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Regulatory interplay between soybean root and soybean cyst nematode during a resistant and susceptible reaction



Plant–parasitic nematodes (PPNs) are obligate parasites that feed on the roots of living host plants. Often, these nematodes can lay hundreds of eggs, each capable of surviving without a host for as long as 12 years. When it comes to wreaking havoc on agricultural yield, few nematodes can compare to the soybean cyst nematode (SCN). Quantifying soybean (Glycine max) transcription factor binding sites (TFBSs) during a late–stage SCN resistant and susceptible reaction can shed light onto the systematic interplay between host and pathogen, thereby elucidating underlying cis–regulatory mechanisms.


We sequenced the soybean root transcriptome at 6 and 8 days upon independent inoculation with a virulent and avirulent SCN population. Genes such as β–1,4 glucanase, chalcone synthase, superoxide dismutase and various heat shock proteins (HSPs) exhibited reaction–specific expression profiles. Several likely defense–response genes candidates were also identified which are believed to confer SCN resistance. To explore magnitude of TFBS representation during SCN pathogenesis, a multivariate statistical software identified 46 over–represented TFBSs which capture soybean regulatory dynamics across both reactions.


Our results reveal a set of soybean TFBSs which are over–represented solely throughout a resistant and susceptible SCN reaction. This set furthers our understanding of soybean cis–regulatory dynamics by providing reaction–specific levels of over–representation at 6 and 8 days after inoculation (dai) with SCN.


Obligate parasites, such as plant–parasitic nematodes (PPNs), are infamously known for their ability to suppress host defense mechanisms and cripple yield of many agricultural crops. Such devastation is tightly orchestrated by nematode effector proteins that commandeer host–plant metabolic machinery. One of the most destructive PPNs to soybean yield is the soybean cyst nematode (SCN; Heterodera glycines). Worldwide, approximately 1.5 billion dollars in soybean yield is lost annually due to SCN infestations [1],[2]. In SCN susceptible soybeans, this devastation begins when the female juvenile–stage 2 (J2) nematode penetrates the host root. J2 effector proteins are injected into the root, dissolving plant cell walls and driving formation of a metabolically–active, multinucleated feeding site known as a syncytium [3]. Newly–molted J3 males and females feed from this nutrient–rich syncytium, subsequently molt into J4 larvae and copulate [4]. After approximately 30 days post–copulation, a hardened sac of SCN eggs known as a cyst becomes visible to the naked–eye. In the resistant reaction however, cysts are not visible since J2 nematodes can neither form a nutrient–rich syncytium nor copulate. Thus, J2 nematodes starve to death.

With next–generation sequencing (NGS) now becoming a central assay in transcriptomics, entire transcriptomes can now be sequenced at unprecedented resolution. Fueled by the economic impact of SCN infestations, numerous studies have utilized NGS assays to sequence and quantify the soybean transcriptome [5]-[8].

In this study, we extend such works by conducting transcriptomic and regulatory analyses on soybean roots (Peking cv.) inoculated with SCN. We sequence the soybean root transcriptome and contrast resistant and susceptible SCN reactions at 6 and 8 days after inoculation (dai). Our findings reveal likely defense–response gene candidates and a potential regulatory “signature” that captures TFBS over–representation throughout both resistant and susceptible reactions.

Results and discussion

Illumina sequencing and read alignment

cDNA libraries from soybean roots were generated after independently inoculating roots for both 6 and 8 dai in two SCN populations, NH1-RHg (confers resistant reaction in Peking; Race 3) and TN8 (confers susceptible reaction in Peking; Race 14). A baseline control cDNA library was also created from roots uninoculated with SCN. RNA was prepared using the Illumina TruSeq sample preparation kit. Single–end RNA–sequencing (RNA–Seq) was performed on the Illumina GAIIx, producing a total of 30 million reads 80 bp in length. Across all sequenced libraries, quality assessment subtracted between 10%–19% of reads for being either a contaminent sequence or of low quality (Table 1). Using the BWA aligner [9], quality reads were mapped against the soybean transcriptome build version 1.1 [10]. Reads aligning to multiple transcripts were identified and assigned to the transcript with the highest quality score. In total, 59% to 69% of quality–assessed reads mapped to the soybean transcriptome.

Table 1 Soybean–SCN pathogenesis RNA–Seq summary

Soybean transcript abundance and profiling during SCN pathogenesis

Differential expression tests were performed using the R package DESeq [11]. Soybean transcripts were functionally annotated using both Gene Ontology (GO) [12] and PFAM [13]. Both fold change and log2 fold change of expression profiles (as RPKM) were computed between experimental and uninoculated samples. To render a soybean transcript differentially expressed (DE), the transcript had to have a log2 fold change greater than or equal to ±1.0 and have atleast 5 mapped reads across all replicates. A total of 12,377 soybean transcripts were identified to be DE in at least one of the samples (Additional file 1). To disseminate the plant–pathogen defense–response landscape, a subset of 181 DE transcripts were mined and classified given their GO and PFAM functional annotations (Table 2, Additional file 2). Interestingly, virtually all of these annotation classifications exhibited induced expression profiles exclusive to the resistant reaction. For instance, all 12 transcripts of β–1,4–glucanase (β–1,4–G) were generally induced throughout the resistant but suppressed in the susceptible reaction. Numerous studies reveal how a pathogenic nematode can commandeer not only β–1,4–glucanase but other cellulases to drive formation of a nematode feeding site [14]-[16]. Both Tucker et al. [16] and Ibrahim et al. [14] quantified this destructive commandeering capability by quantifying the soybean transcriptome using high–throughout microarrays. This latter study, though examining soybean–root knot nematode interplay, reveals cell–wall modeling, defense response, and metabolism, to be the most impacted host pathways following pathogenic nematode infection. Critical genes encoding isoflavonoid and flavonoid biosynthesis such as chalcone synthase (ChS), chalcone reductase (ChR), and chalcone isomerase (ChI) also exhibited similar induced expression profiles. Glutathione S-transferase (GST) genes were also induced in the resistant reaction. GST is a class of enzymes involved in reactions leading to xenobiotic degradation [17], and has been shown to be induced during an SCN resistant reaction [18]-[20].

