BACKGROUND
nicotiana attenuata is an annual native to southwestern usa that germinates from seed banks in response to factors in wood smoke after fires  <cit> .
because of this germination behavior and a strong intra-specific competition, n. attenuata allocates resources primarily to sustain rapid growth and seed setting and as a consequence, it has developed a large number of induced defense responses to ward off the unpredictable attacks from herbivores  <cit> .
hence, when n. attenuata is attacked by insect folivores, an extensive reprogramming of its transcriptome, proteome and metabolome takes place  <cit> .
previous studies estimated that more than  <dig> n. attenuata genes respond to manduca sexta larval feeding  <cit>  and demonstrated that the plant readjusts its metabolism for de novo synthesis of direct and indirect defense responses and to induce tolerance mechanisms  <cit> .
activation of these defensive mechanisms requires energy and resources from primary metabolism and involves therefore a complex rearrangement of resource allocation in the plant, including altered photosynthesis and sink/source relations  <cit> .
how plants decode insect feeding and trigger defense and tolerance responses is starting to be understood.
for example, a snrk <dig> kinase complex has been found to regulate tolerance mechanisms associated to the leaf/root partition of photoassimilates  <cit>  and two mapks, wipk and sipk , were shown to be critical for the induction of direct defense responses in n. attenuata  <cit> .
herbivore attack induces in plants the coordinated activation of several signal cascades including those of jasmonic acid , salicylic acid , and ethylene   <cit> .
among them, ja plays a major and essential role in the induction of a large number of the plant's protective responses against insect herbivory and wounding  <cit> .
thus, having ja as a common signal, a large number of the plant responses to these two stimuli overlap, however, plants can differentiate between mechanical damage and insect herbivory to tailor their responses.
the perception of components in the oral secretions  of feeding larvae is one mechanism by which plants can decode insect feeding.
fatty acid-amino acid conjugates  are major components in the os of m. sexta larvae and they are necessary and sufficient to induce most of the defense responses triggered by feeding m. sexta caterpillar in n. attenuata  <cit> .
hence, previous studies suggest the existence of central herbivore-activated regulators in n. attenuata leaves, which, in turn, are regulated by minute amounts of facs in the insect's os.
disentangling the effect of mechanical tissue damage and fac elicitation will provide critical information on how plants control changes in its metabolism to more efficiently reduce the negative fitness consequences of herbivore attack.
one of the earliest known molecular events differentially induced by os and facs in tobacco is the activation of wipk and sipk.
activation of these protein kinases occur within the first minutes after wounding  <cit>  and the activation is enhanced several-fold by applying m. sexta os to wounds  <cit> .
importantly, not only their activities but also sipk and wipk transcript levels are rapidly  and transiently induced after elicitation  <cit> , indicating that these regulators are under positive feedback control at the transcriptional level.
one of the early targets of the fac signal transduction pathway in n. attenuata is the wrky <dig> gene.
its transcript levels are also rapidly and transiently induced after wounds have been supplemented with m. sexta os or synthetic facs but only marginally by mechanical damage alone  <cit> .
this rapid and transient kinetic of mrna accumulation is characteristic of regulatory components and differs from that showed by, for example, transcripts encoding for defense components  which is characterized by a slower and more persistent rate of mrna accumulation, reaching maximum levels after hours to days  <cit> .
however, not all regulatory components are under positive feedback control at the transcriptional level: the coronatine insensitive  <dig>  gene is an example in the ja transduction pathway  <cit> .
however, some of the recently identified jaz proteins that interact with coi <dig> and participate in ja-ile perception are rapidly and transiently induced at the mrna level by wounding in arabidopsis  <cit> .
the rapid advances in high throughput sequencing capacity in combination with new "open-architecture" techniques for quantification of gene expression has opened the possibility of performing genome-wide transcriptome studies in organisms from which massive nucleotide sequence information is not yet available.
serial analysis of gene expression  is a technique that allows for the absolute quantification of mrna abundance by quantifying the relative frequencies of individual short  transcripts signatures tags  <cit> .
further development of the technique allowed for the generation of  <dig> nt tags   <cit>  which substantially improved the annotation of tags when aligned to sequences in public nucleotide databases  <cit> .
with these techniques, the detection of transcripts is proportionally correlated to the scale of dna sequencing and their combination with next generation sequencing  allows for the detection and analysis of very low abundant transcripts  which have been estimated to account for more than 90% of mrnas in eukaryotic cells  <cit> .
here we used supersage in combination with ngs for the quantification of the early changes  occurring in the transcriptome of n. attenuata plants after a single event of 18:3-glu elicitation.
the major objective of the study was to identify genes encoding for potential regulatory components of the fac-mediated responses by looking for low abundant transcripts that were rapidly and transiently induced after 18:3-glu elicitation.
RESULTS
generation of supersage libraries from wounded and fac elicited n. attenuata leaves
two supersage libraries were generated from the second fully expanded leaf of n. attenuata plants either mechanically wounded or wounded and supplemented with 18:3-glu as a single fac elicitor.
leaf samples were harvested after  <dig> min of the treatments .
wounding is a prerequisite for fac-elicitation; hence, analysis of wounded leaves was used to differentiate between genes regulated by mechanical damage from those regulated more specifically or deferentially by facs.
additionally, elicitation by a single elicitor  and a single wound event were used to eliminate the effects of other os components and repeated wounding on gene expression.
the total number of supersage tags obtained after sequencing the libraries in a single  <dig> plate and eliminating i) incomplete reads, ii) twin-ditags, and iii) ditags without complete library-identification dna linkers was  <dig> ; comprising  <dig>  tags from the wounding  library and  <dig>  from the fac-elicited  library .
these tags represented  <dig>  unique sequences with  <dig>   detected only once  in the combined libraries, and  <dig>  detected at least twice in the combined libraries .
these latter tags are referred as unitags throughout the manuscript  <cit>  and will be considered for further analysis.
singletons represented thus ~60% of unique sequences, in agreement with previous studies  <cit> .
the complete supersage dataset is available in additional file  <dig> .
* values normalized to  <dig> million tags
** w: wounded; f: 18:3-glu elicited
abundance of unitags and annotation to public databases
the unitags were first classified in abundance groups according to their number of copies  <cit> .
unitags present at ≤  <dig>  >  <dig> - ≤  <dig> , >  <dig>  - ≤  <dig>  and > <dig>  copies per million  were considered as low-, mid-, high- and very high-abundant tags, respectively .
the frequency distribution of the  <dig>  unitags showed that the number of copies in low and mid abundance groups  represented > 98% of the unitags while high- and very high-abundant tags  represented only  <dig> % .
however, although the latter group represented only a small fraction of the  <dig>  unitags, together they accounted for ~47% of the total number of tag copies in both the w and f libraries .
these values were in agreement with previously reported data  <cit> .
annotation of the  <dig>  unitags using basic local alignments  gave  <dig>  tags  that matched with a maximum of  <dig> mismatches  to sequences deposited in genbank plant nucleotide databases .
