BACKGROUND
solanum torvum sw  is worldwide employed as rootstock for eggplant cultivation because of its vigor and resistance/tolerance to heavy metal- and salt-contaminated soil and, specially, to the most serious soil-borne diseases   <cit> .
however, despite the fact that identification of the effectors of torvum resistance would be extremely valuable for other solanaceous crops, particularly eggplant, very little studies have been conducted to date regarding torvum resistance mechanism and have mainly dealt with its resistance to bacteria  <cit> .
this scenario is mostly attributable to the paucity of database information on torvum which, in turn is a consequence of the lack of high-throughput genomic tools ).
few expression databases were available for torvum.
only recently,  <dig>  unigenes from torvum cv.
torubamubiga, a low cadmium -accumulating variety, have been obtained in the context of studies on cd acclimation process  <cit> .
this gene catalogue represents a significant improvement in terms of sequence information; however, given the current high-throughput standards, is unsuitable to pose the basis for global transcript profiling  due to its small size.
an approach to overcome such typical non-model species constraints would be undertaking an rna-sequencing approach, since no prior knowledge of transcript sequences is mandatory.
an alternative is represented by microarray hybridizations, a robust and well-assessed technique, about ten-fold less expensive per sample when compared to the cheapest rna-seq approaches  <cit> .
microarray can be considered as a viable alternative to rna-seq provided that the number of available sequences for the species of interest is considered satisfactory.
thus, in order to extend the number of probes for torvum, we combined 454-based pyrosequencing with microarrays as reported by  <cit> .
a further example of this strategy has been recently presented  <cit> .
root-knot nematodes, meloidogyne spp.
determine substantial losses on horticultural and field crops  <cit> .
in the tropical and sub-tropical regions, crop production losses due to nematodes were estimated at  <dig> % compared with  <dig> % in developed countries  <cit> .
the american phytopathological society has reported that root-knot nematodes are the most common and destructive nematode pathogens, accounting for an estimated of 14% of all worldwide plant losses, which translates into almost $  <dig> billion dollars annually  <cit> .
meloidogyne incognita belongs to the order tylenchida, a very large and diverse group of nematodes, which contains a majority of the known plant parasitic species.
representatives of this order have a worldwide distribution and are encountered more frequently than any other group of nematodes.
within tylenchida, members of the family heteroderidae are by far the most damaging to world agriculture, among which the root knot nematodes   and the cyst nematodes .
to date, more than  <dig> rkn species are described, and m. incognita is unquestionably the most important one in terms of distribution and damages.
nematodes establish complex interactions with hosts, and, upon successful infection, cause the reprogramming of host cell developing structures as multinucleate giant feeding cells.
more than  <dig> proteins have been found to be secreted by m. incognita  <cit>  and several of these proteins are thought to play a role in modulating infection and reprogramming host metabolism.
nematode resistant  genes are an obvious topic of interest for crop protection.
the first cloned nem-r gene was the sugar beet hs1pro- <dig> conferring resistance against the sugar beet cyst nematode  <cit> .
however, hs1pro- <dig> appears poorly related to typical plant r genes.
several other nem-r genes  belong to the nbs-lrr class of r-genes  <cit> .
with respect to the subclass of rkn r-genes , few resistance genes have been identified and only two have been cloned, namely mi- <dig> from tomato  <cit>  and, very recently, ma from the myrobalan plum prunus cerasifera  <cit> .
both mi- <dig> and ma confer broad-spectrum resistance against several root knot nematode.
not only a single pathogen-related gene in response to nematodes attack is described in the literature.
more than one major gene is often involved in the resistance mechanisms commonly referred as horizontal resistance.
wang et al.
<cit>  identified one major recessive gene with strong additive effect against m. incognita in cotton, together with a major qtl with strong dominant effect in different crosses using different resistance sources.
transgressive segregation was previously documented from the same authors  <cit>  as an epistatic interaction that strengthened the resistance phenotype.
the determinants of torvum resistance to nematodes are yet to be identified, and, in particular, the contribution of a specific gene-for-gene interaction rather than an horizontal, quantitative resistance, or even a “non-host” resistance phenomena  is far from being clear.
a number of studies have investigated transcriptional changes of m. incognita-challenged compatible and incompatible plants in a variety of species.
two early studies described the transcript profiling of about  <dig> transporter genes in a thaliana <cit>  and of  <dig>  selected genes in tomato  <cit> .
the latter work was carried out on resistant and susceptible tomato genotypes and, by comparing transcript abundance patterns over a time-course, several clusters of genes associated to compatible and incompatible interactions were accurately defined.
furthermore, a glycosyltransferase was shown to be necessary for conferring the resistant phenotype  <cit> .
a larger number of genes  was tested by jammes et al.
<cit>  in arabidopsis, using giant cell-enriched root tissues infected by m. incognita along three time-points.
about  <dig>  genes were called as differentially expressed between uninfected root tissues and galls at different developmental stages.
major classes of modulated genes included metabolism-associated genes and transcription factors.
a substantial down-regulation of defense genes, including phenylpropanoids, was found.
more recently  <cit> , the expression profiling of about  <dig>  genes from est libraries obtained from m. incognita-challenged resistant and susceptible cotton varieties, revealed differential modulation in resistant and susceptible genotypes of genes falling in the broad categories of pathogen recognition, signal transduction and pathogen defense.
this latter category included superoxide dismutases, chitinases and isoflavone reductases.
a further recent work  <cit>  have described the changes in the soybean transcriptome using the affymetrix® soybean genome array  in galls formed in m. incognita-challenged roots at  <dig> days and  <dig> weeks after infection.
genes encoding enzymes involved in carbohydrate and cell wall metabolism, cell-cycle control and plant defense were significantly modulated.
among plant defense genes, several lipoxygenases, pathogenesis-related proteins and phenylpropanoid genes were found to be differentially expressed.
the aim of this work was, by global transcriptome profiling, getting insights on the mechanisms underpinning torvum resistance against m. incognita.
toward this end, we deployed an approach which takes advantage of both next generation sequencing  and microarray techniques.
in particular, we targeted previously uncharacterized transcripts by rna-seq and exploited reliability and cost-effectiveness of well established microarray technologies for transcript quantification.
we also explored the “chip extension” procedure, an approach allowing to increase the reliability of heterologous hybridizations by defining subset of probes less likely to be prone to expression artifacts.
the availability of a 3′ transcript catalogue for torvum and transcript profiling upon nematode infection provides molecular tools for identifying torvum resistance mechanism.
RESULTS
de novo assembly
since only  <dig>  unigenes from torvum are available to date  <cit> , we undertook an rna-seq pyrosequencing approach  to extend the number of torvum genes and thus pose the basis to perform a global transcriptome profiling in a cost-effective manner.
according to  <cit>  we produced a comprehensive normalized catalogue of the 3’ mrna regions  tailored at the generation of a custom chip.
as starting material, torvum roots were subjected to a wide range of environmental stresses  to maximize the number of expressed genes.
sequencing was confined to the 3’ in order to: i) minimize the number of  <dig> reads mapping to the same transcript but assembling in different contigs due to the lack of uniform coverage in low-abundance transcripts ; ii) allow for designing highly specific probes by encompassing 3’ regions which are known to be subjected to lower selection pressure.
the library was normalized as this treatment has been shown to greatly improve rare transcript coverage as well as other quality features  <cit> .
one-quarter of the  <dig> plate was used to sequence the normalized 3’cdna library yielding  <dig>  reads with an average length of  <dig> bases for a total of   <dig> , <dig> bases.
assuming an average number of transcripts of  <dig>  with an average length of  <dig> kb and thus a transcriptome length of  <dig> mb, an yield of  <dig> mb confined to 500– <dig> bp at the 3’ regions represents a coverage of about 4x.
this coverage, while not exhaustive, poses the basis, at least within the context of a normalized library, for quantification of a high number of transcripts in comparison to the few thousands of unigenes to date available for torvum.
de novo assembly of torvum reads was undertaken with mira  <dig> .