Table 2 Various genes perceived during defense response are expressed during SCN inoculation

Transcripts of genes encoding two lipoxygenase (LOX) gene family members, arachidonate 8-lipoxygenase (A–8 LOX; EC: and linoleate 13S-lipoxygenase (L–13S LOX (LOX2); EC: were also induced throughout both 6 dai and 8 dai resistant reactions. The role A–8 LOX plays during a nematode reaction has yet to be elucidated, however lipoxygenases in–general are consistently induced throughout a resistant SCN reaction [21]-[24]. This raises speculation that A–8 LOX may be perceived during SCN pathogenesis.

Ribonucleoside-diphosphate reductase (RnDR; EC: and protein disulfide-isomerase (PDI; EC: were induced in the resistant reaction. Both RnDR and PDI are thioredoxins, a family of reductases known to play defense–response roles upon perception of a pathogen [25]-[27]. Little is known about the role RnDR plays in SCN pathogenesis, however an earlier microarray study examined abaxial and adaxial soybean embryo expression profiles upon exposure to auxin 2,4-dichlorophenoxyacetic acid (2,4–D). Microarray results revealed differentially expressed levels of RnDR 21 days after auxin inoculation [28]. PDI on the other hand, is a well–studied thioreductase expressed during plant defense [29],[30], especially in soybean roots undergoing a resistant SCN reaction [31].

Pathogenesis–Related (PR) transcripts, namely PR5 and PR10, were induced in the resistant reaction. PR genes were expressed not just during SCN nematode pathogenesis [32]-[38] but also throughout abiotic stress [39], phytohormone signaling [40] and drought [41].

Glyoxalase I (GLY I; lactoylglutathione lyase, EC: was also induced throughout the resistant reaction. GLY I has been shown to exhibit an induced expression profile in pumpkin seeds exposed to numerous abiotic stresses [42]. Lastly, little is known about the role phytochelatin synthetase (PCS) plays throughout SCN pathogenesis, however PCS has been shown in a prior study to be induced during aphid herbivory [43].

Following quantification of the SCN–inoculated soybean root transcriptome, our analyses support earlier works by Klink et al. ([44],[45]), Kandoth et al. ([20]), and Li et al. ([33]). We build–on such studies by identifying a small subset of potentially novel defense–response candidate genes as well as a biologically–sound proximal regulatory landscape that captures host–SCN pathogenesis interplay.

Gene Ontology enrichment in resistant and susceptible reactions

To identify statistically significant Gene Ontology (GO) annotations, the top 750 induced and 750 suppressed genes across for all SCN samples each independently underwent GO Process enrichment using the AgriGO server [46]. Numerous GO Processes were statistically significant across resistant and susceptible reactions (Table 3). GO Process p–values were adjusted using Bonferroni False Discovery Rate (FDR) and all GO Processes with adjusted p–values less than 0.05 were selected.

Table 3 Abundance of enriched Gene Ontology annotations

The top 30 most statistically significant GO Processes within induced genes were identified (Table 4). Processes such as “defense response”, “syncytium formation”, “response to other organism”, “response to oxidative stress”, and “response to stress”, were revealed to be statistically significant mainly in the resistant reaction when compared to the susceptible. Processes associated with organelle modification and intracellular organization also exhibited similar reaction–specific significance. This race–exclusivity exposes the crucial role basal operations play during pathogen perception.

Table 4 GO Process enrichment of induced soybean genes

Similarly, the top 30 most statistically significant GO Processes within suppressed genes were also identified (Table 5). Contrasting GO Processes in suppressed genes to that of induced genes reveals an entirely different catalog of annotations. For instance, 20 of the 30 GO Processes in suppressed genes are statistically significant across both resistant and susceptible reactions. This indicates that nematode effectors are generally operable in a race–independent manner and capable of effortlessly suppressing a majority of crucial basal processes.

Table 5 GO Process enrichment of suppressed soybean genes

The most suppressed GO Processes were “photosynthesis”, “photosynthesis, light harvesting”, “photosynthesis, light reaction”, and “generation of precursor metabolites and energy”. Interestingly, it has been shown in prior studies that PPNs can suppress photosynthesis in tomato plants by disrupting cytokinin and gibberellin signaling [47],[48]. Aside from photosynthetic processes, those associated with metabolism and biosynthesis were highly suppressed across both reactions. This suggests that both resistant and susceptible SCN populations share a common goal of crippling basal metabolic machinery and suppressing the host machinery responsible for photosynthesis.

Derivation of over–represented TFBSs

The 1,000 most induced and 1,000 most suppressed genes were identified for each sample and the promoter sequence 2 kb upstream from each genes transcription start site was retrieved and appended to a FASTA file (Additional file 3). To quantify abundance of cis–regulatory TFBSs within promoter sequences, we used a collection of 68 plant Position Weight Matrices (PWMs) from AthaMap [49] and JASPAR [50]. PWMs are multi–dimensional matrices frequently used to model regulatory elements, namely TFBSs. Each cell in a PWM represents a weight as to the likelihood a particular base at a specific index is a regulatory element. Thus, mapping PWMs onto promoter sequences and statistically quantifying its abundance reveals insight into the magnitude of TFBS over–representation. To efficiently execute such mapping, we had developed a multivariate statistical software named Marina [51]. Marina maps TFBS models such as PWMs onto promoter sequences and infers magnitude of TFBS over–representation using 7 knowledge–discovery metrics. The Iterative Proportional Fitting (IPF) algorithm [52] normalizes output produced from each of the 7 metrics, enabling unanimous agreement across the metrics as to the magnitude of TFBS over–representation. IPF scores range from 1 to N whereby N is the total number of over–represented TFBSs. Scores in the range of 1 represent over–represented TFBSs while scores in the range of N represent highly under–represented TFBSs.