<dig> % of these  <dig>  unitags matched perfectly  with sequences in the databases while  <dig> % did it with one mismatch ,  <dig> % with two mismatches  and  <dig> % with three mismatches .
moreover, 88% of the annotated unitags matched sequences corresponding to nicotiana spp, 5% to solanum spp and 8% to other plant species .
fac elicitation induces differential expression of  <dig> unitags
statistically significant changes in tag copy number between the f and w libraries were analyzed by calculating a probability -value according to  <cit>  .
although small changes in expression levels may have biological significance  <cit> , in this study we focused primarily on genes which showed strong changes in expression levels with arbitrary fold-change  values ≥  <dig>  or ≤  <dig>  .
based on the calculated -values and using a 95% confidence level,  <dig> unitags were identified as differentially expressed after fac elicitation .
among these unitags,  <dig> had fc ≥  <dig>  and  <dig> fc ≤  <dig>  .
most of the differentially expressed unitags presented fc values between  <dig>  and  <dig>  with  <dig> and  <dig> unitags presenting fc values ≥  <dig> and ≤  <dig> , respectively .
the majority  of the differentially up-regulated unitags and all of the down-regulated unitags corresponded to low- and mid-abundance groups , indicating that the strongest changes in expression levels occurred primarily in genes expressed at low to intermediate levels.
assignment of differentially expressed unitags to gene ontology : biological and functional categories
to obtain gene function categories of the differentially expressed unitags, gene ontology  annotation was performed by blastx  against the non-redundant genbank and uniprotkb/trembl protein databases .
for this analysis, we used unitags that showed a maximum of  <dig> mismatches  with entries in the genbank nucleotide database .
of the  <dig> differentially expressed unitags,  <dig> had an associated nucleotide sequence and  <dig> matched to an amino acid sequence entry  in the genbank and uniprotkb/trembl databases .
go annotations  could be assigned to  <dig> of these  <dig> unitags with the remaining entries corresponding to uncharacterized proteins .
among the most prevalent go biological processes, ~25% of the unitags classified into metabolism, ~12% into regulation of gene expression , ~10% into amino acid phosphorylation/dephosphorylation, ~8% into translation , ~8% into defense and stress responses, ~7% into transport, ~6% into protein degradation and folding and ~6% into signal transduction components .
the preponderance of changes in transcripts corresponding to metabolism, signaling, transcription, translation and transport associated processes after  <dig> min of 18:3-glu elicitation emphasized the fact that at this early time point a substantial reprogramming of the leaf metabolism is already in progress.
based on changes in metabolic genes, hallmarks of this reprogramming included an increased capacity for protein synthesis and the generation of c skeletons and reducing power .
these changes in the expression of metabolic genes are consistent with a substantial shift in primary metabolism to support secondary metabolism and tolerance mechanisms  <cit>  and are consistent with previous gene expression studies  <cit>  .
the identification of regulatory factors controlling these changes in metabolism and defense and tolerance processes against insects is one of the major challenges for the future and some potential candidates are described below.
changes in the expression of unitags/mrnas encoding for regulatory components
the most prevalent go molecular function with regulatory activity corresponded to transcriptional regulators and protein kinases, represented by  <dig> and  <dig> unitags, respectively.
the protein phosphatase category contained  <dig> unitags, the signal transduction category  <dig> unitags and the nucleic acid binding category  <dig> unitags .
thus, a total of  <dig> annotated unitags corresponded to factors with potential regulatory function.
among transcriptional regulators, unitags corresponding to wrky transcription factors  were the most predominant  and tag- <dig> was the most up-regulated  after 18:3-glu elicitation within this group.
other unitags for wrkys were up-regulated between  <dig> and  <dig>  fold .
within the wrky domain containing family, a wizz tf   <cit>  was up-regulated  <dig> fold.
other prevalent up-regulated tfs included ap2-like factors , rav factors , ethylene-responsive element binding proteins  and ccr4-not transcription complex proteins  .
single up-regulated unitags in this category corresponded to a bzip tf , his <dig> , s1fa , rna polymerase ii  and a sigma subunit for a plastidial rna polymerase .
among down-regulated transcriptional regulators were a gata- <dig> zinc finger protein and rna polymerase iii .
within the protein kinase and phosphatase classes, three unitags corresponded to mapk , three to cell-wall associated kinases , two to brassinosteroid insensitive 1-associated receptor kinase  <dig>  and three to protein phosphatase 2a  and c .
in addition, this category contained a chloroplast precursor for arabidopsis protein kinase  <dig>   <cit>  up-regulated ~ <dig> fold, a shaggy-like kinase , and a cytokinin-regulated kinase  <dig>  and a calmodulin protein kinase  <dig>  among others .
within the signal transduction class, the most predominant unitags corresponded to "avr9/cf- <dig> rapidly elicited proteins"  up-regulated between  <dig> and  <dig>  fold.
single up-regulated unitags corresponded to a hs1pro-1-like protein ;  <dig>  fold), sgt <dig> , a lipid phosphate phosphatase  and an extra-large g protein  were also contained in this category .
validation of the supersage data by qpcr
a subset of  <dig> differentially expressed unitags  was selected for further analysis based on the fulfillment of at least two of the following criteria: 1) strong and significant changes in their fc values ; 2) abundance of < <dig>  copies.million- <dig> ; 3) matched known regulatory components in the databases.
fc: fold change .
nm: no match in genbank
1unitags selected for vigs analysis are depicted in bold.
the selected unitags were first elongated by amplification of their corresponding cdnas and blasted against the genbank plant nucleotide databases to confirm their identities.
all of the elongated sequences  matched to the same entries as the original  <dig> bp tags .
secondly, the elongated sequences were used to design gene-specific primers to i) validate the supersage data and ii) to study the kinetic of mrna induction by real time quantitative pcr .
total rna was extracted from both wounded and 18:3-glu elicited leaves of wt plants after different times of the stimuli.
the accumulation of  <dig> mrnas corresponding to the selected unitags was consistent with a rapid increase  after fac elicitation and a rapid decrease  to basal or lower levels after the stimuli .
interestingly, several transcripts showed either no or minimal induction by wounding, representing therefore genes activated almost specifically by facs .
for some transcripts mechanical damage induced an increase in their corresponding mrna levels which was potentiated several fold by 18:3-glu elicitation .
for transcripts corresponding to four unitags , the differential regulation by 18:3-glu elicitation could not be confirmed  and they may represent false positives in the supersage analysis  <cit> .
finally, mrnas for three unitags  were differentially repressed by 18:3-glu elicitation .
functional characterization of candidate regulatory components of insect mediated responses by vigs
to validate the use of the supersage approach for the identification of candidate regulatory components of the interaction between n. attenuata and m. sexta larvae, six genes were selected for preliminary gene function characterization by virus-induced gene silencing .
the selection of these genes was based on: 1) their kinetic of mrna induction and 2) their fold-change compared to wounding .
some of the genes encoded for putative regulatory components and two presented no similarity to any other protein of known or predicted function .
the selected unitags corresponded to a hs1pro-1-like protein ; tag-6205), lipid phosphate phosphatase , nicotiana elicitor induced gene , cell wall-associated protein kinase , unka  and unkb   .
to evaluate whether these genes participate in fac- and insect defense-mediated responses, gene-specific silenced plants and plants transformed with the empty vector  were assessed for m. sexta larval performance and the accumulation of ja and ja-ile after 18:3-glu elicitation and wounding.
gene silencing efficiency in these plants was analyzed by qpcr in 18:3-glu-elicited leaves after  <dig> h of the treatment .
the morphological phenotype of the silenced-plants was indistinguishable from ev control plants .