<dig>  <cit> .
the assembly led to  <dig>  contigs plus  <dig>  singletons .
various parameters including n <dig> were calculated to describe the average size of the contigs.
in additional file  <dig>  the distribution of contig length  and contig coverage  is shown.
as a consequence of our 3’ sequencing design, the most enriched bin for unigenes  was, as expected, in the 500– <dig> bp region.
contig coverage  was relatively uniform as a result of the normalization step  <cit> .
to further assess the assembly, we compared the contigs plus singletons  against selected public assemblies, including the recently released  <dig>  unigene catalogue from solanum torvum cv.
torubamubiga  <cit> .
further queried databases  were the current releases in tc database from the phylogenetically related species eggplant , tobacco , tomato , potato  and pepper .
at last, we tested arabidopsis  as a phylogenetically distant reference.
as expected, a limited number of torvum queries showed hits against the small  torvum torubamubiga dataset , while the larger tc solanaceous datasets as potato, tomato, eggplant and tobacco exhibited between  <dig> and 80% hits.
however, when these results are corrected for the number of entries of the queried databases , eggplant and s. torvum cv.
torubamubiga clearly emerged as the most correlated to torvum database.
on the other hand, the phylogenetically distant species arabidopsis shows a barely detectable ratio of percent hits to database extent .
overall, the blast data closely mirror known phylogenetic relationships within solanaceous species  <cit>  with torvum having its closest counterpart in eggplant and, in order of decreasing relatedness, potato, tomato, pepper and tobacco.
noteworthy, at an expect value of 10- <dig>  more than 60% of torvum unigenes had no hits against cv.
torubamubiga database, indicating that a majority of torvum unigenes in our catalogue are not represented in the small torubamubiga dataset .
on the other hand, when torubamubiga database was queried against our torvum unigene catalogue, only 18% of the  <dig>  torubamubiga unigenes had no hits, indicating that our torvum transcript tags catalogue is likely to represent the most complete dataset for torvum available to date.
custom chip design
oligoarray  <dig>  software  <cit>  was used to compute gene-specific oligonucleotides corresponding to torvum unigenes.
oligoarray output, besides microarray design, provides hints on the quality  of input sequences by declaring how many specific probes can be designed based on input sequences.
about 80% of oligos  turned out to be specific for one torvum unigene, while 15% oligos were specific for 1– <dig> unigenes,  indicating efficient normalization and substantial lack of redundancy in the torvum unigene set.
a final filtering step over torvum unigenes was conducted to exclude the less specific probes .
this also allowed to contain the number of probes in the chip to maximum  <dig> , consistent with a triplicate probe layout in the 90k-features combimatrix chip design.
the final layout consisted in  <dig>  probes representative of contigs and  <dig>  probes derived from singletons.
the significant reduction  of singleton representation in the final chip layout likely reflects the constraints experienced by oligoarray software in designing specific probes for singletons due to the shorter length and/or lower qualities of sequences when compared to contigs.
differentially expressed genes in nematode-infected torvum
two-months-old torvum plants were infected with meloidogyne incognita and left to proceed for  <dig> days.
neither root-galls  nor egg-masses  were never collected on torvum stained root  even in longer infection stages .
torvum rna samples  were used for array hybridizations.
pearson correlation coefficients for biological replicates were all above  <dig>  .
genes were considered differentially expressed if exhibiting at least a 2-fold change and a false discovery rate  <=  <dig> .
figure  <dig> depicts transcript abundance values in control vs. infected roots as ma-plot.
differentially expressed genes  fulfilling the two criteria  are plotted in red.
the full list of deg accompanied by expression ratio, fdr values, blast hits, blast2go annotations and go mappings are reported in additional file  <dig>
annotation of torvum gene catalogue
the software suite blast2go  <cit>  was used to annotate torvum unigenes.
as a first blast step, we run blastx against ncbi non-redundant database using as query the  <dig>  unigenes included in the chip layout for which an hybridization signal could be obtained.
a total of  <dig>  go annotations were retrieved.
the full catalogue of torvum genes including blast2go annotations and complementary information  are available in additional file  <dig>  while the full set of torvum unigene sequences  is provided in fasta format in additional file  <dig>
go enrichment analysis of torvum deg genes
major expression trends in infected torvum were investigated to test whether the  <dig> degs could be grouped in some enriched go terms.
towards this end, we used as a reference set the  <dig>  annotations obtained with blast2go and as test set the  <dig> degs.
the fisher’s exact test results  for the entire set of enriched go terms and corresponding torvum unigenes is reported in additional file  <dig>  in figure  <dig>  the most specific enriched terms are combined in a chart.
among enriched go terms, several fall within a generalized biotic stress, namely “cell wall macromolecule metabolic processes”, “extracellular region”, “response to stress”, “polysaccharide catabolic process” and “response to fungus”.
further enriched go terms, including chitin and isoprenoid –associated processes appear of special interest within the context of nematode infection and will be analyzed in more detail.
chip-extension to eggplant
while the focus of this study was to investigate torvum defense responses upon nematode infection, we reasoned that the custom chip designed in this work may as well provide valuable complementary information concerning eggplant, a phylogenetically close, nematode-susceptible species.
eggplants are most seriously affected by meloidogyne, when plants of the breeding line 1f <dig> were infected with root knot nematodes a very high susceptibility with rg-index =  <dig> and em =  <dig> was scored.
to our knowledge, no genome-wide expression studies have been published to date for nematode-challenged eggplant.
heterologous hybridizations are based on interrogation of a chip specifically designed for a species  with transcripts from a phylogenetically-related species .
heterologous hybridizations have in many cases shown to be valuable resources for non-model species, especially when the primary and secondary species are closely related .
furthermore, in recent years, validation procedures have been developed allowing the definition in the secondary species of subsets of probes where expression data are predicted to be highly reliable  <cit> .
these procedures exploit the fact that the probes designed for a primary species  can be tested for matching to database transcripts of a secondary, interrogating species.
this allows assessment of expression data reliability and eventually definition of subsets of genes in the secondary species which can be evaluated with reduced concerns of expression artifacts.
to validate the heterologous expression data, we followed an approach conceptually similar to that presented in bagnaresi et al.
<cit> .
toward this end, we first pooled several expression  eggplant databases and queried the merged eggplant database with torvum chip probe sequences using local blastn at a relaxed stringency.
alignment results were parsed to filter probes based on alignment parameters expected to influence hybridization strength.
the following parameters were considered : i) ratio of alignment length to oligo length ; ii) maximum number of mismatches; and iii) distance from the start of oligo/transcript alignment to oligo 5’ end.
the rationale for the choice of these parameters is based on data presented in additional file  <dig>  the mean of all expression values  for all  <dig>  probes  was  <dig> .
when probes are selected by filtering with the above parameters, the mean of all expression values reaches the maximum for probes filtered for percent alignment = 100% and  <dig> mismatches  .
on the other hand, when less stringent values were tested, down to the very relaxed >= 30% alignment and up to  <dig> mismatches, mean expression values drop to about  <dig>  .
this variation in expression values is consistent with the fact that, when the homology between chip oligos and eggplant transcripts is high, high hybridization values are detectable.
additionally, the influence of the distance between the starting point of oligo alignment and the respective blast hit within 5′ end was monitored .
in fact, oligos are attached to the chip in the 3’ side and this causes steric hindrance in the crowded 3’ regions to interfere with hybridization  <cit> .
therefore, poor homology along oligo 3’ region against interrogating transcripts can be expected to be less influential on hybridization intensity, as confirmed in our data no effect could be detected when selection was made for distance of alignment from 3’ side .