For all SCN samples, Marina mapped all 68 plant PWMs onto promoter sequences of both induced and suppressed genes. In total, 46 TFBSs were over–represented in atleast one of the four samples (Figure 1). To reveal which TFBSs exhibited variations in their IPF scores, we computed the percent change of IPF scores across both Race 3 and Race 14 timepoints. The difference in Race 3 and Race 14 percent change was derived and partitioned into 2 bins: TFBSs with a Race 3 and Race 14 IPF score percent difference of at least 50% (Figure 1a), and TFBSs with a Race 3 and Race 14 IPF score percent difference under 50% (Figure 1b). Thus, such computation allows for identification of which TFBSs vary greatly not with respect to 6 dai or 8 dai, but with respect to Race 3 and Race 14 inoculations.

Figure 1
figure 1

A heatmap of Marina IPF scores. Across the four SCN samples, over–represented TFBSs were identified given promoter sequences from the 1,000 most induced and 1,000 most suppressed genes. In total, 46 TFBSs were over–represented in one of the inoculations and 29 TFBSs were over–represented across all inoculations. IPF scores range from 1 to N whereby 1 represents over–represented TFBSs and N represents under–represented TFBSs. (a) Enriched TFBSs within Race 3 and Race 14 reactions with IPF scores having percent difference of at least 50%. (b) Enriched TFBSs within Race 3 and Race 14 reactions with IPF scores having percent difference less than 50%.

There were 29 TFBSs over–represented across all four samples (Additional file 4). If a TFBS was not over–represented in a specific sample, that TFBS was assigned an score of N+1 so as to serve as a proxy for being highly under–represented.

Many TFBSs are over/under–represented in both resistant and susceptible reactions

Contrasting TFBS IPF scores across samples reveals that 30 of the 46 TFBSs either increase or decrease in IPF score regardless of the reaction (Figure 1). For instance, the TFBS for STF1 exhibits a relatively modest increase in its IPF score across both reactions. Interestingly, STF1 IPF score increases from 11th to 1st from 6 dai to 8 dai respectively in the resistant reaction. Besides the role STF1 plays in plant development [53], little is known of the role this transcription factor plays in plant defense.

IPF score for the HAHB4 TFBS greatly increased in the resistant reaction and susceptible reaction. A prior study found HAHB4 to contribute to jasmonic acid and ethylene signaling crosstalk [54]. Similarly, TFBSs for DOF2 and DOF3 exhibited relatively weak increases in IPF scores across resistant and susceptible samples. DOF transcripts have not been explicitly quantified as–far as their gene expression during SCN pathogenesis, however such proteins have been detected during auxin signaling [55]. In contrast to DOF2 and DOF3, the TFBS for TEIL had a near–50% jump in IPF scores across both reactions. Being the tobacco homolog of ethylene insensitive (EIN3), TEIL gene products have been shown to bind directly to the promoter sequence of PR1a, a central contributor in plant defense dynamics [56]. Interestingly, across both resistant and susceptible reactions, TEIL scores appear to be relatively equal to one another.

The A. thaliana MYB77 homolog, AtMYB77, exhibits a mild change in IPF score across both resistant and susceptible reactions. Across both reactions, AtMYB77 IPF scores were generally under–represented at 6 dai but become slightly over–represented at 8 dai. An earlier study revealed interaction between MYB77 and auxin response factor 7 (ARF7) [57], further accentuating the role AtMYB77 could play in host–pathogen interplay [58]. The OsCBT TFBS exhibited pronounced IPF scores across all four treatments. In both the resistant and susceptible reaction, OsCBT was highly over–represented only at 6 dai. It was shown that OsCBT mutants conferred increased pathogen resistance upon inoculation with Magnaporthe grisea, revealing that OsCBT suppresses defense response [59].

Several TFBSs are over–represented in a race–dependent manner

The remaining 16 TFBSs were over–represented in one reaction compared to the other. Such TFBSs can expose novel insight into TFBSs over–representation patterns respective to a specific reaction.

ZAP1, a WRKY1 TFBS [60], appears to be highly over–represented during the resistant reaction but slightly under–represented in the susceptible reaction. Being a WRKY TFBS, it comes as no surprise that enrichment of this TFBS in the resistant reaction captures the need to host a significant, systematic plant defense response. Similarly, PIF3–1 and PIF3–2 were both under–represented during the susceptible reaction however slightly over–represented in the resistant reaction. It has been shown that PIF plays roles in phytochrome signaling [61]. Due to its photomorphogenic regulatory capabilities, Since photosynthetic processes are heavily suppressed within resistant and susceptible reactions (Table 5), such suppression explains why PIF3–1 and PIF3–2 have such severely under–represented IPF scores. Indeed SCN pathogenesis does not only disrupt the photosynthetic machinery but also the plants ability to execute sound phytochrome signaling.


We used RNA–Seq to sequence soybean whole–root (Peking cv.) at both 6 and 8 dai upon inoculation with a resistant (NH1–RHg; Race 3) and susceptible (TN8; Race 14) population. Contrasting TFBSs over–represented in promoter sequences of DE soybean genes across 6 and 8 dai time points exposed underlying transcriptomic and cis–regulatory dynamics within the soybean root during pathogenesis. In–total, over 30 million reads from soybean whole–root was sequenced and differential expression analysis revealed 181 transcripts to be statistically and biologically significant during defense–response. Several viable defense–response gene candidates joined these ranks, including glyoxalase I, arachidonate–8 lipoxygenase, phytochelatin synthetase, and ribonucleoside-diphosphate reductase.