<dig> the silencing efficiency is expressed as the reduction  of the mrna levels in the vigs-silenced plants relative to the levels of the corresponding mrna in ev  plants.
in all cases the reductions were statistically significant .
m. sexta larva feeding on plants silenced in the expression of lpp or unka showed significant increases in mass gained after  <dig> and/or  <dig> days compared to ev plants .
in contrast, larval performance was similar between ev and plants silenced in nrp, neig, wak and unkb .
the rate of ja and ja-ile accumulation after wounding was similar between ev and lpp-silenced plants .
after 18:3-glu elicitation, the accumulation of ja and ja-ile was significantly slower in lpp-silenced plants however after  <dig> min the levels were similar to ev plants .
plants silenced in unka expression had similar rates of ja and ja-ile accumulation to ev plants after both 18:3-glu elicitation and wounding .
likewise, induced levels of ja and ja-ile in nrp-, wak-, neig- and unkb-silenced plants were similar to ev plants .
discussion
in this study we exploited the combined capacities of supersage and ngs to quantify the expression of thousands of genes in n. attenuata leaves elicited by one of the major elicitors  present in the os of m. sexta larvae.
we analyzed the expression of > <dig>  supersage tags, representing  <dig>  unique transcript sequences with the main objective of identifying factors with potential regulatory functions during the m. sexta-n. attenuata interaction.
the analysis disclosed  <dig> annotated putative regulatory factors and from a subset of  <dig> selected we could confirm that the kinetic of mrna induction for  <dig> of them followed the expected profile, a rapid and transient up-regulation.
because the supersage generates  <dig> nt tags, dna sequence databases are a prerequisite to warrant efficient gene annotation of the tags.
consistent with the presence of > <dig>  nicotiana spp nucleotide sequences publicly available in genbank, ~88% of the n. attenuata unitags matched to nicotiana species .
however, only  <dig> % of the  <dig>  unitags matched -with a maximum of  <dig> mismatches- to sequences in genbank .
with a tolerance of  <dig> mismatches ,  <dig>  unitags  found a hit in this database .
most supersage tags are derived from the 3' utr of each mrna molecule  <cit>  which has been shown to be allele-specific in plants  <cit> .
since most of the nicotiana spp nucleotide entries in genbank correspond to n. tabacum, a percentage of the mismatches may be attributed to polymorphisms in the 3' utr of mrnas from n. attenuata and this tobacco species.
regarding the  <dig> differentially expressed unitags, 60% could be assigned to a protein entry  in genbank and uniprotkb/trembl protein databases and 25% of this fraction represented fully uncharacterized protein entries , a fact that partially handicapped the functional characterization of the n. attenuata transcription profiles.
nevertheless, a total of  <dig> unitags were reliably assigned to a go category.
however, since these  <dig> unitags represented < 50% of the differentially regulated mrnas , we expect that improved gene annotation will increase  the number of putative regulators that change expression after 18:3-glu elicitation.
changes in the expression of mrnas encoding for regulatory components
wrky transcription factors  occur in large gene families in plants and orchestrate different responses including those for pathogen resistance and wound healing  <cit> .
for example, wrkys bind to w-box elements in pr <dig> genes and regulate their expression after salicylic acid  induction and pathogen elicitation  <cit> .
wrky <dig> and  <dig> in n. attenuata have been involved in responses against insect herbivores  <cit> .
wizz  was identified as an early and transiently activated wound-responsive gene in tobacco  <cit>  and contains a leucine-zipper motif and a wrky domain in its structure.
after wounding, wizz transcripts accumulate within  <dig> min reaching maximal levels by  <dig> min and decreasing thereafter to basal levels  <cit> .
our results suggested that several wrky members including wizz may play critical roles in the coordination of m. sexta-n. attenuata interactions.
ap2/erf is a large family of tfs in plants, encoding transcriptional regulators with a variety of functions in the control of developmental and physiological processes including the integration of ja and et signals  <cit> .
the ap2/erf family is classified into subfamilies containing ap <dig>  dreb, erebp and rav tfs.
three ap2-like, two erebp and two rav tfs were rapidly up-regulated after 18:3-glu elicitation , suggesting that this family of tf may also play important roles in the orchestration of some of the plant's responses to insect feeding.
two unitags corresponding to the ccr4-associated factor  <dig>  were up-regulated by 18:3-glu elicitation.
caf <dig> is a subunit of the ccr4-not complex involved in mrna degradation and arabidopsis plants mutated in caf1a and b genes are more susceptible to pseudomonas syringae infection  <cit> .
these authors hypothesized that the caf1-containing complex controls the expression of a repressor of defense genes during pathogenesis.
our results suggested that the ccr4-not complex may also plays a role in defense responses against insects.
several unitags corresponding to putative cell wall-associated protein kinases  were rapidly up-regulated after 18:3-glu elicitation .
waks are transmembrane proteins containing a cytoplasmic ser/thr kinase domain and an extracellular domain in contact with components of the plant cell walls  <cit> .
waks play important roles in cell expansion, pathogen resistance, and heavy-metal stress tolerance  <cit> .
these protein kinases may associate changes in the cell wall structure after insect attack with downstream responses.
indirect evidence for rapid changes in cell wall structure and metabolism comes from the substantial number of genes associated with these processes that were up-regulated after 18:3-glu elicitation , beta-glucan-binding protein , cellulose synthase , α-expansin , cell wall peroxidase , raffinose synthase , xyloglucan endotransglycosylases , udp-glcua 4-epimerase  and xylose isomerase .
changes in the cell wall structure trigger ja- and et-mediated defense responses in arabidopsis as evidenced by the cev <dig> mutant, carrying a genetic lesion in a cellulose synthase gene  <cit> .
thus, changes in cell wall structure or homeostasis after mechanical damage and fac elicitation might influence defensive signaling in a manner analogous to the cev <dig> mutant of arabidopsis.
gsk3/shaggy-like kinase is a highly conserved ser/thr kinase involved in several signaling pathways.
