The soybean line PI 595099 presents high resistance to the main RKNs, as well as to H. glycines. The low-level of galls and egg masses observed in PI 595099 in the present study indicates that this genotype has a resistance mechanism that limited RKN infection, which is consistent with preceding histological report [4, 27, 28]. Previous molecular studies using this soybean line showed differential expression of a set of genes when inoculated with M. javanica in comparison to the mock-inoculated [29, 30]. However, they were not fully able to provide insights into the mechanisms operating in the host’s resistance. To provide an extensive characterization of the distinctive responses of PI 595099-RKN interaction we present large-scale transcript sequence data generated by NGS technology.
PI 595099 comparative transcriptome analysis reveals complex stress signaling upon RKN infection
The gene expression profile analysis carried out using next generation sequencing data and bioinformatics analysis shows that PI595099 triggers a broad set of plant stress genes in response to nematode invasion between 0 to 8 DAI (Additional file 4). Our findings indicate that several members of Lipid transfer proteins (LTPs) family, also known as PR–14, which are important players in the general plant stress response, were induced during pathogen infection. Recently, it has been shown that LTPs are also involved in salt and drought stress , and have been implanted in Ca2+ signaling networks, due to the presence of a calmodulin binding region within this protein family . This particular observation is in contrast to what was observed using microarrays to study the gene expression of a susceptible soybean cultivar and M. incognita, where PR–1, PR–2 and PR–5 protein families increased in expression, but not PR–14 members. Most noteworthy, the induction of expression of genes encoding PR-1 or plant defensin (PDF1.2), globally known as marker genes to study SA-dependent and ET/JA-mediated defense responses, did not take place in our analysis.
Another protein class that was identified include Arabinogalactan-proteins (AGPs), which contain a glycosylphosphatidylinositol-(GPI-) membrane anchor and have established roles in plant development and signaling , specifically cellulose deposition and cell wall plasticity . Furthermore, AGPs were seen to be implicated in the production of mucilage during the incompatible interaction with parasitic plant  and their expression was found to increase in response to salt stress . AGPs involvement in the protection against nematode infection has not previously been established and the observed up–regulation of AGPs can be the result of the plant’s efforts to recover from stress and resumption of its growth. Proteins containing the BURP domain (PF03181) were also significantly up–regulated. Although there is no precise functional characterization of this large family, recent reports corroborate over-expression following cyst nematode infection in tomato  and following abiotic stresses such as abscisic acid and NaCl treatment .
As LTPs, AGPs and BURP are thought to be related to salt stress tolerance, they may be involved in the common denominator of our findings named the increase of ROS production, which notably also occurs in situations like water deficit or salinity [39, 40]. Furthermore, it has been shown that salt stress signaling shares several events with biotic defense in plants, including the formation of ROS and induction of JAZ/TIFY transcript levels, known as negative regulator of jasmonate signaling  also observed in the present study. Another interesting aspect concerning the suggestive overlapping between salt stress signaling and biotic defense in PI505999 may be the increase plant annexin abundance noted by the up-regulation of two genes putatively encoding members of this protein family (PF00191). Annexins abundance was already observed to increase in stress conditions like salinity, drought, metal stress, and exogenous abscisic acid and it was recently suggested that they could function in integrating ROS and Ca2+ in stress signaling .
The expression profiling analysis based on the associations between genes and their GO annotation also suggests a key role of glycosyltransferases, and gibberellin signal transduction proteins (incl. DELLA-like proteins, GA20ox and GA2ox) in the resistance reaction. In addition these results suggest the probable participation of these gene products in jasmonate signaling and redox homeostasis by mediating aspects of plant growth and biotic stress responses (Additional file 4). It clearly reinforces the recent findings demonstrating that hormonal signaling can also function as a mechanism regulating growth to adverse conditions, for example by the reduction in ROS levels [13, 26].
ROS generation as a defense against nematode infection
Activation of various oxidases and peroxidases in response to adverse environmental factors led to the production of ROS, comprising superoxide anion radical, singlet oxygen, hydrogen peroxide, and hydroxyl radicals. Respiratory burst oxidases (NADPH-dependent oxidases) are thought to be important sources of ROS in response to pathogen attack . A total of 17 members of this family were detected without sign of expression modulation. NADPH-oxidases activity has been also attributed to apoplastic peroxidases in some plant species challenged by pathogens and after elicitor treatment. Peroxibase  reports 19 records of soybean peroxidases with homology to respiratory burst oxidases but none of them were found with a sign of expression modulation in our study.