46 TFBSs were rendered over/under–represented across all resistant and susceptible samples. Interestingly, 30 of these TFBSs were either over or under–represented across both reactions. Thus, our results reveal presence of a biologically–sound regulatory “signature” that identifies reaction–specific soybean regulatory patterns during both resistant and susceptible SCN reactions.


Plant procurement and SCN inoculation

Glycine max cv. Peking seeds were surface–sterilized by treating the seeds with 10% bleach (0.6% sodium hypochlorite) for ten minutes, followed by several washes with distilled water. Seeds were planted in sterile sand in 20 × 20 cm flats. Eight days later, seedlings were gently lifted out of the sand and rinsed clean. Five seedlings for each time point were placed on moistened germination paper in 8 × 12 × 3.5 cm plastic trays. The SCN populations NH1–RHg and TN8, were independently harvested from stock plants [62]. Females were crushed with a rubber stopper and eggs were washed through a 250 micron screen and collected on a 25 micron screen. Eggs were rinsed into a small covered tray and left to hatch for three days. J2 stage nematodes were further purified by passing them through a 30 micron cloth into deionized, distilled water and gently centrifuged at 250 relative centrifugal force (RCF) for one minute to concentrate to 2,000 J2/ml. Roots from four plants were inoculated with one ml of inoculum. Roots were covered with a second piece of moistened germination paper and the trays were placed in a larger tray with 0.5 cm water below to add humidity and wrapped in a semi-clear plastic bag for the duration of the time points. Three uninoculated control plants were also placed trays and collected separately. Per plant, four plant roots, following 6 and 8 days after inoculation (dai), were harvested and immediately frozen in liquid nitrogen and ground to a fine powder in a mortar and pestle and stored in microfuge tubes at –80°C until RNA extraction. The fifth root was stained for visualization of nematode infection with acid fuchsin [63]. RNA was extracted at 6 dai and 8 dai by phenol/chloroform and lithium chloride precipitation [64]. RNA was treated with DNase to remove any genomic DNA remaining in the samples. RNA integrity was checked by visualizing the intact 18S and 28S ribosomal bands on an agarose gel and concentrations were measured on a Nanodrop spectrophotometer (Thermo Scientific; Waltham, MA).

RNA extraction and cDNA isolation

cDNA libraries were prepared using the TruSeq RNA Prep Kit according to the manufacturer instruction (Illumina). Briefly, mRNA was purified from four micrograms of total RNA diluted in fifty microliters of nuclease–free ultra pure water using magnetic beads. Resulting mRNA was fragmented at 94°C for eight minutes. Seventeen microliters of fragmented mRNA was used as template for cDNA synthesis performed by a Superscript II Reverse Transcriptase. Second–strand synthesis was immediately performed and fifty microliters of double stranded DNA was transferred to a new tube and submitted to end repair followed by adenylation of 3’ ends. Once adenylation of 3’ reached completion, adapters containing different indexes were ligated to each library. DNA fragments having adapter molecules on both ends were amplified and enriched. Quantification and quality control were performed by loading one microliter of cDNA libraries on an Agilent DNA–1000 chip and running it on an Agilent Technologies 2100 Bioanalyzer.

Deep–sequencing and transcriptome quantification

For both NH1–RHg (Race 3) and TN8 (Race 14) reactions, cDNA libraries were sequenced from 8 day old soybean whole–root independently inoculated with SCN at 6 dai and 8 dai. Two biological replicates were sequenced for each inoculation and timepoint. Single–end RNA–sequencing was performed on the Illumina GAIIx at the United States Department of Agriculture (USDA), Beltsville, MD. An uninoculated whole–root single–replicate control was also sequenced using the same sequencing protocol. To remove low quality reads across all sequencing runs, custom bash scripts filtered all reads should its 3’ tail have a quality score of less than 22. To remove contaminent reads, sequences were subtracted if they mapped atleast once to both the Ensembl human genome (Hg19) or the JCVI Microbial Resource [65]. Remaining sequences were mapped to the soybean transcriptome (build 1.1) using BWA [9]. Across all SCN inoculated samples, transcript counts underwent normalization and variance estimation using the DESeq R package. To infer magnitude of differential expression, RPKM was computed for all inoculated and uninoculated samples and lo g 2 RPK M inoculated RPK M uninoculated was subsequently derived. All transcripts with a log 2 RPKM less than 1 and fewer than 5 mapped reads were rendered not differentially expressed.

Functional annotation & Gene Ontology (GO) enrichment

Functional annotation comprised of homology–based analysis of all sequences in the Phytozome soybean transcriptome. Of these 73,320 soybean transcriptome sequences, 7,810 sequences were subtracted for being either a scaffold or duplicate sequence. BLASTX [66] aligned the remaining 65,510 query sequences onto all UniProt plant proteins [67]. The top–scoring UniProt function annotation was assigned to the query if it did not contain ambiguous keywords, namely “Hypothetical”, “Uncharacterized” or “Unknown”.

For all samples, soybean Phytozome accessions for the top 750 induced and top 750 suppressed transcripts were identified. Gene Ontology (GO) enrichment on each accession–set was performed using the AgriGO web–server [46]. AgriGO settings were modified to quantify GO annotations using the hypergeometric distribution and Bonferroni p–value false–discovery rate (FDR) correction. To measure GO Process statistical significance in both resistant and susceptible reactions, the –log 10FDR per GO Process was summed across both 6 and 8 dai time points. Subsequently, the top 30 most statistically significant GO Processes from the top 750 induced and suppressed transcript sets were identified.

Availability of supporting data

All RNA–Seq FASTQ raw data is available from NCBI SRA. Please refer to Table 1 for such accessions.