the arabidopsis brassinosteroid-insensitive  <dig>  gene encodes a gsk3/shaggy-like kinase and was identified as a negative regulator of brassinosteroid  signaling  <cit> .
changes in the expression of transcripts for this kinase together with brassinosteroid insensitive 1-associated receptor kinase  <dig>  suggested that br play a role in the regulation of m. sexta-n. attenuata interaction.
br induces resistance against tmv, p. syringae and oidium spp in tobacco plants  <cit> .
evidence for cytokinins also playing a role in this interaction came from the strong up-regulation of the cytokonin-regulated kinase  <dig> gene   <cit> .
unitags corresponding to pp2a and c were up-regulated while one unitag corresponding to a pp2c isoform was down-regulated .
these regulators are known to play a central role in the control of defense-associated mechanisms.
for example, the arabidopsis ap2c <dig>  inactivates mpk <dig> and mpk <dig> and the mutant ap2c <dig> produces higher amounts of ja after wounding and is more resistant to phytophagous mites than wt.
in contrast, plants with increased ap2c <dig> activity produced less et and had compromised immunity against necrotrophic pathogens  <cit> .
among components associated to signal transduction processes, genes encoding for "avr9-cf <dig> rapidly elicited proteins" were the most predominant.
the go molecular function associated with these proteins was either protein kinase or receptor activity  and some may correspond to r  genes in n. attenuata.
the avr9-cf <dig> elicitor-receptor system is involved in the race-specific resistance of tomato  against cladosporium fulvum  <cit> .
interestingly, similar to wounding and os elicitation, the cf <dig> receptor induces the activation of wipk and sipk upon avr <dig> binding and it has been proposed that these protein kinases are hubs in the integration of signals for diverse elicitors  <cit> .
thus, "avr9/cf <dig> rapidly elicited proteins" may be either important components in defense responses against insects or they may reflect redundancy  in the signal transduction pathway activated by wounding and facs.
an additional regulator that shows high homology to protein kinase receptors and r genes is the putative hs1pro-1-like receptor .
the hs1pro- <dig> gene confers resistance to the beet cyst nematode heterodera schachtii in sugar beet   <cit> .
the transcript of hs1pro- <dig> is present at low levels in uninfected roots and it is induced specifically after nematode infection independently of sa, ja, aba or wounding  <cit> .
its induction by 18:3-glu in leaves on n. attenuata suggested that this gene may play a role in some plant responses against lepidopteran larvae in aboveground tissue.
up-regulation of unitags corresponding to sgt <dig>  a component of the skp1-cullin-f-box protein  ubiquitin ligases previously linked to early plant defenses responses conferred by r genes  <cit> , also suggested that signaling pathways connected to r genes may be induced by insect elicitors in n. attenuata.
early changes in gene expression after 18:3-glu elicitation reflect a rapid reprogramming of leaf metabolism
as a validation of the supersage approach, several unitags identified as differentially regulated by 18:3-glu elicitation corresponded to transcripts previously identified as differentially regulated by m. sexta larval feeding or os/fac elicitation  <cit> .
most of these genes corresponded to proteins involved in primary metabolism .
changes in the expression of these genes supported the idea of a shift in primary metabolism to supply energy, c skeletons and reducing power for the synthesis of defensive compounds and to induce tolerance mechanisms  <cit> .
most of the putative regulatory factors identified in the present study were not identified in previous studies except for a wrky transcription factor  <cit> .
changes occurring at the level of metabolism were among the most prevalent comprising more than  <dig> annotated unitags.
the up-regulation of twelve unitags corresponding to transcripts for ribosomal structural proteins, four to translation initiation factors, two to trna synthases and several corresponding to amino acid biosynthesis  suggested that, within  <dig> min, 18:3-glu elicitation stimulated an increased capacity for protein biosynthesis.
additionally, several annotated unitags corresponding to genes involved in the generation of energy and c skeletons  and in the generation and metabolism of reducing power + reductase, nad kinase, nicotinamidase) showed strong up-regulations .
nad-dependent malic enzymes  catalyze the oxidative decarboxylation of malate to produce pyruvate, co <dig>  and nadh in mitochondria  <cit>  while nadp-dependent plastidic and cytosolic isoforms provide c skeletons and reducing power for defense responses, lignin biosynthesis and reactive oxygen species formation  <cit> .
changes in the expression of these genes are probably necessary to meet large requirements for nadph to supply anabolic processes.
interestingly, several unitags corresponding to low abundant mrnas for isoforms of photosynthetic proteins were found up-regulated between ~ <dig> and 7-fold, including an oxygen-evolving protein, ribulose bisphosphate carboxylase activase, ribulose- <dig> -bisphosphate carboxylase/oxygenase  large subunit, chlorophyll a/b-binding protein, ferredoxin -nadp reductase and a psi-h precursor .
in contrast to the up-regulation of these low abundant unitags, high abundant unitags corresponding to similar genes did not change significantly within  <dig> min after 18:3-glu elicitation .
why these low abundant isoforms of photosynthetic genes are up-regulated after elicitation is unknown.
one possibility is that they play some specific roles during defense responses.
in previous studies, it has been observed that after lepidopteran larvae feeding or os/fac elicitation transcripts encoding for photosynthetic enzymes  in attacked n. attenuata leaves tend to be down-regulated, with the lowest expression after several hours  <cit> .
long-term reductions in the synthesis of these proteins have been proposed as a mechanism that attacked plants use to reinvest resources into other processes such as the synthesis of secondary defense pathways or tolerance  <cit> .
within  <dig> min of fac elicitation there were no significant reductions in the copy number of high abundance unitags corresponding to mrnas for photosynthetic proteins, suggesting that repressive mechanisms of major leaf isoforms act later during the fac-induced response.
identification of two mediators of m. sexta-n. attenuata interaction
the analysis of six candidate genes by vigs identified two putative regulatory components of resistance mechanisms against lepidopteran larval feeding.
caterpillars that fed for two weeks on plants silenced in the expression of a putative lipid phosphate phosphatase  and a protein of unknown function  gained ~2-fold and ~ <dig> -fold more mass, respectively, than larvae grown on ev control plants .
gain of mass by the larvae can be achieved by increased foliar consumption by stimulatory mechanisms, increased efficiency of food intake by increased food quality, or a combination both  <cit> .
by which mechanism m. sexta larvae grew larger on these plants is at present unknown and the subject of future investigations using stable transformed plants silenced in the expression of lpp and unka by rna interference .
lpps are signal transduction components that utilize a variety of lipid phosphate substrates including phosphatidic acid , diacylglycerol pyrophosphate , lyso-pa, ceramide 1-phosphate, and sphingosine 1-phosphate and it has been proposed that their function is to attenuate the signaling functions of these molecules  <cit> .
both lpp- and unka-silenced plants accumulated similar levels of ja and ja-ile after wounding and fac elicitation , indicating that the effects of llp and unka on m. sexta caterpillar performance was not the result of impaired ja biosynthesis.
together, these results suggested that mechanisms acting independently of ja biosynthesis must be affected in these plants to confer reduced resistance to m. sexta larval performance.