we further checked that the variation in mean hybridization is not a mere consequence of the varying number of probes filtered at the different imposed stringencies.
a plot where a number of random probes corresponding to the number of probes resulting by setting stringency conditions is shown in additional file 8f.
as expected, no meaningful variation in signal strength is detectable in this case, ruling out that simply the number of probes, irrespective of probe vs. interrogating sequence homology, is influential.
all these observations are summarized in additional file  <dig>  where percent alignment, number of mismatches within the alignment and oligo alignment start position are plotted versus mean hybridization values and number of retained probes.
based on the above data, in order to define an high confidence pool of probes for eggplant, we selected a reasonable tradeoff between stringency and number of preserved probes: i) ratio of alignment length to oligo length >=  <dig> ; ii) maximum number of mismatches <= 2; iii) start of oligo alignment within the first  <dig> bases from 5’ oligo end.
these parameters lead to  <dig>  retained oligos with a mean signal of  <dig> , a value well above the mean of  <dig> obtained when only probes with an alignment <= 40% were considered  and is still higher than  <dig>  the mean of all probes in the chip .
to finally verify that our probe validation procedure was beneficial, we computed the pearson’s product moment correlation values for eggplant samples.
the correlation increased in most cases  when comparing validated versus total probes  as expected by removal of probes where, due to the absence of a matching transcript target, random noise prevails leading to inconsistencies among replicates  <cit> .
pearson’s product moment correlation values for eggplant samples are compared.
overall comparison between torvum vs. eggplant degs
as for torvum samples, eggplant total rna samples were extracted from control and 14-d inoculated roots  and used for array hybridization.
in contrast with torvum, numerous galls and egg-masses were visible in eggplant at the end of the infection process confirming a successful establishment of the infection and reproduction.
degs calling using the same values as for torvum  resulted in  <dig>  genes over the entire  <dig>  chip gene set, a value approximately 3-fold larger than found for torvum genes .
when the  <dig>  validated genes were considered,  <dig> eggplant degs were obtained.
thus,  <dig>  and  <dig> % degs were respectively obtained if the validated or total gene sets are chosen.
only  <dig> and  <dig> of these eggplant deg were in common with torvum degs for total and validated dataset, respectively .
we further conducted go enrichment analysis with both the raw and validated eggplant dataset.
for validated dataset, prior to fisher’s test, non-validated genes were subtracted from test and reference sets.
no enriched go terms were revealed using either total or validated dataset.
eggplant responses to infection at  <dig> dpi appear to involve modulation of a substantial amount of genes whose large majority is distinct from torvum degs.
furthermore, such modulation appears more sparse and heterogeneous as indicated by a lack of go enrichment.
this scenario is likely attributable to successful infection in eggplant as well as to the late infection stage monitored .
in fact, it is expected that wide gene modulation  occurs due to the establishment of infection in eggplant.
indeed, compatible interaction involves complex rearrangements as development of multinucleate feeding sites, reallocation of nutritional resource to fulfill pathogen needs, root-galling circuitry and further morpho-physiological alterations to cope with enzyme-rich nematode secretions  <cit> .
indeed, more degs were found in compatible vs. incompatible interaction of meloydogine spp.
in cowpea hosts  <cit> .
the lack of go enrichment for eggplant deg as opposed to torvum deg may reflect a more targeted response of torvum genes towards activation of specific defense responses against nematodes.
in the following sections, the heatmaps of expression data where torvum and eggplant are compared include the information of eggplant validation .
this allows to evaluate eggplant expression data with additional reliability hints based on the existence in eggplant databases of a transcript with satisfactory homology to torvum probes.
it should be stressed, however, that the absence of a validated probe for eggplant may also indicate that the expression of this gene is torvum-specific; alternatively, the gene may be expressed at very low levels in eggplant causing its absence in expression repositories.
both of these information may be valuable to pinpoint torvum-specific expression responses.
chitinases
as shown in the bar chart in figure  <dig>  chitin binding, chitin-catabolic process and chitinase activity are enriched go terms in torvum degs.
figure  <dig> depicts an heatmap of the  <dig> torvum genes annotated with the go term ‘chitinase activity’ .
eight of these  <dig> genes are modulated in torvum .
six of these are induced by infection and cluster together .
intriguingly, only one probe  is among eggplant validated probes.
this finding, while inviting caution on eggplant expression data for this cluster, indicates that, to date, no other transcripts have been reported for eggplant in this cluster suggesting that these genes might be absent or expressed at very low levels in eggplant.
thus, those chitinases may represent a group of nematode-responsive genes whose presence and/or inducibility recruitment is an exquisite feature of nematode-challenged torvum.
chitinases have long been known to be involved in defense responses against nematodes .
in particular, soybean cultivars exhibiting enhanced resistance to m. incognita displayed earlier induction and higher overall chitinase activity when compared to susceptible cultivars  <cit> .
furthermore, chitinases transcripts have been found to be upregulated in transcript profiling experiments of m. incognita-challenged cotton roots  <cit>  although only the egg shell of nematodes contains chitin as a constituent, chitinases may interfere with nematode vitality at various stages of infection  <cit> .
these previous findings have prompted transgenesis approaches aiming at introducing exogenous chitinase in crops.
in preliminary attempts with chitinases from manduca sexta <cit>  and the fungus t. harzianum <cit> , chitinases did not provided enhanced resistance to nematodes when introduced in soybean and tobacco, respectively.
this may indicate that native, specific chitinase isoforms and/or appropriate conditions  as experienced by the native chitinases may be required to exert nematode protection activity.
for example, concomitant action of protease may be required to effectively disrupt nematode chitin  <cit> .
more recently, an active chitinase, pjchi- <dig>  has been identified in paecilomyces javanicus, a non-nematophagous fungus.
when expressed in tomato, pjchi- <dig> has been shown to reduce egg masses and repress the embryonic development of m. incognita <cit> .
pjchi- <dig> belongs to class v of chitinase family  <dig>  <cit>  and, intriguingly, tor5_c <dig> shows as best blast hit tobacco class v chitinase .
details on induced torvum chitinases are listed in table  <dig>  induced chitinases  appear to belong to distinct classes, probably reflecting that efficient chitinase activity may require a pool of various chitinase classes acting in concert.
identification at the sequence level of torvum nematode-induced chitinase may thus help to narrow down the choice of chitinase isoforms to effective ones for introgression into susceptible crops.
sesquiterpene biosynthetic enzymes
go analysis of torvum degs revealed an enrichment for ‘isoprenoid biosynthetic process’ term .
figure  <dig> provides an overview of expression pattern of  <dig> torvum genes annotated with this term.
noteworthy, torvum samples cluster together, while eggplant samples  appear to respond less coherently, as control and infected samples do not cluster together.
figure  <dig> details expression patterns for the  <dig> modulated torvum genes , while fold change data and the top blast hit for each of the modulated genes are reported in table  <dig>  intriguingly, within this group of ‘isoprenoid biosynthetic process’ degs, sesquiterpene and diterpenoid biosynthetic genes undergo opposite modulation, i.e.
induced the former and repressed the latter.
in plants, diterpenes  are produced via the plastid pathway , whereas sesquiterpenes  derive from cytosolic mevalonate pathway  <cit> .
based on blast2go annotation and blastx hits as reported in table  <dig>  tor5_c <dig> encodes for a cytp <dig> enzyme with as best hits several cyp <dig>  including epi-aristolochene  <dig>  dihydroxylase and premnaspirodiene oxygenase   <cit> .
this p <dig> enzyme catalyzes several hydroxylations for sesquiterpene substrates including phytoalexins as solavetivone.