Alternatively, increased levels of ROS may also be a consequence of the action of plant hormones, altered sugar levels and fatty acids . Auxins were hypothesized to induce cell-specific ROS formation and affect cell antioxidant content in response to environmentally unfavorable conditions. Accordingly, cells of the quiescent centre can accumulate high auxin levels and contribute to the overproduction of ROS affecting the ROS/antioxidant balance in the root apical meristem. This mechanism of action comprises auxin binding to the TIR1 receptor leading to removal of transcriptional repression of a large array of genes possessing the Auxin Response Factor (ARF) signature and causing the generation of H2O2 and superoxide ions .
Apparently, high levels of auxins, mainly in the form of free indole-3-acetic acid (IAA), can arise in the interplay between IAA biosynthesis and conjugation in the maintenance of IAA gradients . In the inoculated sample we found an over-expressed gene (GH3.6) which the encoded enzyme functions in the synthesis of IAA-amino acid conjugates. Accordingly, the over-expression of GH3 might lead to the accumulation of IAA-Asp conjugates which is a potential mechanism for plant cells to cope with presence of auxin excess in its bioactive form . This GH3-mediated auxin homeostasis also was proposed as an essential constituent of auxin actions that regulate stress adaptation responses in plants . Furthermore, our study also found a number of up-regulated genes, encoding family domains associated with induction by the plant hormone auxin, such as AUX/IAA and small auxin-up RNA (SAUR).
Jasmonic acid (JA) and its methyl ester (methyl jasmonate, MeJa) were reported to enhance the production of ROS, especially H202, and to induce cell death synergistically with other plant hormones such as ethylene and salicylic acid . It also has been suggested that the amplitude and onset of elevated ROS levels in tolerant or resistant plant genotypes might behave antagonistically to events such as induction of JAZ/TIFY transcripts (jasmonate ZIM/tify-domain) and apoplastic alkalinization . In this scenario, jasmonate action might correlate with adaptive responses that modulate ROS accumulation instead of promoting the accumulation of ROS that could lead to cellular damage. This latter interpretation of jasmonate’s action as part of a defense reaction seems also to occur in the studied pathosystem, since we could observe enhanced activity of one gene coding for allene oxide cyclase (AOC) class of enzymes in the inoculated sample, which acts in the primary steps of jasmonate biosynthesis, and also observed the induction of two genes encoding JAZ/TIFY proteins. It is also noteworthy in our study that the production of jasmonates might be rate limited by the down-regulation of the acy-CoA oxidase (ACX) gene, which gene product is one of the core enzymes that catalyze the final steps of JA synthesis. ACX is also known as an H202-generating enzyme that acts into the peroxisome.
In parallel, an enhancement was observed of the activity of the enzyme ascorbate (ASC) oxidase, which is known to oxidize ascorbate to dehydroascorbate (DHA), a molecule able to directly interact with reduced glutathione (GSH) and thiol-containing proteins. ASC are potentially important components regulating redox-sensitive proteins via auxin functioning. Between the up-regulated genes containing multicopper oxidase domains, we observed two genes encoding the enzyme ASC oxidase (EC 184.108.40.206). Thiol-containing proteins were also found up-regulated in the infected sample at significant expression levels.
Antioxidant status as part of defense against nematode infection and signaling pathways
Given the over–representation of genes containing various oxidases and peroxidases domains in the infected sample, it was done a more detailed analysis of transcripts related to ROS antioxidant activity since the ROS release may in turn induce ROS scavengers and other protective mechanisms.
Peroxidases are key player in the detoxification of reactive oxygen species during cellular metabolism and oxidative stress. The annotation of all up–regulated genes containing the peroxidase domain reveals that they were found to belong to class III plant peroxidases (EC 220.127.116.11). This class is known to participate in many different plant processes, such as auxin metabolism, cell wall elongation and stiffening and protection against pathogens . Recently it was demonstrated that these class III peroxidases are located at the tonoplast and plasma membrane and are able to catalyze the reduction of hydrogen peroxide by taking electrons to various donor molecules, such as phenolic compounds, lignin precursors, auxin or secondary metabolites .