Additional files



4–Coumarate–CoA ligase

A–8 LOX:

Arachidonate 8-lipoxygenase

L–13S LOX:

Linoleate 13S-lipoxygenase


Chalcone isomerase


Chalcone reductase


Glutathione S–transferase


Glyoxalase I


Gene Ontology


Phytochelatin synthetase


Protein disulfide–isomerase


Plant Parasitic Nematode




Position weight matrix


Ribonucleoside-diphosphate reductase


Super–oxide dismutase


Soybean cyst nematode


Starch–Free 1


Transcription factor


Transaction factor binding site


  1. Wrather J, Anderson T, Arsyad D, Tan Y, Ploper L, Porta-Puglia A, HH R, Yorinori J: Soybean disease loss estimates for the top ten soybean-producing counries in 1998 . Can J Plant Pathol. 2001, 23 (2): 115-121. 10.1080/07060660109506918.

    Article  Google Scholar 

  2. Matsye P, Lawrence G, Youssef R, Kim K, Lawrence K, Matthews B, Klink V: The expression of a naturally occurring, truncated allele of an α-SNAP gene suppresses plant parasitic nematode infection. Plant Mol Biol. 2012, 80 (2): 131-155. 10.1007/s11103-012-9932-z.

    Article  CAS  PubMed  Google Scholar 

  3. Endo B: Penetration and development of Heterodera glycines in soybean roots and related anatomical changes . Phytopath. 1964, 54: 79-88.

    Google Scholar 

  4. Klink V, Hosseini P, MacDonald M, Alkharouf N, Matthews B: Population-specific gene expression in the plant pathogenic nematode Heterodera glycines exists prior to infection and during the onset of a resistant or susceptible reaction in the roots of the Glycine max genotype Peking . BMC Genomics. 2009, 10: 111-10.1186/1471-2164-10-111.

    Article  PubMed Central  PubMed  Google Scholar 

  5. Li X, Wang X, Zhang S, Liu D, Duan Y, Dong W: Comparative profiling of the transcriptional response to soybean cyst nematode infection of soybean roots by deep sequencing . Chin Sci Bull. 2011, 56 (18): 1904-1911. 10.1007/s11434-011-4510-3.

    Article  CAS  Google Scholar 

  6. Li X, Wang X, Zhang S, Liu D, Duan Y, Dong W: Identification of soybean micrornas involved in soybean cyst nematode infection by deep sequencing . PLoS ONE. 2012, 7 (6): e39650-10.1371/journal.pone.0039650.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  7. Hamamouch N, Li C, Hewezi T, Baum T, Mitchum M, Hussey R, Vodkin L, Davis E: The interaction of the novel 30C02 cyst nematode effector protein with a plant β-1,3-endoglucanase may suppress host defence to promote parasitism . J Exp Bot. 2012, 63 (10): 3683-3695. 10.1093/jxb/ers058.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  8. Guttikonda S, Trupti N, Bisht J, Xu S: Whole genome co-expression analysis of soybean cytochrome P450 genes identifies nodulation-specific P450 monooxygenases . BMC Plant Biol. 2010, 10: 243-10.1186/1471-2229-10-243.

    Article  PubMed Central  PubMed  Google Scholar 

  9. Li H: Fast and accurate short read alignment with Burrows-Wheeler transform . Bioinformatics. 2009, 25 (14): 1754-1760. 10.1093/bioinformatics/btp324.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  10. Goodstein DM, Shu S, Howson R, Neupane R, Hayes RD, Fazo J, Mitros T, Dirks W, Hellsten U, Putnam N: Phytozome: a comparative platform for green plant genomics. Nucleic Acids Res. 2012, 40 (Database issue): D1178–D1186

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  11. Anders S: Differential expression analysis for sequence count data . Genome Biol. 2010, 11 (10): R106-10.1186/gb-2010-11-10-r106.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  12. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, Harris MA, Hill DP, Issel-Tarver L, Kasarskis A, Lewis S, Matese JC, Richardson JE, Ringwald M, Rubin GM, Sherlock G: Gene Ontology: tool for the unification of biology . Nat Genet. 2000, 25: 25-29. 10.1038/75556.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  13. Bateman A, Birney E, Cerruti L, Durbin R, Etwiller L, Eddy SR, Griffiths-Jones S, Howe KL, Marshall M, Sonnhammer ELL: The Pfam Protein Families Database . Nucleic Acids Res. 2002, 30: 276-280. 10.1093/nar/30.1.276.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  14. Ibrahim HM, Hosseini P, Alkharouf NW, Hussein EH, Gamal ED AEKE, Aly MA: Analysis of gene expression in soybean (Glycine max) roots in response to the root-knot nematode Meloidogyne incognita using microarrays and KEGG pathways . BMC Genomics. 2011, 12: 220-10.1186/1471-2164-12-220.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  15. Goellner M, Wang X: Endo- β-1,4-glucanase expression in compatible plant-nematode interactions. Plant Cell. 2001, 13 (10): 2241-2255.

    PubMed Central  CAS  PubMed  Google Scholar 

  16. Tucker M, Burke A, Murphy C, Thai V: Gene expression profiles for cell wall-modifying proteins associated with soybean cyst nematode infection, petiole abscission, root tips, flowers, apical buds, and leaves . J Exp Bot. 2007, 58 (12): 3395-3406. 10.1093/jxb/erm188.

    Article  CAS  PubMed  Google Scholar 

  17. Dalton D, Boniface C, Turner Z, Lindahl A, Kim H, Jelinek L, Govindarajulu M, Finger R, Taylor C: Physiological roles of glutathione s-transferases in soybean root nodules . Plant Physiol. 2009, 150: 521-530. 10.1104/pp.109.136630.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  18. Mazarei M, Liu W, Al-Ahmad H, Arelli P, Pantalone V, Stewart CJ: Gene expression profiling of resistant and susceptible soybean lines infected with soybean cyst nematode . Theor Appl Genet. 2011, 123 (7): 1193-1206. 10.1007/s00122-011-1659-8.