in turn, tor5_rep_c <dig> and tor5_rep_c <dig> show both as best hit sesquiterpene synthase  <dig>  while tor5_c <dig> shows as best hit potato vetispiradiene synthase.
the tor5_rep_c <dig>  and tor5_c <dig>  are the only two genes within torvum degs present in the validated eggplant dataset although they are not differentially expressed.
hmgr plays a critical role in isoprenoid biosynthesis as catalyzes the first committed and rate limiting step in isoprenoid biosynthesis  <cit> .
thus, its presence in validated eggplant is a bona fide comparative clue towards the fact that the isoprenoid biosynthetic pathway is not up-regulated  following nematode infection in eggplant.
while an in-depth understanding of sesquiterpenoids biochemical reactions undergoing in nematode-challenged torvum will require obtaining full-length transcripts from the torvum transcripts, which in our dataset are limited by design to the 3’ region, the fact that the biosynthetic sesquiterpenoid pathway is stimulated points to sesquiterpenoids as critical effectors of torvum resistance mechanisms.
sesquiterpenoids include several compounds of well-established nematotoxic and nematostatic effects as gossypol, solavetivone, rishitin and lubumin  <cit> , and references therein.
gossypol is a very effective sesquiterpene aldehyde shown to cause resistance to meloydogine incognita <cit>  in cotton and solavetivone, rishitin and lubumin are bicyclic sesquiterpenoids expressed in solanaceous species as tomato and potato capable of causing nematostatic effects  <cit> .
high content of solavetivone  was found to be critical for conferring resistance to globodera rostochiensis <cit> .
rishitin was shown to be induced by plant-parasitic nematodes and higher rishitin contents in various potato species were associated to higher nematode resistance.
in particular, the action of rishitin or of a rishitin-like compound causing nematode to migrate away from plants, would be consistent with observed the poor establishment on parasites on torvum plants  <cit> .
it should be noted, however, that a substantial number of plant phytochemicals exhibit antinematodal activity and that evidence on the antinematodal activity of sesquiterpenoids is largely based on in vitro studies  <cit> .
few studies monitored the accumulation of sesquiterpenoids upon elicitation and, in only a subset of them, as in cotton roots  <cit> , elicitation was driven by m. incognita infection, more frequently, general and unspecific elicitors as arachidonic acid were employed.
a further work based on six cotton cultivars, did not support a correlation between resistance and sesquiterpenoid content  <cit> .
nevertheless, it should be noted that in the above mentioned elicitation studies, extraction and detection techniques were tailored to reveal a narrow range of compounds as low-molecular weight, non-polar chemicals  <cit> .
this approach prevents and unbiased, global overview of the whole spectrum of defense strategies mounted by the host to counteract infection, and thus hampers an assessment of their relative contribution to actual resistance.
prior to our report, no large-scale transcriptional profiling of m. incognita-infected resistant vs. susceptible genotypes, had revealed transcriptional induction of sesquiterpenoid genes  <cit> .
this fact may be attributable to the limited number of genes tested in previous transcriptional profiling studies, and/or to further factors as genotypes under investigation and sampling time.
transcriptional induction of sesquiterpenoid genes associated to the resistant genotype torvum, but not to the susceptible counterpart eggplant, strongly points to sesquiterpenoids as key effectors of torvum resistance against m. incognita and sheds lights on their activation mode and relative contribution to the battery of torvum defense strategies.
overall, it appears that torvum biosynthetic resources are channeled towards sesquiterpene biosynthesis at the expense of distinct biosynthetic branches.
further work based on the torvum sequences reported here will likely provide the tools for an in-depth understanding of torvum responses ultimately pointing to key phytoalexin products.
identification of torvum resistance gene analogs
several resistance genes against plant-parasitic nematodes have been cloned  <cit> .
among them the best-studied is the tomato gene mi, which exhibits a broad resistance pattern to root-knot nematodes as well as to phloem-feeding insects, including macrosiphum euphorbiae  and to the white fly bemisia tabaci.
further nematode r genes cloned are: hs1pro- <dig>  hero, gpa <dig>  gro1- <dig>  rhg <dig> and rhg4r <cit> .
several aspects of the interaction among torvum and meloydogine would be consistent with an early rejection mechanism as ensured by vertical resistance .
in the additional file  <dig> we report the  <dig> torvum transcripts with annotations referring to candidate r genes analogs .
among these,  <dig>  and  <dig>  showed enhanced expression in infected vs. control samples, respectively, in eggplant and torvum.
while no transcriptional modulation is strictly required for a r gene to be considered a candidate r gene  several active r genes show moderate level of pathogen responsiveness in terms of transcription , e.g.
xa <dig>  xa <dig>  <cit> .
thus, monitoring the expression patterns of torvum candidate r genes analogs  can help pointing to rgas of interest  <cit> .
table  <dig> enlists  <dig> torvum transcripts annotated as disease resistance genes showing an expression ratio  of at least  <dig> .
only one of these torvum induced transcripts has a validated counterpart in eggplant  pointing to major sequence divergence and/or lack of counterparts in eggplants for this set of induced torvum candidate resistance genes.
intriguingly, among the candidate induced rgas one shows as best hit a homologous to mi nematode resistance gene .
figure  <dig> depicts a multiple alignment and associated dendrogram encompassing selected torvum rgas and the most c-terminal  <dig> residues of known r genes .
protein alignment was made for torvum rgas by selecting the longest orf.
as expected, torvum sequences by design align to the most c-terminal regions of r genes  and only the most c-terminal 100– <dig> aa of alignments are shown.
despite the fact the aligned regions are the poorly conserved lrr  regions, several torvum transcripts show homology and cluster close to distinct r-prototype resistance genes and appear good candidates for future assessment of their role as true r genes.
qpcr validation of selected genes
in order to validate our microarray expression results, we choose six sequences  among both upregulated and downregulated deg of particular relevance as discussed above.
overall, despite the correlation among qpcr and array data  was non-significant according to pearson’s product–moment correlation , the direction of change in expression of qpcr and microarray was in agreement for all the tested transcripts .
CONCLUSIONS
we conducted a cost-effective global transcriptome profiling in solanum torvum, a non-model species, by combining ngs pyrosequencing and microarray technology.
as a first step, we generated a 3’ transcript catalogue for torvum by assembling 500– <dig> bp reads from a normalized library.
by limiting the sequencing to the 3’ region we improved average coverage while conserving specificity .
this catalogue represents a substantial advancement along characterization of torvum transcriptome, since even at the relaxed stringency of an 10- <dig> expect value more than 60% of torvum unigenes in our catalogue do not have blast hits in available torvum databases.
the catalogue was subsequently used to design a custom chip for profiling transcriptome changes as a consequence of nematode infection in nematode-resistant species torvum and the related nematode-susceptible species eggplant.
to assess expression results for the heterologous hybridization conducted with eggplant samples, we developed an in silico validation strategy which can be applied in general to extend the use of species-specific chips to samples from phylogenetically related species devoid of a chip.
this allows to define, for such heterologous hybridizations, pool of genes whose hybridization data are expected to be more reliable.
finally, the expression profiling of torvum responses to nematode infection reveals a focused upregulation of various classes of chitinases and, for the first time, of sesquiterpenoids biosynthetic genes and.
on the other hand, no such responses are detectable in eggplant, where a large but sparse and incoherent gene modulation occurs, probably as a consequence of successful establishment of infection.
the availability of the long sequence tags in torvum catalogue will allow precise identification of active nematocide/nematostatic compounds and associated enzymes posing the basis for the exploitation of these resistance mechanisms in other species.