CONCLUSIONS
the analysis of fac-elicited n. attenuata plants by combined supersage and ngs enabled the identification of multiple factors with potential regulatory activity during the m. sexta-n. attenuata interaction.
together with the use of vigs to analyze candidate gene function we provided experimental evidence for the participation of two of these potential regulators in this interaction.
the further characterization of genetically stable llp- and unka-silenced plants in addition to the identification and characterization of novel regulators based on the supersage data will shed light on mechanisms used by plants to control a large reorganization of their metabolism and physiology to adjust defense and tolerance mechanisms with growth and reproduction.

 nicotiana attenuata is an annual native to southwestern usa that germinates from seed banks in response to factors in wood smoke after fires. because of this germination behavior and a strong intra-specific competition, n. attenuata allocates resources primarily to sustain rapid growth and seed setting and as a consequence, it has developed a large number of induced defense responses to ward off the unpredictable attacks from herbivores. hence, when n. attenuata is attacked by insect folivores, an extensive reprogramming of its transcriptome, proteome and metabolome takes place. previous studies estimated that more than n. attenuata genes respond to manduca sexta larval feeding and demonstrated that the plant readjusts its metabolism for de novo synthesis of direct and indirect defense responses and to induce tolerance mechanisms. activation of these defensive mechanisms requires energy and resources from primary metabolism and involves therefore a complex rearrangement of resource allocation in the plant, including altered photosynthesis and sink/source relations. how plants decode insect feeding and trigger defense and tolerance responses is starting to be understood. for example, a snrk kinase complex has been found to regulate tolerance mechanisms associated to the leaf/root partition of photoassimilates and two mapks, wipk and sipk, were shown to be critical for the induction of direct defense responses in n. attenuata. herbivore attack induces in plants the coordinated activation of several signal cascades including those of jasmonic acid, salicylic acid, and ethylene. among them, ja plays a major and essential role in the induction of a large number of the plant's protective responses against insect herbivory and wounding. thus, having ja as a common signal, a large number of the plant responses to these two stimuli overlap, however, plants can differentiate between mechanical damage and insect herbivory to tailor their responses. the perception of components in the oral secretions of feeding larvae is one mechanism by which plants can decode insect feeding. fatty acid-amino acid conjugates are major components in the os of m. sexta larvae and they are necessary and sufficient to induce most of the defense responses triggered by feeding m. sexta caterpillar in n. attenuata. hence, previous studies suggest the existence of central herbivore-activated regulators in n. attenuata leaves, which, in turn, are regulated by minute amounts of facs in the insect's os. disentangling the effect of mechanical tissue damage and fac elicitation will provide critical information on how plants control changes in its metabolism to more efficiently reduce the negative fitness consequences of herbivore attack. one of the earliest known molecular events differentially induced by os and facs in tobacco is the activation of wipk and sipk. activation of these protein kinases occur within the first minutes after wounding and the activation is enhanced several-fold by applying m. sexta os to wounds. importantly, not only their activities but also sipk and wipk transcript levels are rapidly and transiently induced after elicitation, indicating that these regulators are under positive feedback control at the transcriptional level. one of the early targets of the fac signal transduction pathway in n. attenuata is the wrky gene. its transcript levels are also rapidly and transiently induced after wounds have been supplemented with m. sexta os or synthetic facs but only marginally by mechanical damage alone. this rapid and transient kinetic of mrna accumulation is characteristic of regulatory components and differs from that showed by, for example, transcripts encoding for defense components which is characterized by a slower and more persistent rate of mrna accumulation, reaching maximum levels after hours to days. however, not all regulatory components are under positive feedback control at the transcriptional level: the coronatine insensitive gene is an example in the ja transduction pathway. however, some of the recently identified jaz proteins that interact with coi and participate in ja-ile perception are rapidly and transiently induced at the mrna level by wounding in arabidopsis. the rapid advances in high throughput sequencing capacity in combination with new "open-architecture" techniques for quantification of gene expression has opened the possibility of performing genome-wide transcriptome studies in organisms from which massive nucleotide sequence information is not yet available. serial analysis of gene expression is a technique that allows for the absolute quantification of mrna abundance by quantifying the relative frequencies of individual short transcripts signatures tags. further development of the technique allowed for the generation of nt tags which substantially improved the annotation of tags when aligned to sequences in public nucleotide databases. with these techniques, the detection of transcripts is proportionally correlated to the scale of dna sequencing and their combination with next generation sequencing allows for the detection and analysis of very low abundant transcripts which have been estimated to account for more than 90% of mrnas in eukaryotic cells. here we used supersage in combination with ngs for the quantification of the early changes occurring in the transcriptome of n. attenuata plants after a single event of 18:3-glu elicitation. the major objective of the study was to identify genes encoding for potential regulatory components of the fac-mediated responses by looking for low abundant transcripts that were rapidly and transiently induced after 18:3-glu elicitation.  generation of supersage libraries from wounded and fac elicited n. attenuata leaves two supersage libraries were generated from the second fully expanded leaf of n. attenuata plants either mechanically wounded or wounded and supplemented with 18:3-glu as a single fac elicitor. leaf samples were harvested after min of the treatments. wounding is a prerequisite for fac-elicitation; hence, analysis of wounded leaves was used to differentiate between genes regulated by mechanical damage from those regulated more specifically or deferentially by facs. additionally, elicitation by a single elicitor and a single wound event were used to eliminate the effects of other os components and repeated wounding on gene expression. the total number of supersage tags obtained after sequencing the libraries in a single plate and eliminating i) incomplete reads, ii) twin-ditags, and iii) ditags without complete library-identification dna linkers was; comprising tags from the wounding library and from the fac-elicited library. these tags represented unique sequences with detected only once in the combined libraries, and detected at least twice in the combined libraries. these latter tags are referred as unitags throughout the manuscript and will be considered for further analysis. singletons represented thus ~60% of unique sequences, in agreement with previous studies. the complete supersage dataset is available in additional file. * values normalized to million tags ** w: wounded; f: 18:3-glu elicited abundance of unitags and annotation to public databases the unitags were first classified in abundance groups according to their number of copies. unitags present at ≤ > - ≤, > - ≤ and > copies per million were considered as low-, mid-, high- and very high-abundant tags, respectively. the frequency distribution of the unitags showed that the number of copies in low and mid abundance groups represented > 98% of the unitags while high- and very high-abundant tags represented only %. however, although the latter group represented only a small fraction of the unitags, together they accounted for ~47% of the total number of tag copies in both the w and f libraries. these values were in agreement with previously reported