    Article  CAS  PubMed  Google Scholar 

  19. Alkharouf N, Khan R, Matthews B: Analysis of expressed sequence tags from roots of resistant soybean infected by the soybean cyst nematode . Genome. 2004, 47 (2): 380-388. 10.1139/g03-114.

    Article  CAS  PubMed  Google Scholar 

  20. Kandoth P, Ithal N, Recknor J, Maier T, Nettleton D, Baum T, Mitchum M: The Soybean Rhg1 locus for resistance to the soybean cyst nematode Heterodera glycines regulates the expression of a large number of stress- and defense-related genes in degenerating feeding cells . Plant Physiol. 2011, 155 (4): 1960-1975. 10.1104/pp.110.167536.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  21. Klink VP: Emerging approaches to broaden resistance of soybean to soybean cyst nematode as supported by gene expression studies . Plant Physiol. 2009, 151 (3): 1017-1022. 10.1104/pp.109.144006.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  22. Klink VP, Matsye PD, Lawrence KS, Lawrence GW: Engineered soybean cyst nematode resistance. In Soybean - Pest Resistance. Edited by El-Shemy H: InTech; 2013. doi:10.5772/54514. ISBN: 978-953-51-0978-5, Available from: [], []

    Google Scholar 

  23. Veronico P, Giannino D, Melillo M, Leone A, Reyes A, Kennedy M, Bleve-Zacheo T: A novel lipoxygenase in pea roots: its function in wounding and biotic stress . Plant Physiol. 2006, 141 (3): 1045-1055. 10.1104/pp.106.081679.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  24. Ithal N, Recknor J, Nettleton D, Maier T, Baum TJ, Mitchum MG: Developmental transcript profiling of cyst nematode feeding cells in soybean roots . Mol Plant Microbe Interact. 2007, 20 (5): 510-525. 10.1094/MPMI-20-5-0510.

    Article  CAS  PubMed  Google Scholar 

  25. Vieira Dos Santos C, Rey P: Plant thioredoxins are key actors in the oxidative stress response . Trends Plant Sci. 2006, 11 (7): 329-334. 10.1016/j.tplants.2006.05.005.

    Article  CAS  PubMed  Google Scholar 

  26. Laloi C, Mestres-Ortega D, Marco Y, Meyer Y, Reichheld J: The Arabidopsis cytosolic thioredoxin h5 gene induction by oxidative stress and its W-box-mediated response to pathogen elicitor . Plant Physiol. 2004, 134 (3): 1006-1016. 10.1104/pp.103.035782.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  27. Wang D, Weaver ND, Kesarwani M, Dong X: Induction of protein secretory pathway is required for systemic acquired resistance . Science. 2005, 308 (5724): 1036-1040. 10.1126/science.1108791.

    Article  CAS  PubMed  Google Scholar 

  28. Thibaud-Nissen F, Shealy R, Khanna A, Vodkin L: Clustering of microarray data reveals transcript patterns associated with somatic embryogenesis in soybean . Plant Physiol. 2003, 132: 118-136. 10.1104/pp.103.019968.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  29. Ray S, Anderson J, Urmeev F, Goodwin S: Rapid induction of a protein disulfide isomerase and defense-related genes in wheat in response to the hemibiotrophic fungal pathogen Mycosphaerella graminicola . Plant Mol Biol. 2003, 53 (5): 701-714. 10.1023/B:PLAN.0000019120.74610.52.

    Article  CAS  PubMed  Google Scholar 

  30. Gruber C, Cemazar M, Clark R, Horibe T, Renda R, Anderson M, Craik D: A novel plant protein-disulfide isomerase involved in the oxidative folding of cystine knot defense proteins . J Biol Chem. 2007, 282 (28): 20435-20446. 10.1074/jbc.M700018200.

    Article  CAS  PubMed  Google Scholar 

  31. Klink V, Hosseini P, Matsye P, Alkharouf N, Matthews B: Syncytium gene expression in Glycine max([PI 88788]) roots undergoing a resistant reaction to the parasitic nematode Heterodera glycines . Plant Physiol Biochem. 2010, 48 (2–3): 176-193. 10.1016/j.plaphy.2009.12.003.

    Article  CAS  PubMed  Google Scholar 

  32. Afzal A, Natarajan A, Saini N, Iqbal M, Geisler M, El Shemy H, Mungur R, Willmitzer L, Lightfoot D: The nematode resistance allele at the rhg1 locus alters the proteome and primary metabolism of soybean roots . Plant Physiol. 2009, 151 (3): 1264-1280. 10.1104/pp.109.138149.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  33. Li X, Wang X, Zhang S, Liu D, Duan Y, Dong W: Comparative profiling of the transcriptional response to soybean cyst nematode infection of soybean roots by deep sequencing . Chin Sci Bull. 2011, 56 (18): 1904-1911. 10.1007/s11434-011-4510-3.

    Article  CAS  Google Scholar 

  34. Matthews BF, Ibrahim HMM, Klink VP: Changes in the expression of genes in soybean roots infected by nematodes. In Soybean - Genetics and Novel Techniques for Yield Enhancement: InTech; 2011. doi:10.5772/20883, ISBN: 978-953-307-721-5. Available from: [], []

    Google Scholar 

  35. Alkharouf N, Klink V, Chouikha I, Beard H, MacDonald M, Meyer S, Knap H: Timecourse microarray analyses reveal global changes in gene expression of susceptible Glycine max (soybean) roots during infection by Heterodera glycines (soybean cyst nematode) . Planta. 2006, 224 (4): 838-852. 10.1007/s00425-006-0270-8.

    Article  CAS  PubMed  Google Scholar 

  36. Ithal N, Recknor J, Nettleton D, Hearne L, Maier T, Baum T, Mitchum M: Parallel genome-wide expression profiling of host and pathogen during soybean cyst nematode infection of soybean . Mol Plant Microbe Interact. 2007, 20 (3): 293-305. 10.1094/MPMI-20-3-0293.