Other genes encoding enzymes with known antioxidant activity, such as catalases and ascorbate peroxidases were found in our survey, but without sign of expression modulation. Only one germin-like protein coding gene with putative superoxide dismutase (SOD) activity was found up-regulated in the infected sample, as well only one gene coding for glutathione peroxidase enzyme. Up-regulation of gene expression of SNARE complex proteins and SYP111, SYP121 and SYP132 members of SYP1 of plasma membrane-localized syntaxins family  was found in our study. This suggest that further investigations on the intracellular localization of ROS is needed to elucidate whether membrane trafficking is an important characteristic in cellular responses to nematode invasion.
Our study also showed that transcriptional activity of glutathione S-transferases (GST) genes increased in the inoculated sample, as well as many PR protein encoding genes, mainly group 5 (PR-5; Pfam: PF00314) and LTPs (PR-14; Pfam: PF00234), and a phenylalanine ammonia-lyase (PAL) gene (Pfam:PF00221; EC:18.104.22.168). GST is known to function in hydroperoxide detoxification through reduction of peroxides with the help of GSH and PR. PAL proteins were identified as inhibitors of H2O2 production . Taken together, these observations may suggest that the host plants have the ability to adapt very well to low activities of both catalase and ascorbate peroxidase by induction of other defense systems, probably by signaled induction.
Increasing evidence indicates that ROS function in plants also as signaling molecules involved in regulating development and pathogen defense responses [53, 54]. Pathways of ROS signaling are reported to participate in homeostatic regulation by antioxidant redox buffering, which provides robust protection against oxidative stress. The ability of GSH to act as redox buffer is one of the most important attributes of plant cells. In the cytoplasm, signaling linked to increased availability of ROS may be caused, limited, or mediated by changes in the redox buffering capacity. Therefore such way, any stimulus that perturbs cellular redox balance may serve as an inducer for a set of defense-related genes, including PR proteins. Key redox signaling components are thioredoxins (TRX) and glutaredoxins (GRX), which are reduced by ferredoxin, NADPH thioredoxin reductase (TR), or glutathione . Members of thiol-containing families were found at increased expression levels in the infected samples and may have an important role in the redox signal transduction. Another interesting finding is the significant up-regulation of one gene encoding an NADPH thioredoxin reductase (TR) enzyme (EC: 22.214.171.124). Recently, it was found that purified TRX can reduce oxidized glutathione (GSSG) to GSH in the presence of TR and NADPH in a reconstituted in vitro system in yeast .
Unlike the cytoplasm, the apoplast is deficient in glutathione and therefore its redox buffering capacity is considered weaker. However, the apoplast is considered crucial in facilitating ROS-mediated signaling by maintaining a balance of reduced and oxidized forms of ascorbate (ASC). This ascorbate-based system is reported as important in driving plasma membrane and tonoplast electron transport chains by influencing cell wall composition. Moreover, low apoplastic antioxidant buffering capacity establishes a steep redox gradient across both the plasma membrane and the tonoplast. Low buffering capacity also permits further reactions to be triggered by secondary oxidant-induced signaling events in the cell wall, such as release of small oligosaccharides that are generated during the breakdown of pectins (pectic polysaccharides) . Many differentially expressed genes encoding transferase enzymes (PF02458) involved in the biosynthesis of soluble phenolic compounds (PCs) were found, which were that accumulated in the inoculated sample.
A PAL gene found under up-regulation in our study encodes a key enzyme for the biosynthesis of anthocyanin and other PCs. Although it is widely recognized that PCs are involved in the H2O2 scavenging cascade in plant cells , it was only recently that their accumulation was postulated to form part of an integrated redox system, quenching ROS and contributing to stress tolerance [45, 58]. The model proposed to integrate PCs into a redox system is supposed to largely depend on cellular nucleotide sugar concentrations. Nucleotide sugars linked to a nucleotide-diphosphate (NDP-sugars) can serve as donor substrates for glycosyltransferases (GT) that transfer sugar to a wide range of acceptors and can directly affect bioactivity of diverse plant hormones, as well as defense-related small molecules . Transcriptomic analysis  revealed a number of potential GT transcripts up-regulated in response to methyl jasmonate, and their co-expression relative to that of β-amyrin synthase.