 solanum torvum sw is worldwide employed as rootstock for eggplant cultivation because of its vigor and resistance/tolerance to heavy metal- and salt-contaminated soil and, specially, to the most serious soil-borne diseases. however, despite the fact that identification of the effectors of torvum resistance would be extremely valuable for other solanaceous crops, particularly eggplant, very little studies have been conducted to date regarding torvum resistance mechanism and have mainly dealt with its resistance to bacteria. this scenario is mostly attributable to the paucity of database information on torvum which, in turn is a consequence of the lack of high-throughput genomic tools ). few expression databases were available for torvum. only recently, unigenes from torvum cv. torubamubiga, a low cadmium -accumulating variety, have been obtained in the context of studies on cd acclimation process. this gene catalogue represents a significant improvement in terms of sequence information; however, given the current high-throughput standards, is unsuitable to pose the basis for global transcript profiling due to its small size. an approach to overcome such typical non-model species constraints would be undertaking an rna-sequencing approach, since no prior knowledge of transcript sequences is mandatory. an alternative is represented by microarray hybridizations, a robust and well-assessed technique, about ten-fold less expensive per sample when compared to the cheapest rna-seq approaches. microarray can be considered as a viable alternative to rna-seq provided that the number of available sequences for the species of interest is considered satisfactory. thus, in order to extend the number of probes for torvum, we combined 454-based pyrosequencing with microarrays as reported by. a further example of this strategy has been recently presented. root-knot nematodes, meloidogyne spp. determine substantial losses on horticultural and field crops. in the tropical and sub-tropical regions, crop production losses due to nematodes were estimated at % compared with % in developed countries. the american phytopathological society has reported that root-knot nematodes are the most common and destructive nematode pathogens, accounting for an estimated of 14% of all worldwide plant losses, which translates into almost $ billion dollars annually. meloidogyne incognita belongs to the order tylenchida, a very large and diverse group of nematodes, which contains a majority of the known plant parasitic species. representatives of this order have a worldwide distribution and are encountered more frequently than any other group of nematodes. within tylenchida, members of the family heteroderidae are by far the most damaging to world agriculture, among which the root knot nematodes and the cyst nematodes. to date, more than rkn species are described, and m. incognita is unquestionably the most important one in terms of distribution and damages. nematodes establish complex interactions with hosts, and, upon successful infection, cause the reprogramming of host cell developing structures as multinucleate giant feeding cells. more than proteins have been found to be secreted by m. incognita and several of these proteins are thought to play a role in modulating infection and reprogramming host metabolism. nematode resistant genes are an obvious topic of interest for crop protection. the first cloned nem-r gene was the sugar beet hs1pro- conferring resistance against the sugar beet cyst nematode. however, hs1pro- appears poorly related to typical plant r genes. several other nem-r genes belong to the nbs-lrr class of r-genes. with respect to the subclass of rkn r-genes, few resistance genes have been identified and only two have been cloned, namely mi- from tomato and, very recently, ma from the myrobalan plum prunus cerasifera. both mi- and ma confer broad-spectrum resistance against several root knot nematode. not only a single pathogen-related gene in response to nematodes attack is described in the literature. more than one major gene is often involved in the resistance mechanisms commonly referred as horizontal resistance. wang et al. identified one major recessive gene with strong additive effect against m. incognita in cotton, together with a major qtl with strong dominant effect in different crosses using different resistance sources. transgressive segregation was previously documented from the same authors as an epistatic interaction that strengthened the resistance phenotype. the determinants of torvum resistance to nematodes are yet to be identified, and, in particular, the contribution of a specific gene-for-gene interaction rather than an horizontal, quantitative resistance, or even a “non-host” resistance phenomena is far from being clear. a number of studies have investigated transcriptional changes of m. incognita-challenged compatible and incompatible plants in a variety of species. two early studies described the transcript profiling of about transporter genes in a thaliana and of selected genes in tomato. the latter work was carried out on resistant and susceptible tomato genotypes and, by comparing transcript abundance patterns over a time-course, several clusters of genes associated to compatible and incompatible interactions were accurately defined. furthermore, a glycosyltransferase was shown to be necessary for conferring the resistant phenotype. a larger number of genes was tested by jammes et al. in arabidopsis, using giant cell-enriched root tissues infected by m. incognita along three time-points. about genes were called as differentially expressed between uninfected root tissues and galls at different developmental stages. major classes of modulated genes included metabolism-associated genes and transcription factors. a substantial down-regulation of defense genes, including phenylpropanoids, was found. more recently, the expression profiling of about genes from est libraries obtained from m. incognita-challenged resistant and susceptible cotton varieties, revealed differential modulation in resistant and susceptible genotypes of genes falling in the broad categories of pathogen recognition, signal transduction and pathogen defense. this latter category included superoxide dismutases, chitinases and isoflavone reductases. a further recent work have described the changes in the soybean transcriptome using the affymetrix® soybean genome array in galls formed in m. incognita-challenged roots at days and weeks after infection. genes encoding enzymes involved in carbohydrate and cell wall metabolism, cell-cycle control and plant defense were significantly modulated. among plant defense genes, several lipoxygenases, pathogenesis-related proteins and phenylpropanoid genes were found to be differentially expressed. the aim of this work was, by global transcriptome profiling, getting insights on the mechanisms underpinning torvum resistance against m. incognita. toward this end, we deployed an approach which takes advantage of both next generation sequencing and microarray techniques. in particular, we targeted previously uncharacterized transcripts by rna-seq and exploited reliability and cost-effectiveness of well established microarray technologies for transcript quantification. we also explored the “chip extension” procedure, an approach allowing to increase the reliability of heterologous hybridizations by defining subset of probes less likely to be prone to expression artifacts. the availability of a 3′ transcript catalogue for torvum and transcript profiling upon nematode infection provides molecular tools for identifying torvum resistance mechanism.  de novo assembly since only unigenes from torvum are available to date, we undertook an rna-seq pyrosequencing approach to extend the number of torvum genes and thus pose the basis to perform a global transcriptome profiling in a cost-effective manner. according to we produced a comprehensive normalized catalogue of the 3’ mrna regions tailored at the generation of a custom chip. as starting material, torvum roots were subjected to a wide range of environmental stresses to maximize the number of expressed genes. sequencing was confined to the 3’ in order to: i) minimize the number of reads mapping to the same transcript but assembling in different contigs due to the lack of uniform coverage in low-abundance transcripts; ii) allow for designing highly specific probes by encompassing 3’ regions which are known to be subjected to lower selection pressure. the library was normalized as this treatment has been shown to greatly improve rare transcript coverage as well as other quality features. one-quarter of the plate was used to sequence the normalized 3’cdna library yielding reads with an average length of bases for a total of, bases. assuming an average number of transcripts of with an average length of kb and thus a transcriptome length of mb, an yield of mb confined to 500– bp at the 3’ regions represents a coverage of about 4x. this coverage, while not exhaustive, poses the basis, at least within the context of a normalized library, for quantification of a high number of transcripts in comparison to the few thousands of unigenes to date available for torvum. de novo assembly of torvum reads was undertaken with mira.. the assembly led to contigs plus singletons. various parameters including n were calculated to describe the average size of the contigs. in additional file the distribution of contig length and contig coverage is shown. as a consequence of our 3’ sequencing design, the most enriched bin for unigenes was, as expected, in the 500– bp region. contig coverage was relatively uniform as a result of the normalization step. to further assess the assembly, we compared the contigs plus singletons against selected public assemblies, including the recently released unigene catalogue from solanum torvum cv. torubamubiga. further queried databases were the current releases in tc database from the phylogenetically related species eggplant, tobacco, tomato, potato and pepper. at last, we