    Article  CAS  PubMed  Google Scholar 

  37. Mitchum MG, Baum TJ: Genomics of the soybean cyst nematode-soybean interaction. In Genetics and Genomics of Soybean, Volume 2 of Plant Genetics and Genomics: Crops and Models. Edited by Stacey G. New York: Springer; 2008:321–341.

    Chapter  Google Scholar 

  38. Matthews B, Beard H, MacDonald MH, Kabir S, Youssef RH, Hosseini P, Brewer E: Engineered resistance and hypersusceptibility through functional metabolic studies of 100 genes in soybean to its major pathogen, the soybean cyst nematode . Planta. 2013, 237 (5): 1337-1357. 10.1007/s00425-013-1840-1.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  39. Hashimoto M, Kisseleva L, Sawa S, Furukawa T, Komatsu S, Koshiba T: A novel rice PR10 protein, RSOsPR10, specifically induced in roots by biotic and abiotic stresses, possibly via the jasmonic acid signaling pathway . Plant Cell Physiol. 2004, 45 (5): 550-559. 10.1093/pcp/pch063.

    Article  CAS  PubMed  Google Scholar 

  40. Kitajima S, Sato F: Plant pathogenesis-related proteins: molecular mechanisms of gene expression and protein function . J Biochem. 1999, 125: 1-8. 10.1093/oxfordjournals.jbchem.a022244.

    Article  CAS  PubMed  Google Scholar 

  41. Dubos C, Plomion C: Drought differentially affects expression of a PR-10 protein, in needles of maritime pine (Pinus pinaster Ait.) seedlings . J Exp Bot. 2001, 52 (358): 1143-1144. 10.1093/jexbot/52.358.1143.

    Article  CAS  PubMed  Google Scholar 

  42. Hossain MA, Hossain MZ, Fujita M: Stress-induced changes of methylglyoxal level and glyoxalase I activity in pumpkin seedlings and cDNA cloning of glyoxalase I gene . Austr JCrop Sci. 2009, 3 (2): 53-64.

    CAS  Google Scholar 

  43. Michael Smith C, Boyko EV: The molecular bases of plant resistance and defense responses to aphid feeding: current status . Entomologia Experimentalis et Applicata. 2007, 122: 1-16. 10.1111/j.1570-7458.2006.00503.x.

    Article  Google Scholar 

  44. Klink VP, Hosseini P, Matsye P, Alkharouf NW, Matthews BF: A gene expression analysis of syncytia laser microdissected from the roots of the Glycine max (soybean) genotype PI 548402 (Peking) undergoing a resistant reaction after infection by Heterodera glycines (soybean cyst nematode) . Plant Mol Biol. 2009, 71 (6): 525-567. 10.1007/s11103-009-9539-1.

    Article  CAS  PubMed  Google Scholar 

  45. Klink VP, Overall CC, Alkharouf NW, MacDonald MH, Matthews BF: A time–course comparative microarray analysis of an incompatible and compatible response by Glycine max (soybean) to Heterodera glycines (soybean cyst nematode) infection . Planta. 2007, 226 (6): 1423-1447. 10.1007/s00425-007-0581-4.

    Article  CAS  PubMed  Google Scholar 

  46. Du Z, Zhou X, Ling Y, Zhang Z: Su Z: agriGO: a GO analysis toolkit for the agricultural community . Nucleic Acids Res. 2010, 38 (Web-Server-Issue): 64-70. 10.1093/nar/gkq310.

    Article  Google Scholar 

  47. Brueske C, Bergeson G: Investigation of growth hormones in xylem exudate and root tissue of tomato infected with root-knot nematode . J Exp Bot. 1972, 23 (74): 14-22. 10.1093/jxb/23.1.14.

    Article  CAS  Google Scholar 

  48. Loveys BR, Bird AF: The influence of nematodes on photosynthesis in tomato plants. Physiol Plant Pathol. 1973, 3 (4): 525-529. 10.1016/0048-4059(73)90063-5.

    Article  CAS  Google Scholar 

  49. Bülow L, Engelmann S, Schindler M, Hehl R: AthaMap, integrating transcriptional and post-transcriptional data. Nucleic Acids Res. 2009, 37 (Database-Issue): D983–D986

    Article  PubMed Central  PubMed  Google Scholar 

  50. Sandelin A, Alkema W, Engstrom P, Wasserman WW, Lenhard B: JASPAR: an open-access database for eukaryotic transcription factor binding profiles. Nucleic Acids Res. 2007, 32: D91–D94

    Article  Google Scholar 

  51. Hosseini P, Ovcharenko I, Matthews BF: Using an ensemble of statistical metrics to quantify large sets of plant transcription factor binding sites . Plant Methods. 2013, 9: 12-10.1186/1746-4811-9-12.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  52. Deming WE, Stephan FF: On a least squares adjustment of a sampled frequency table when the expected marginal totals are known . Ann Math Stat. 1940, 11 (4): 427-444. 10.1214/aoms/1177731829.

    Article  Google Scholar 

  53. Cheong YH, Yoo CM, Park JM, Ryu GR, Goekjian VH, Nagao RT, Key JL, Cho MJ, Hong JC: STF1 is a novel TGACG-binding factor with a zinc-finger motif and a bZIP domain which heterodimerizes with GBF proteins . Plant J. 1998, 15 (2): 199-209. 10.1046/j.1365-313X.1998.00197.x.

    Article  CAS  PubMed  Google Scholar 

  54. Manavella PA, Dezar CA, Bonaventure G, Baldwin IT: HAHB4, a sunflower HD-Zip protein, integrates signals from the jasmonic acid and ethylene pathways during wounding and biotic stress responses . Plant J. 2008, 56 (3): 376-388. 10.1111/j.1365-313X.2008.03604.x.