data. annotation of the unitags using basic local alignments gave tags that matched with a maximum of mismatches to sequences deposited in genbank plant nucleotide databases. % of these unitags matched perfectly with sequences in the databases while % did it with one mismatch, % with two mismatches and % with three mismatches. moreover, 88% of the annotated unitags matched sequences corresponding to nicotiana spp, 5% to solanum spp and 8% to other plant species. fac elicitation induces differential expression of unitags statistically significant changes in tag copy number between the f and w libraries were analyzed by calculating a probability -value according to. although small changes in expression levels may have biological significance, in this study we focused primarily on genes which showed strong changes in expression levels with arbitrary fold-change values ≥ or ≤. based on the calculated -values and using a 95% confidence level, unitags were identified as differentially expressed after fac elicitation. among these unitags, had fc ≥ and fc ≤. most of the differentially expressed unitags presented fc values between and with and unitags presenting fc values ≥ and ≤, respectively. the majority of the differentially up-regulated unitags and all of the down-regulated unitags corresponded to low- and mid-abundance groups, indicating that the strongest changes in expression levels occurred primarily in genes expressed at low to intermediate levels. assignment of differentially expressed unitags to gene ontology: biological and functional categories to obtain gene function categories of the differentially expressed unitags, gene ontology annotation was performed by blastx against the non-redundant genbank and uniprotkb/trembl protein databases. for this analysis, we used unitags that showed a maximum of mismatches with entries in the genbank nucleotide database. of the differentially expressed unitags, had an associated nucleotide sequence and matched to an amino acid sequence entry in the genbank and uniprotkb/trembl databases. go annotations could be assigned to of these unitags with the remaining entries corresponding to uncharacterized proteins. among the most prevalent go biological processes, ~25% of the unitags classified into metabolism, ~12% into regulation of gene expression, ~10% into amino acid phosphorylation/dephosphorylation, ~8% into translation, ~8% into defense and stress responses, ~7% into transport, ~6% into protein degradation and folding and ~6% into signal transduction components. the preponderance of changes in transcripts corresponding to metabolism, signaling, transcription, translation and transport associated processes after min of 18:3-glu elicitation emphasized the fact that at this early time point a substantial reprogramming of the leaf metabolism is already in progress. based on changes in metabolic genes, hallmarks of this reprogramming included an increased capacity for protein synthesis and the generation of c skeletons and reducing power. these changes in the expression of metabolic genes are consistent with a substantial shift in primary metabolism to support secondary metabolism and tolerance mechanisms and are consistent with previous gene expression studies. the identification of regulatory factors controlling these changes in metabolism and defense and tolerance processes against insects is one of the major challenges for the future and some potential candidates are described below. changes in the expression of unitags/mrnas encoding for regulatory components the most prevalent go molecular function with regulatory activity corresponded to transcriptional regulators and protein kinases, represented by and unitags, respectively. the protein phosphatase category contained unitags, the signal transduction category unitags and the nucleic acid binding category unitags. thus, a total of annotated unitags corresponded to factors with potential regulatory function. among transcriptional regulators, unitags corresponding to wrky transcription factors were the most predominant and tag- was the most up-regulated after 18:3-glu elicitation within this group. other unitags for wrkys were up-regulated between and fold. within the wrky domain containing family, a wizz tf was up-regulated fold. other prevalent up-regulated tfs included ap2-like factors, rav factors, ethylene-responsive element binding proteins and ccr4-not transcription complex proteins. single up-regulated unitags in this category corresponded to a bzip tf, his, s1fa, rna polymerase ii and a sigma subunit for a plastidial rna polymerase. among down-regulated transcriptional regulators were a gata- zinc finger protein and rna polymerase iii. within the protein kinase and phosphatase classes, three unitags corresponded to mapk, three to cell-wall associated kinases, two to brassinosteroid insensitive 1-associated receptor kinase and three to protein phosphatase 2a and c. in addition, this category contained a chloroplast precursor for arabidopsis protein kinase up-regulated ~ fold, a shaggy-like kinase, and a cytokinin-regulated kinase and a calmodulin protein kinase among others. within the signal transduction class, the most predominant unitags corresponded to "avr9/cf- rapidly elicited proteins" up-regulated between and fold. single up-regulated unitags corresponded to a hs1pro-1-like protein; fold), sgt, a lipid phosphate phosphatase and an extra-large g protein were also contained in this category. validation of the supersage data by qpcr a subset of differentially expressed unitags was selected for further analysis based on the fulfillment of at least two of the following criteria: 1) strong and significant changes in their fc values; 2) abundance of < copies.million-; 3) matched known regulatory components in the databases. fc: fold change. nm: no match in genbank 1unitags selected for vigs analysis are depicted in bold. the selected unitags were first elongated by amplification of their corresponding cdnas and blasted against the genbank plant nucleotide databases to confirm their identities. all of the elongated sequences matched to the same entries as the original bp tags. secondly, the elongated sequences were used to design gene-specific primers to i) validate the supersage data and ii) to study the kinetic of mrna induction by real time quantitative pcr. total rna was extracted from both wounded and 18:3-glu elicited leaves of wt plants after different times of the stimuli. the accumulation of mrnas corresponding to the selected unitags was consistent with a rapid increase after fac elicitation and a rapid decrease to basal or lower levels after the stimuli. interestingly, several transcripts showed either no or minimal induction by wounding, representing therefore genes activated almost specifically by facs. for some transcripts mechanical damage induced an increase in their corresponding mrna levels which was potentiated several fold by 18:3-glu elicitation. for transcripts corresponding to four unitags, the differential regulation by 18:3-glu elicitation could not be confirmed and they may represent false positives in the supersage analysis. finally, mrnas for three unitags were differentially repressed by 18:3-glu elicitation. functional characterization of candidate regulatory components of insect mediated responses by vigs to validate the use of the supersage approach for the identification of candidate regulatory components of the interaction between n. attenuata and m. sexta larvae, six genes were selected for preliminary gene function characterization by virus-induced gene silencing. the selection of these genes was based on: 1) their kinetic of mrna induction and 2) their fold-change compared to wounding. some of the genes encoded for putative regulatory components and two presented no similarity to any other protein of known or predicted function. the selected unitags corresponded to a hs1pro-1-like protein; tag-6205), lipid phosphate phosphatase, nicotiana elicitor induced gene, cell wall-associated protein kinase, unka and unkb. to evaluate whether these genes participate in fac- and insect defense-mediated responses, gene-specific silenced plants and plants transformed with the empty vector were assessed for m. sexta larval performance and the accumulation of ja and ja-ile after 18:3-glu elicitation and wounding. gene silencing efficiency in these plants was analyzed by qpcr in 18:3-glu-elicited leaves after h of the treatment. the morphological phenotype of the silenced-plants was indistinguishable from ev control plants. the silencing efficiency is expressed as the reduction of the mrna levels in the vigs-silenced