tested arabidopsis as a phylogenetically distant reference. as expected, a limited number of torvum queries showed hits against the small torvum torubamubiga dataset, while the larger tc solanaceous datasets as potato, tomato, eggplant and tobacco exhibited between and 80% hits. however, when these results are corrected for the number of entries of the queried databases, eggplant and s. torvum cv. torubamubiga clearly emerged as the most correlated to torvum database. on the other hand, the phylogenetically distant species arabidopsis shows a barely detectable ratio of percent hits to database extent. overall, the blast data closely mirror known phylogenetic relationships within solanaceous species with torvum having its closest counterpart in eggplant and, in order of decreasing relatedness, potato, tomato, pepper and tobacco. noteworthy, at an expect value of 10- more than 60% of torvum unigenes had no hits against cv. torubamubiga database, indicating that a majority of torvum unigenes in our catalogue are not represented in the small torubamubiga dataset. on the other hand, when torubamubiga database was queried against our torvum unigene catalogue, only 18% of the torubamubiga unigenes had no hits, indicating that our torvum transcript tags catalogue is likely to represent the most complete dataset for torvum available to date. custom chip design oligoarray software was used to compute gene-specific oligonucleotides corresponding to torvum unigenes. oligoarray output, besides microarray design, provides hints on the quality of input sequences by declaring how many specific probes can be designed based on input sequences. about 80% of oligos turned out to be specific for one torvum unigene, while 15% oligos were specific for 1– unigenes, indicating efficient normalization and substantial lack of redundancy in the torvum unigene set. a final filtering step over torvum unigenes was conducted to exclude the less specific probes. this also allowed to contain the number of probes in the chip to maximum, consistent with a triplicate probe layout in the 90k-features combimatrix chip design. the final layout consisted in probes representative of contigs and probes derived from singletons. the significant reduction of singleton representation in the final chip layout likely reflects the constraints experienced by oligoarray software in designing specific probes for singletons due to the shorter length and/or lower qualities of sequences when compared to contigs. differentially expressed genes in nematode-infected torvum two-months-old torvum plants were infected with meloidogyne incognita and left to proceed for days. neither root-galls nor egg-masses were never collected on torvum stained root even in longer infection stages. torvum rna samples were used for array hybridizations. pearson correlation coefficients for biological replicates were all above. genes were considered differentially expressed if exhibiting at least a 2-fold change and a false discovery rate <=. figure  depicts transcript abundance values in control vs. infected roots as ma-plot. differentially expressed genes fulfilling the two criteria are plotted in red. the full list of deg accompanied by expression ratio, fdr values, blast hits, blast2go annotations and go mappings are reported in additional file annotation of torvum gene catalogue the software suite blast2go was used to annotate torvum unigenes. as a first blast step, we run blastx against ncbi non-redundant database using as query the unigenes included in the chip layout for which an hybridization signal could be obtained. a total of go annotations were retrieved. the full catalogue of torvum genes including blast2go annotations and complementary information are available in additional file while the full set of torvum unigene sequences is provided in fasta format in additional file go enrichment analysis of torvum deg genes major expression trends in infected torvum were investigated to test whether the degs could be grouped in some enriched go terms. towards this end, we used as a reference set the annotations obtained with blast2go and as test set the degs. the fisher’s exact test results for the entire set of enriched go terms and corresponding torvum unigenes is reported in additional file in figure  the most specific enriched terms are combined in a chart. among enriched go terms, several fall within a generalized biotic stress, namely “cell wall macromolecule metabolic processes”, “extracellular region”, “response to stress”, “polysaccharide catabolic process” and “response to fungus”. further enriched go terms, including chitin and isoprenoid –associated processes appear of special interest within the context of nematode infection and will be analyzed in more detail. chip-extension to eggplant while the focus of this study was to investigate torvum defense responses upon nematode infection, we reasoned that the custom chip designed in this work may as well provide valuable complementary information concerning eggplant, a phylogenetically close, nematode-susceptible species. eggplants are most seriously affected by meloidogyne, when plants of the breeding line 1f were infected with root knot nematodes a very high susceptibility with rg-index = and em = was scored. to our knowledge, no genome-wide expression studies have been published to date for nematode-challenged eggplant. heterologous hybridizations are based on interrogation of a chip specifically designed for a species with transcripts from a phylogenetically-related species. heterologous hybridizations have in many cases shown to be valuable resources for non-model species, especially when the primary and secondary species are closely related. furthermore, in recent years, validation procedures have been developed allowing the definition in the secondary species of subsets of probes where expression data are predicted to be highly reliable. these procedures exploit the fact that the probes designed for a primary species can be tested for matching to database transcripts of a secondary, interrogating species. this allows assessment of expression data reliability and eventually definition of subsets of genes in the secondary species which can be evaluated with reduced concerns of expression artifacts. to validate the heterologous expression data, we followed an approach conceptually similar to that presented in bagnaresi et al.. toward this end, we first pooled several expression eggplant databases and queried the merged eggplant database with torvum chip probe sequences using local blastn at a relaxed stringency. alignment results were parsed to filter probes based on alignment parameters expected to influence hybridization strength. the following parameters were considered: i) ratio of alignment length to oligo length; ii) maximum number of mismatches; and iii) distance from the start of oligo/transcript alignment to oligo 5’ end. the rationale for the choice of these parameters is based on data presented in additional file the mean of all expression values for all probes was. when probes are selected by filtering with the above parameters, the mean of all expression values reaches the maximum for probes filtered for percent alignment = 100% and mismatches. on the other hand, when less stringent values were tested, down to the very relaxed >= 30% alignment and up to mismatches, mean expression values drop to about. this variation in expression values is consistent with the fact that, when the homology between chip oligos and eggplant transcripts is high, high hybridization values are detectable. additionally, the influence of the distance between the starting point of oligo alignment and the respective blast hit within 5′ end was monitored. in fact, oligos are attached to the chip in the 3’ side and this causes steric hindrance in the crowded 3’ regions to interfere with hybridization. therefore, poor homology along oligo 3’ region against interrogating transcripts can be expected to be less influential on hybridization intensity, as confirmed in our data no effect could be detected when selection was made for distance of alignment from 3’ side. we further checked that the variation in mean hybridization is not a mere consequence of the varying number of probes filtered at the different imposed stringencies. a plot where a number of random probes corresponding to the number of probes resulting by setting stringency conditions is shown in additional file 8f. as expected, no meaningful variation in signal strength is detectable in this case, ruling out that simply the number of probes, irrespective of probe vs. interrogating sequence homology, is influential. all these observations are summarized in additional file where percent alignment, number of mismatches within the alignment and oligo alignment start position are plotted versus mean hybridization values and number of retained probes. based on the above data, in order to define an high confidence pool of probes for eggplant, we selected a reasonable tradeoff between stringency and number of preserved probes: i) ratio of alignment length to oligo length >=; ii) maximum number of mismatches <= 2; iii) start of oligo alignment within the first bases from 5’ oligo end. these parameters lead to retained oligos with a mean signal of, a value well above the mean of obtained when only probes with an alignment <= 40% were considered and is still higher than the mean of all probes in the chip. to finally verify that our probe validation procedure was beneficial, we computed the pearson’s product moment correlation values for eggplant samples. the correlation increased in most cases when comparing validated versus total probes as expected by removal of probes where, due to the absence of a matching transcript target, random noise prevails leading to inconsistencies among replicates. pearson’s product moment correlation values for eggplant samples are compared. overall comparison between torvum vs. eggplant degs as for torvum samples, eggplant total rna samples were extracted from control and 14-d inoculated