    Article  CAS  PubMed  Google Scholar 

  55. Yanagisawa S: Dof domain proteins: plant-specific transcription factors associated with diverse phenomena unique to plants . Plant Cell Physiol. 2004, 45 (4): 386-391. 10.1093/pcp/pch055.

    Article  CAS  PubMed  Google Scholar 

  56. Hibi T, Kosugi S, Iwai T, Kawata M, Seo S, Mitsuhara I, Ohashi Y: Involvement of EIN3 homologues in basic PR gene expression and flower development in tobacco plants . J Exp Bot. 2007, 58 (13): 3671-3678. 10.1093/jxb/erm216.

    Article  CAS  PubMed  Google Scholar 

  57. Shin R, Burch AY, Huppert KA, Tiwari SB, Murphy AS, Guilfoyle TJ, Schachtman DP: The Arabidopsis transcription factor MYB77 modulates auxin signal transduction . Plant Cell Online. 2007, 19 (8): 2440-2453. 10.1105/tpc.107.050963.

    Article  CAS  Google Scholar 

  58. Fu J, Wang S: Insights into auxin signaling in plant–pathogen interactions . Front Plant Sci. 2011, 2: 74-10.3389/fpls.2011.00074.

    Article  PubMed Central  PubMed  Google Scholar 

  59. Koo SC, Choi MS, Chun HJ, Shin DB, Park BS, Kim YH, Park H, Seo HS, Song JT, Kang KY, Yun D, Chung WS, Cho MJ, Kim MC: The calmodulin-binding transcription factor OsCBT suppresses defense responses to pathogens in rice . Mol Cells. 2009, 27 (5): 563-570. 10.1007/s10059-009-0081-4.

    Article  CAS  PubMed  Google Scholar 

  60. Eulgem T, Rushton PJ, Robatzek S, Somssich IE: The WRKY superfamily of plant transcription factors . Trends Plant Sci. 2000, 5 (5): 199-206. 10.1016/S1360-1385(00)01600-9.

    Article  CAS  PubMed  Google Scholar 

  61. Kim J, Yi H, Choi G, Shin B, Song P, Choi G: Functional characterization of phytochrome interacting factor 3 in phytochrome-mediated light signal transduction . Plant Cell. 2003, 15 (10): 2399-2407. 10.1105/tpc.014498.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  62. Sardanelli S, Kenworthy WJ: Soil moisture control and direct seeding for bioassay of Heterodera glycines on soybean . J Nematol. 1997, 29 (4S): 625-634.

    PubMed Central  CAS  PubMed  Google Scholar 

  63. Bybd DW, Kirkpatrick T, Barker KR: An improved technique for clearing and staining plant tissues for detection of nematodes . J Nematol. 1983, 15: 142-143.

    PubMed Central  CAS  PubMed  Google Scholar 

  64. Mujer C, Andrews DL, Manhart J, Pierce S, Rumpho M: Chloroplast genes are expressed during intracellular symbiotic association of Vaucheria litorea plastids with the sea slug Elysia chlorotica . Proc Nat Acad Sci. 1996, 93 (22): 12333-12338. 10.1073/pnas.93.22.12333.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  65. Peterson JD, Umayam LA, Dickinson TM, Hickey EK, White O: The comprehensive microbial resource . Nucleic Acids Res. 2001, 29: 123-125. 10.1093/nar/29.1.123.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  66. Altschul S, Madden T, Schäffer A, Zhang J, Zhang Z, Miller W, Lipman D: Gapped Blast and PsiBlast: a new generation of protein database search programs. Nucleic Acids Res. 1997, 25 (17): 3389-3402. 10.1093/nar/25.17.3389.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  67. The UniProt Consortium: Reorganizing the protein space at the Universal Protein Resource (UniProt). Nucleic Acids Res. 2012, 40 (D1): D71-D75. 10.1093/nar/gkr981.

    Article  PubMed Central  Google Scholar 

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We wish to thank the United States Department of Agriculture – Soybean Genomics and Improvement Laboratory (USDA – SGIL) for research funding and support. Our appreciations go out to Ivan Ovcharenko for advice on GO enrichment analysis and TFBS over–representation derivation. We wish to thank Arianne Tremblay for overseeing cDNA derivation and RNA extraction. We wish to thank Margaret H. MacDonald for inoculation of soybean roots with SCN. We also wish to thank Patrick Gillevet and James Willett for numerous thought–provoking discussions. This research was supported in part by the Intramural Research Program of the National Institutes of Health, National Library of Medicine.

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Correspondence to Parsa Hosseini.

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The authors declare that they have no competing interests.

Authors’ contributions

PH wrote the manuscript, performed RNA–Seq analysis and quantification of over–represented TFBSs. BFM conceived of the study and oversaw TFBS quantification. Both authors read, critiqued and approved the final manuscript.

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Additional file 1: Differentially expressed transcripts across all inoculations. A table of 12,377 transcripts that are DE across all four SCN inoculations. (XLSX 3 MB)


Additional file 2: Differentially expressed transcripts annotated to be involved in plant defense. A set of 181 transcripts collectively annotated by GO and PFAM annotations to contribute to plant defense. (XLSX 59 KB)

Additional file 3: TFBSs over–represented across all inoculations. A collection of 46 TFBSs over–represented in atleast one inoculation. (ZIP 4 MB)


Additional file 4: Promoter sequences of induced and suppressed transcripts. FASTA sequences representing promoter sequences of induced and suppressed transcripts following 6 dai and 8 dai with SCN virulent and avirulent populations. (XLSX 12 KB)

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Hosseini, P., Matthews, B.F. Regulatory interplay between soybean root and soybean cyst nematode during a resistant and susceptible reaction. BMC Plant Biol 14, 300 (2014).

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