complexity in terms of the potential ligands that this receptor protein could recognize.
doi:10.4161/psb.6.12.18324
PMCID: PMC3356243  PMID: 22105024
defense responses; insect elicitor; jasmonic acid; lectin receptor kinase; plant-insect interactions; salicylic acid

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