roots and used for array hybridization. in contrast with torvum, numerous galls and egg-masses were visible in eggplant at the end of the infection process confirming a successful establishment of the infection and reproduction. degs calling using the same values as for torvum resulted in genes over the entire chip gene set, a value approximately 3-fold larger than found for torvum genes. when the validated genes were considered, eggplant degs were obtained. thus, and % degs were respectively obtained if the validated or total gene sets are chosen. only and of these eggplant deg were in common with torvum degs for total and validated dataset, respectively. we further conducted go enrichment analysis with both the raw and validated eggplant dataset. for validated dataset, prior to fisher’s test, non-validated genes were subtracted from test and reference sets. no enriched go terms were revealed using either total or validated dataset. eggplant responses to infection at dpi appear to involve modulation of a substantial amount of genes whose large majority is distinct from torvum degs. furthermore, such modulation appears more sparse and heterogeneous as indicated by a lack of go enrichment. this scenario is likely attributable to successful infection in eggplant as well as to the late infection stage monitored. in fact, it is expected that wide gene modulation occurs due to the establishment of infection in eggplant. indeed, compatible interaction involves complex rearrangements as development of multinucleate feeding sites, reallocation of nutritional resource to fulfill pathogen needs, root-galling circuitry and further morpho-physiological alterations to cope with enzyme-rich nematode secretions. indeed, more degs were found in compatible vs. incompatible interaction of meloydogine spp. in cowpea hosts. the lack of go enrichment for eggplant deg as opposed to torvum deg may reflect a more targeted response of torvum genes towards activation of specific defense responses against nematodes. in the following sections, the heatmaps of expression data where torvum and eggplant are compared include the information of eggplant validation. this allows to evaluate eggplant expression data with additional reliability hints based on the existence in eggplant databases of a transcript with satisfactory homology to torvum probes. it should be stressed, however, that the absence of a validated probe for eggplant may also indicate that the expression of this gene is torvum-specific; alternatively, the gene may be expressed at very low levels in eggplant causing its absence in expression repositories. both of these information may be valuable to pinpoint torvum-specific expression responses. chitinases as shown in the bar chart in figure  chitin binding, chitin-catabolic process and chitinase activity are enriched go terms in torvum degs. figure  depicts an heatmap of the torvum genes annotated with the go term ‘chitinase activity’. eight of these genes are modulated in torvum. six of these are induced by infection and cluster together. intriguingly, only one probe is among eggplant validated probes. this finding, while inviting caution on eggplant expression data for this cluster, indicates that, to date, no other transcripts have been reported for eggplant in this cluster suggesting that these genes might be absent or expressed at very low levels in eggplant. thus, those chitinases may represent a group of nematode-responsive genes whose presence and/or inducibility recruitment is an exquisite feature of nematode-challenged torvum. chitinases have long been known to be involved in defense responses against nematodes. in particular, soybean cultivars exhibiting enhanced resistance to m. incognita displayed earlier induction and higher overall chitinase activity when compared to susceptible cultivars. furthermore, chitinases transcripts have been found to be upregulated in transcript profiling experiments of m. incognita-challenged cotton roots although only the egg shell of nematodes contains chitin as a constituent, chitinases may interfere with nematode vitality at various stages of infection. these previous findings have prompted transgenesis approaches aiming at introducing exogenous chitinase in crops. in preliminary attempts with chitinases from manduca sexta and the fungus t. harzianum, chitinases did not provided enhanced resistance to nematodes when introduced in soybean and tobacco, respectively. this may indicate that native, specific chitinase isoforms and/or appropriate conditions as experienced by the native chitinases may be required to exert nematode protection activity. for example, concomitant action of protease may be required to effectively disrupt nematode chitin. more recently, an active chitinase, pjchi- has been identified in paecilomyces javanicus, a non-nematophagous fungus. when expressed in tomato, pjchi- has been shown to reduce egg masses and repress the embryonic development of m. incognita. pjchi- belongs to class v of chitinase family and, intriguingly, tor5_c shows as best blast hit tobacco class v chitinase. details on induced torvum chitinases are listed in table  induced chitinases appear to belong to distinct classes, probably reflecting that efficient chitinase activity may require a pool of various chitinase classes acting in concert. identification at the sequence level of torvum nematode-induced chitinase may thus help to narrow down the choice of chitinase isoforms to effective ones for introgression into susceptible crops. sesquiterpene biosynthetic enzymes go analysis of torvum degs revealed an enrichment for ‘isoprenoid biosynthetic process’ term. figure  provides an overview of expression pattern of torvum genes annotated with this term. noteworthy, torvum samples cluster together, while eggplant samples appear to respond less coherently, as control and infected samples do not cluster together. figure  details expression patterns for the modulated torvum genes, while fold change data and the top blast hit for each of the modulated genes are reported in table  intriguingly, within this group of ‘isoprenoid biosynthetic process’ degs, sesquiterpene and diterpenoid biosynthetic genes undergo opposite modulation, i.e. induced the former and repressed the latter. in plants, diterpenes are produced via the plastid pathway, whereas sesquiterpenes derive from cytosolic mevalonate pathway. based on blast2go annotation and blastx hits as reported in table  tor5_c encodes for a cytp enzyme with as best hits several cyp including epi-aristolochene dihydroxylase and premnaspirodiene oxygenase. this p enzyme catalyzes several hydroxylations for sesquiterpene substrates including phytoalexins as solavetivone. in turn, tor5_rep_c and tor5_rep_c show both as best hit sesquiterpene synthase while tor5_c shows as best hit potato vetispiradiene synthase. the tor5_rep_c and tor5_c are the only two genes within torvum degs present in the validated eggplant dataset although they are not differentially expressed. hmgr plays a critical role in isoprenoid biosynthesis as catalyzes the first committed and rate limiting step in isoprenoid biosynthesis. thus, its presence in validated eggplant is a bona fide comparative clue towards the fact that the isoprenoid biosynthetic pathway is not up-regulated following nematode infection in eggplant. while an in-depth understanding of sesquiterpenoids biochemical reactions undergoing in nematode-challenged torvum will require obtaining full-length transcripts from the torvum transcripts, which in our dataset are limited by design to the 3’ region, the fact that the biosynthetic sesquiterpenoid pathway is stimulated points to sesquiterpenoids as critical effectors of torvum resistance mechanisms. sesquiterpenoids include several compounds of well-established nematotoxic and nematostatic effects as gossypol, solavetivone, rishitin and lubumin, and references therein. gossypol is a very effective sesquiterpene aldehyde shown to cause resistance to meloydogine incognita in cotton and solavetivone, rishitin and lubumin are bicyclic sesquiterpenoids expressed in solanaceous species as tomato and potato capable of causing nematostatic effects. high content of solavetivone was found to be critical for conferring resistance to globodera rostochiensis. rishitin was shown to be induced by plant-parasitic nematodes and higher rishitin contents in various potato species were associated to higher nematode resistance. in particular, the action of rishitin or of a rishitin-like compound causing nematode to migrate away from plants, would be consistent with observed the poor establishment on parasites on torvum plants. it should be noted, however, that a substantial number of plant phytochemicals exhibit antinematodal activity and that evidence on the antinematodal activity of sesquiterpenoids is largely based on in vitro studies. few studies monitored the accumulation of sesquiterpenoids upon elicitation and, in only a subset of them, as in cotton roots, elicitation was driven by m. incognita infection, more frequently, general and unspecific elicitors as arachidonic acid were employed. a further work based on six cotton cultivars, did not support a correlation between resistance and sesquiterpenoid content. nevertheless, it should be noted that in the above mentioned elicitation studies, extraction and detection techniques were tailored to reveal a narrow range of compounds as low-molecular weight, non-polar chemicals. this approach prevents and unbiased, global overview of the whole spectrum of defense strategies mounted by the host to counteract infection, and thus hampers an assessment of their relative contribution to actual resistance. prior to our report, no large-scale transcriptional profiling of m. incognita-infected resistant vs. susceptible genotypes, had revealed transcriptional induction of sesquiterpenoid genes. this fact may be attributable to the limited number of genes tested in previous transcriptional profiling studies, and/or to further factors as genotypes under investigation