Gibberellin and auxin act as key in integrating ROS signaling pathways during (PI 595099)-RKN interaction
The phytohormone gibberellin (GA) and its signaling components have been shown to play important roles in plant defense [61, 62]. However, little is known about their effects on metabolic adjustments and influence on the fine-tuning ROS levels in response to plant stress. In view of this, H2O2 was observed to be implicated in activation of GA synthesis and signaling . Furthermore, it has been shown that variation in GA levels can provide a mechanism for plant growth regulation, causing ROS levels to remain low after biotic or abiotic stress .
Investigations of GA regulatory mechanism in plants under salt and mannitol stress and pathogen interaction led to identification of a large set of differentially expressed genes DELLA-dependent. Interestingly, a wide range of those genes was found to be responsive to oxidative stress, encoding known antioxidant systems such as SOD, peroxidases or GSTs . Thus, it was suggested that DELLA proteins can accumulate under stress conditions through reduction in GA levels and in turn activate a complex genetic regulation network to control ROS. Additionally, DELLA proteins can positively regulate anthocyanin accumulation related to nutrient stress , providing another link between GA-DELLA and ROS regulation.
Auxin was previously thought to interact positively with gibberellin to promote GA responses by destabilizing DELLA and by inducing the expression of GA biosynthetic genes, such as GA20ox and GA3ox, and leading to the down-regulation of GA catabolism genes such as GA2ox . This interaction was proposed to occur through a DELLA-independent pathway by the removal of transcriptional repression of a large array of genes possessing the Auxin Response Factor (ARF) signature through the degradation of auxin signaling suppressors Aux/IAA proteins. Therefore, the effect of auxins, such as indole-3-acetic acid (IAA), is thought to be at least in part mediated by its effect on GA metabolism . However, under adverse environmental conditions, proteins which belong to a subfamily of the GRAS protein can accumulate in the cell and function as repressors of GA signaling playing a prominent role in the auxin-gibberellin interplay through the maintenance of reduced levels of gibberellic acids .
One interesting protein found in our study regards great similarity to Glyma05g03490, a gene that encodes a protein containing GRASS domain, and exhibits high expression level in the inoculated sample. Similarity analysis using tblastn program indicates that the Glyma05g03490 gene shares suggestive homology (55% identity and 70% similarity) with the rice locus Os06g0127800 gene encoded protein. Further investigation accounts that the Glyma05g03490 encoded protein shares GRAS domains such as LHRI, VHIID, LHR, PFYRE and SAW motifs with the Os06g0127800 encoded protein, but both of them lack DELLA and TVHYNP motifs found conserved in DELLA proteins. Recently, the locus Os06g0127800 was amplified from dwarf 62(d62) rice mutant inducing the dwarf phenotype with increased gene expression levels of both GA biosynthetic and deactivating genes, OsGA20ox2 and OsGA2ox3, respectively .
In our study, the scenario described above depicts a plausible interpretation of the observed expression patterns on the possible interactions between auxin, gibberellin, jasmonate and ROS related responses. As illustrated in the Figure 8 this model involves the participation of auxins, DELLA-like (d62 rice gene related) proteins and JAZ/TIFFY proteins in controlling plant immunity and stress responses through the modulation of the amplitude and onset of elevated ROS levels. Significative findings supporting this view can include the up-regulation of GA biosynthetic genes, GA20ox1 and GA20ox2, in the inoculated sample as well as one GA deactivating gene, GA2ox1. Furthermore, two genes encoding Aux/IAA transcriptional repressors, IAA7 and IAA9, were found up-regulated, while one gene possessing ARF10 was found to be under down-regulation in the inoculated sample. The enhanced activity of these repressor proteins indicates that elevated levels of auxin might be present in biological active form. Furthermore, a number of genes known to show inducible patterns of expression related to auxin hormones were found up-regulated in the inoculated sample, possibly in response to varying regime of these hormones during their interaction. Based on these data, we suggest that the varying levels of both auxin and gibberellins might be crucial to amplify the extent and modulate the levels of ROS in the resistance reaction to nematode invasion. We also believe that the bioactivity of these plant hormones, as well as other defense-related small molecules, might be related to the activity of glycosyltransferases on nucleotide sugars (NDP-sugars). Furthermore, this model reinforces previous ideas that propose the integration of phenolic compounds into the redox system with large dependence on cellular nucleotide sugar concentrations. This latter suggests further effort is needed to investigate pathways that might play an essential role for nucleotide-sugar biosynthesis and for the regulation of the NDP-sugar pool in the host challenged by the RKN– Meloidogyne genus in the incompatible interaction.