plants relative to the levels of the corresponding mrna in ev plants. in all cases the reductions were statistically significant. m. sexta larva feeding on plants silenced in the expression of lpp or unka showed significant increases in mass gained after and/or days compared to ev plants. in contrast, larval performance was similar between ev and plants silenced in nrp, neig, wak and unkb. the rate of ja and ja-ile accumulation after wounding was similar between ev and lpp-silenced plants. after 18:3-glu elicitation, the accumulation of ja and ja-ile was significantly slower in lpp-silenced plants however after min the levels were similar to ev plants. plants silenced in unka expression had similar rates of ja and ja-ile accumulation to ev plants after both 18:3-glu elicitation and wounding. likewise, induced levels of ja and ja-ile in nrp-, wak-, neig- and unkb-silenced plants were similar to ev plants. discussion in this study we exploited the combined capacities of supersage and ngs to quantify the expression of thousands of genes in n. attenuata leaves elicited by one of the major elicitors present in the os of m. sexta larvae. we analyzed the expression of > supersage tags, representing unique transcript sequences with the main objective of identifying factors with potential regulatory functions during the m. sexta-n. attenuata interaction. the analysis disclosed annotated putative regulatory factors and from a subset of selected we could confirm that the kinetic of mrna induction for of them followed the expected profile, a rapid and transient up-regulation. because the supersage generates nt tags, dna sequence databases are a prerequisite to warrant efficient gene annotation of the tags. consistent with the presence of > nicotiana spp nucleotide sequences publicly available in genbank, ~88% of the n. attenuata unitags matched to nicotiana species. however, only % of the unitags matched -with a maximum of mismatches- to sequences in genbank. with a tolerance of mismatches, unitags found a hit in this database. most supersage tags are derived from the 3' utr of each mrna molecule which has been shown to be allele-specific in plants. since most of the nicotiana spp nucleotide entries in genbank correspond to n. tabacum, a percentage of the mismatches may be attributed to polymorphisms in the 3' utr of mrnas from n. attenuata and this tobacco species. regarding the differentially expressed unitags, 60% could be assigned to a protein entry in genbank and uniprotkb/trembl protein databases and 25% of this fraction represented fully uncharacterized protein entries, a fact that partially handicapped the functional characterization of the n. attenuata transcription profiles. nevertheless, a total of unitags were reliably assigned to a go category. however, since these unitags represented < 50% of the differentially regulated mrnas, we expect that improved gene annotation will increase the number of putative regulators that change expression after 18:3-glu elicitation. changes in the expression of mrnas encoding for regulatory components wrky transcription factors occur in large gene families in plants and orchestrate different responses including those for pathogen resistance and wound healing. for example, wrkys bind to w-box elements in pr genes and regulate their expression after salicylic acid induction and pathogen elicitation. wrky and in n. attenuata have been involved in responses against insect herbivores. wizz was identified as an early and transiently activated wound-responsive gene in tobacco and contains a leucine-zipper motif and a wrky domain in its structure. after wounding, wizz transcripts accumulate within min reaching maximal levels by min and decreasing thereafter to basal levels. our results suggested that several wrky members including wizz may play critical roles in the coordination of m. sexta-n. attenuata interactions. ap2/erf is a large family of tfs in plants, encoding transcriptional regulators with a variety of functions in the control of developmental and physiological processes including the integration of ja and et signals. the ap2/erf family is classified into subfamilies containing ap dreb, erebp and rav tfs. three ap2-like, two erebp and two rav tfs were rapidly up-regulated after 18:3-glu elicitation, suggesting that this family of tf may also play important roles in the orchestration of some of the plant's responses to insect feeding. two unitags corresponding to the ccr4-associated factor were up-regulated by 18:3-glu elicitation. caf is a subunit of the ccr4-not complex involved in mrna degradation and arabidopsis plants mutated in caf1a and b genes are more susceptible to pseudomonas syringae infection. these authors hypothesized that the caf1-containing complex controls the expression of a repressor of defense genes during pathogenesis. our results suggested that the ccr4-not complex may also plays a role in defense responses against insects. several unitags corresponding to putative cell wall-associated protein kinases were rapidly up-regulated after 18:3-glu elicitation. waks are transmembrane proteins containing a cytoplasmic ser/thr kinase domain and an extracellular domain in contact with components of the plant cell walls. waks play important roles in cell expansion, pathogen resistance, and heavy-metal stress tolerance. these protein kinases may associate changes in the cell wall structure after insect attack with downstream responses. indirect evidence for rapid changes in cell wall structure and metabolism comes from the substantial number of genes associated with these processes that were up-regulated after 18:3-glu elicitation, beta-glucan-binding protein, cellulose synthase, α-expansin, cell wall peroxidase, raffinose synthase, xyloglucan endotransglycosylases, udp-glcua 4-epimerase and xylose isomerase. changes in the cell wall structure trigger ja- and et-mediated defense responses in arabidopsis as evidenced by the cev mutant, carrying a genetic lesion in a cellulose synthase gene. thus, changes in cell wall structure or homeostasis after mechanical damage and fac elicitation might influence defensive signaling in a manner analogous to the cev mutant of arabidopsis. gsk3/shaggy-like kinase is a highly conserved ser/thr kinase involved in several signaling pathways. the arabidopsis brassinosteroid-insensitive gene encodes a gsk3/shaggy-like kinase and was identified as a negative regulator of brassinosteroid signaling. changes in the expression of transcripts for this kinase together with brassinosteroid insensitive 1-associated receptor kinase suggested that br play a role in the regulation of m. sexta-n. attenuata interaction. br induces resistance against tmv, p. syringae and oidium spp in tobacco plants. evidence for cytokinins also playing a role in this interaction came from the strong up-regulation of the cytokonin-regulated kinase gene. unitags corresponding to pp2a and c were up-regulated while one unitag corresponding to a pp2c isoform was down-regulated. these regulators are known to play a central role in the control of defense-associated mechanisms. for example, the arabidopsis ap2c inactivates mpk and mpk and the mutant ap2c produces higher amounts of ja after wounding and is more resistant to phytophagous mites than wt. in contrast, plants with increased ap2c activity produced less et and had compromised immunity against necrotrophic pathogens. among components associated to signal transduction processes, genes encoding for "avr9-cf rapidly elicited proteins" were the most predominant. the go molecular function associated with these proteins was either protein kinase or receptor activity and some may correspond to r genes in n. attenuata. the avr9-cf elicitor-receptor system is involved in the race-specific resistance of tomato against cladosporium fulvum. interestingly, similar to wounding and os elicitation, the cf receptor induces the activation of wipk and sipk upon avr binding and it has been proposed that these protein kinases are hubs in the integration of signals for diverse elicitors. thus, "avr9/cf rapidly elicited proteins" may be either important components in defense responses against insects or they may reflect redundancy in the signal transduction pathway activated by wounding and facs. an additional regulator that shows high homology to protein kinase receptors and r genes is the putative hs1pro-1-like receptor. the hs1pro- gene confers resistance to the beet cyst nematode heterodera schachtii in sugar beet. the transcript of hs1pro- is present at low levels in uninfected roots and it is induced specifically after nematode infection independently