and sampling time. transcriptional induction of sesquiterpenoid genes associated to the resistant genotype torvum, but not to the susceptible counterpart eggplant, strongly points to sesquiterpenoids as key effectors of torvum resistance against m. incognita and sheds lights on their activation mode and relative contribution to the battery of torvum defense strategies. overall, it appears that torvum biosynthetic resources are channeled towards sesquiterpene biosynthesis at the expense of distinct biosynthetic branches. further work based on the torvum sequences reported here will likely provide the tools for an in-depth understanding of torvum responses ultimately pointing to key phytoalexin products. identification of torvum resistance gene analogs several resistance genes against plant-parasitic nematodes have been cloned. among them the best-studied is the tomato gene mi, which exhibits a broad resistance pattern to root-knot nematodes as well as to phloem-feeding insects, including macrosiphum euphorbiae and to the white fly bemisia tabaci. further nematode r genes cloned are: hs1pro- hero, gpa gro1- rhg and rhg4r. several aspects of the interaction among torvum and meloydogine would be consistent with an early rejection mechanism as ensured by vertical resistance. in the additional file we report the torvum transcripts with annotations referring to candidate r genes analogs. among these, and showed enhanced expression in infected vs. control samples, respectively, in eggplant and torvum. while no transcriptional modulation is strictly required for a r gene to be considered a candidate r gene several active r genes show moderate level of pathogen responsiveness in terms of transcription, e.g. xa xa. thus, monitoring the expression patterns of torvum candidate r genes analogs can help pointing to rgas of interest. table  enlists torvum transcripts annotated as disease resistance genes showing an expression ratio of at least. only one of these torvum induced transcripts has a validated counterpart in eggplant pointing to major sequence divergence and/or lack of counterparts in eggplants for this set of induced torvum candidate resistance genes. intriguingly, among the candidate induced rgas one shows as best hit a homologous to mi nematode resistance gene. figure  depicts a multiple alignment and associated dendrogram encompassing selected torvum rgas and the most c-terminal residues of known r genes. protein alignment was made for torvum rgas by selecting the longest orf. as expected, torvum sequences by design align to the most c-terminal regions of r genes and only the most c-terminal 100– aa of alignments are shown. despite the fact the aligned regions are the poorly conserved lrr regions, several torvum transcripts show homology and cluster close to distinct r-prototype resistance genes and appear good candidates for future assessment of their role as true r genes. qpcr validation of selected genes in order to validate our microarray expression results, we choose six sequences among both upregulated and downregulated deg of particular relevance as discussed above. overall, despite the correlation among qpcr and array data was non-significant according to pearson’s product–moment correlation, the direction of change in expression of qpcr and microarray was in agreement for all the tested transcripts.  we conducted a cost-effective global transcriptome profiling in solanum torvum, a non-model species, by combining ngs pyrosequencing and microarray technology. as a first step, we generated a 3’ transcript catalogue for torvum by assembling 500– bp reads from a normalized library. by limiting the sequencing to the 3’ region we improved average coverage while conserving specificity. this catalogue represents a substantial advancement along characterization of torvum transcriptome, since even at the relaxed stringency of an 10- expect value more than 60% of torvum unigenes in our catalogue do not have blast hits in available torvum databases. the catalogue was subsequently used to design a custom chip for profiling transcriptome changes as a consequence of nematode infection in nematode-resistant species torvum and the related nematode-susceptible species eggplant. to assess expression results for the heterologous hybridization conducted with eggplant samples, we developed an in silico validation strategy which can be applied in general to extend the use of species-specific chips to samples from phylogenetically related species devoid of a chip. this allows to define, for such heterologous hybridizations, pool of genes whose hybridization data are expected to be more reliable. finally, the expression profiling of torvum responses to nematode infection reveals a focused upregulation of various classes of chitinases and, for the first time, of sesquiterpenoids biosynthetic genes and. on the other hand, no such responses are detectable in eggplant, where a large but sparse and incoherent gene modulation occurs, probably as a consequence of successful establishment of infection. the availability of the long sequence tags in torvum catalogue will allow precise identification of active nematocide/nematostatic compounds and associated enzymes posing the basis for the exploitation of these resistance mechanisms in other species. 
a features custom combimatrix chip was then designed and microarray hybridizations were conducted for both control and dpi with meloidogyne incognita-infected roots samples resulting in differentially expressed genes. de novo assembly yielded contigs and singletons. the little information on solanum torvum resistance mechanisms, is mostly attributable to the lack of genomic tools as well as to the paucity of database information limiting high-throughput expression studies in torvum. the availability of the long sequence tags in s. torvum catalogue will allow precise identification of active nematocide/nematostatic compounds and associated enzymes posing the basis for exploitation of these resistance mechanisms in other species. solanum torvum sw is worldwide employed as rootstock for eggplant cultivation because of its vigour and resistance/tolerance to the most serious soil-borne diseases as bacterial, fungal wilts and root-knot nematodes. as a first step towards transcriptome profiling of torvum inoculated with the nematode m. incognita, we built a torvum 3’ transcript catalogue. by combining pyrosequencing and microarray technology we were able to conduct a cost-effective global transcriptome profiling in a non-model species. an in-silico validation strategy was developed based on assessment of sequence similarity among torvum probes and eggplant expressed sequences available in public repositories. in addition, the development of an in silico validation strategy allowed to further extend the use of the custom chip to a related species and to assess by comparison the expression of selected genes without major concerns of artifacts. the genes identified from s. torvum catalogue, bearing high similarity to known nematode resistance genes, were further investigated in view of their potential role in the nematode resistance mechanism. the expression profiling of s. torvum responses to nematode infection points to sesquiterpenoids and chitinases as major effectors of nematode resistance. go term enrichment analyses with the torvum deg revealed enhancement of several processes as chitin catabolism and sesquiterpenoids biosynthesis, while no go term enrichment was found with eggplant deg.
 solanum torvum sw is worldwide employed as rootstock for eggplant cultivation because of its vigour and resistance/tolerance to the most serious soil-borne diseases as bacterial, fungal wilts and root-knot nematodes. the little information on solanum torvum resistance mechanisms, is mostly attributable to the lack of genomic tools as well as to the paucity of database information limiting high-throughput expression studies in torvum.  as a first step towards transcriptome profiling of torvum inoculated with the nematode m. incognita, we built a torvum 3’ transcript catalogue. one-quarter of a full run resulted in quality-filtered reads. de novo assembly yielded contigs and singletons. similarity searches of the s. torvum transcript tags catalogue produced annotations. a features custom combimatrix chip was then designed and microarray hybridizations were conducted for both control and dpi with meloidogyne incognita-infected roots samples resulting in differentially expressed genes. we also tested the chip with samples from the phylogenetically-related nematode-susceptible eggplant species solanum melongena. an in-silico validation strategy was developed based on assessment of sequence similarity among torvum probes and eggplant expressed sequences available in public repositories. go term enrichment analyses with the torvum deg revealed enhancement of several processes as chitin catabolism and sesquiterpenoids biosynthesis, while no go term enrichment was found with eggplant deg. the genes identified from s. torvum catalogue, bearing high similarity to known nematode resistance genes, were further investigated in view of their potential role in the nematode resistance mechanism.  by combining pyrosequencing and microarray technology we were able to conduct a cost-effective global transcriptome profiling in a non-model species. in addition, the development of an in silico validation strategy allowed to further extend the use of the custom chip to a related species and to assess by comparison the expression of selected genes without major concerns of artifacts. the expression profiling of s. torvum responses to nematode infection points to sesquiterpenoids and chitinases as major effectors of nematode resistance. the availability of the long sequence tags in s. torvum catalogue will allow precise identification of active nematocide/nematostatic compounds and associated enzymes posing the basis for exploitation of these resistance mechanisms in other species. torvumnematode resistance pyrosequencingmicroarrayheterologous hybridizations 