of sa, ja, aba or wounding. its induction by 18:3-glu in leaves on n. attenuata suggested that this gene may play a role in some plant responses against lepidopteran larvae in aboveground tissue. up-regulation of unitags corresponding to sgt a component of the skp1-cullin-f-box protein ubiquitin ligases previously linked to early plant defenses responses conferred by r genes, also suggested that signaling pathways connected to r genes may be induced by insect elicitors in n. attenuata. early changes in gene expression after 18:3-glu elicitation reflect a rapid reprogramming of leaf metabolism as a validation of the supersage approach, several unitags identified as differentially regulated by 18:3-glu elicitation corresponded to transcripts previously identified as differentially regulated by m. sexta larval feeding or os/fac elicitation. most of these genes corresponded to proteins involved in primary metabolism. changes in the expression of these genes supported the idea of a shift in primary metabolism to supply energy, c skeletons and reducing power for the synthesis of defensive compounds and to induce tolerance mechanisms. most of the putative regulatory factors identified in the present study were not identified in previous studies except for a wrky transcription factor. changes occurring at the level of metabolism were among the most prevalent comprising more than annotated unitags. the up-regulation of twelve unitags corresponding to transcripts for ribosomal structural proteins, four to translation initiation factors, two to trna synthases and several corresponding to amino acid biosynthesis suggested that, within min, 18:3-glu elicitation stimulated an increased capacity for protein biosynthesis. additionally, several annotated unitags corresponding to genes involved in the generation of energy and c skeletons and in the generation and metabolism of reducing power + reductase, nad kinase, nicotinamidase) showed strong up-regulations. nad-dependent malic enzymes catalyze the oxidative decarboxylation of malate to produce pyruvate, co and nadh in mitochondria while nadp-dependent plastidic and cytosolic isoforms provide c skeletons and reducing power for defense responses, lignin biosynthesis and reactive oxygen species formation. changes in the expression of these genes are probably necessary to meet large requirements for nadph to supply anabolic processes. interestingly, several unitags corresponding to low abundant mrnas for isoforms of photosynthetic proteins were found up-regulated between ~ and 7-fold, including an oxygen-evolving protein, ribulose bisphosphate carboxylase activase, ribulose- -bisphosphate carboxylase/oxygenase large subunit, chlorophyll a/b-binding protein, ferredoxin -nadp reductase and a psi-h precursor. in contrast to the up-regulation of these low abundant unitags, high abundant unitags corresponding to similar genes did not change significantly within min after 18:3-glu elicitation. why these low abundant isoforms of photosynthetic genes are up-regulated after elicitation is unknown. one possibility is that they play some specific roles during defense responses. in previous studies, it has been observed that after lepidopteran larvae feeding or os/fac elicitation transcripts encoding for photosynthetic enzymes in attacked n. attenuata leaves tend to be down-regulated, with the lowest expression after several hours. long-term reductions in the synthesis of these proteins have been proposed as a mechanism that attacked plants use to reinvest resources into other processes such as the synthesis of secondary defense pathways or tolerance. within min of fac elicitation there were no significant reductions in the copy number of high abundance unitags corresponding to mrnas for photosynthetic proteins, suggesting that repressive mechanisms of major leaf isoforms act later during the fac-induced response. identification of two mediators of m. sexta-n. attenuata interaction the analysis of six candidate genes by vigs identified two putative regulatory components of resistance mechanisms against lepidopteran larval feeding. caterpillars that fed for two weeks on plants silenced in the expression of a putative lipid phosphate phosphatase and a protein of unknown function gained ~2-fold and ~ -fold more mass, respectively, than larvae grown on ev control plants. gain of mass by the larvae can be achieved by increased foliar consumption by stimulatory mechanisms, increased efficiency of food intake by increased food quality, or a combination both. by which mechanism m. sexta larvae grew larger on these plants is at present unknown and the subject of future investigations using stable transformed plants silenced in the expression of lpp and unka by rna interference. lpps are signal transduction components that utilize a variety of lipid phosphate substrates including phosphatidic acid, diacylglycerol pyrophosphate, lyso-pa, ceramide 1-phosphate, and sphingosine 1-phosphate and it has been proposed that their function is to attenuate the signaling functions of these molecules. both lpp- and unka-silenced plants accumulated similar levels of ja and ja-ile after wounding and fac elicitation, indicating that the effects of llp and unka on m. sexta caterpillar performance was not the result of impaired ja biosynthesis. together, these results suggested that mechanisms acting independently of ja biosynthesis must be affected in these plants to confer reduced resistance to m. sexta larval performance.  the analysis of fac-elicited n. attenuata plants by combined supersage and ngs enabled the identification of multiple factors with potential regulatory activity during the m. sexta-n. attenuata interaction. together with the use of vigs to analyze candidate gene function we provided experimental evidence for the participation of two of these potential regulators in this interaction. the further characterization of genetically stable llp- and unka-silenced plants in addition to the identification and characterization of novel regulators based on the supersage data will shed light on mechanisms used by plants to control a large reorganization of their metabolism and physiology to adjust defense and tolerance mechanisms with growth and reproduction. 
fatty acid-amino acid conjugates 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. the analysis of the early changes in the transcriptome of n. attenuata after fac elicitation using supersage/ has identified regulatory genes involved in insect-specific mediated responses in plants. the analysis targeted mrnas encoding regulatory components: rare transcripts with very rapid fac-elicited kinetics. based on gene ontology classification, more than 25% of the annotated unitags corresponded to putative regulatory components, including transcriptional regulators and protein kinases. six fac-regulated genes were functionally characterized by vigs and two, a putative lipid phosphate phosphatase and a protein of unknown function, were identified as important mediators of the m. sexta-n. attenuata interaction. plants trigger and tailor defense responses after perception of the oral secretions of attacking specialist lepidopteran larvae. we used supersage combined with sequencing to quantify the early transcriptional changes elicited by the fac n-linolenoyl-glutamic acid and virus induced gene silencing to examine the function of candidate genes in the m. sexta-n. attenuata interaction. from unique tag sequences identified, and were significantly up- and down-regulated ≥ -fold, respectively, after 18:3-glu elicitation compared to wounding.
 plants trigger and tailor defense responses after perception of the oral secretions of attacking specialist lepidopteran larvae. fatty acid-amino acid conjugates 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.  we used supersage combined with sequencing to quantify the early transcriptional changes elicited by the fac n-linolenoyl-glutamic acid and virus induced gene silencing 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. from unique tag sequences identified, and were significantly up- and down-regulated ≥ -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 transcriptional regulators and protein kinases. quantitative pcr analysis was used to analyze the fac-dependent regulation of a subset of 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 and a protein of unknown function, were identified as important mediators of the m. sexta-n. attenuata interaction.  the analysis of the early changes in the transcriptome of n. attenuata after fac elicitation using supersage/ 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. 