BACKGROUND
phytophthora root and stem rot  is one of the most devastating diseases of soybean , causing nearly $ <dig> million in annual yield losses in the u.s. alone  <cit> .
prr is caused by the soil-borne, hemibiotrophic pathogen phytophthora sojae, and is most effectively controlled by rps  genes.
the resistance conferred by an rps gene is race specific, and the interaction between an rps gene and the corresponding avirulence  gene in p. sojae, follows the gene-for-gene model  <cit> .
currently,  <dig> rps genes/alleles from soybean and  <dig> avr genes from p. sojae have been identified  <cit> .
like most resistance  genes, the rps gene family encodes, or is predicted to encode nucleotide binding leucine rich repeat -type proteins  <cit> , which are able to recognize the avr effector proteins of pathogens directly or indirectly to induce the appropriate defense response  <cit> .
the first evidence of a direct interaction between an r and avr protein was reported in the flax-melampsora lini pathosystem  <cit> .
indirect interactions, however, are more common.
in these cases, the r protein appears to require the existence of a ‘guard protein’ or ‘decoy’ in order to recognize an avr protein  <cit> .
a classical example of this type of interaction is the rpm1-interacting protein  <dig> , which is ‘guarded’ by rpm <dig>  and rps <dig>  proteins in arabidopsis <cit> .
the recognition of pathogen effectors triggers an innate immunity response that is mediated by two distinct types of nb-lrr proteins, the toll-interleukin- <dig> receptor -nb-lrr proteins and the coiled-coil -nb-lrr proteins.
the former requires eds <dig> , a central regulator of effector-triggered immunity , which functions together with pad <dig> .
in contrast, the latter nb-lrr proteins are independent of eds <dig> but require ndr <dig>   <cit> .
the interaction among these intracellular proteins results in a regulation network of phytohormone signaling, which is mainly mediated by salicylic acid  for biotrophic and hemibiotrophic pathogens, and jasmonic acid  and ethylene  for necrotrophic pathogens  <cit> .
in addition to sa, ja and et, other phytohormones such as brassinosteroids , abscisic acid , auxin, gibberellins  and cytokinin  also contribute to plant immune responses, with complex crosstalk between one another  <cit> .
it is suggested that the resistance to p. sojae conferred by rps genes is mediated by the sa signaling pathway, with the induction of sa-mediated systemic acquired resistance  occurring several days post infection  via expression of the gene npr <dig> <cit> .
during this process, two putative regulators of the chromosome condensation  <dig>  gene family are down-regulated during the incompatible interaction  <cit> .
besides sa, exogenous treatment of 1-aminocyclopropane-1-carboxylic acid  has been shown to enhance resistance while applications of ga and aba induce susceptibility, highlighting the complexity of phytohormone signaling pathways in response to attack by p. sojae <cit> .
a microarray study of transcriptomes from one susceptible and two partially resistant soybean genotypes indicated that 97-99% of all detectable genes experienced transcriptional modulation five dpi in response to infection by p. sojae <cit> .
however, the majority of these differences were less than two fold.
another microarray study of transcriptomic changes in soybean revealed a peak in most defense related genes at  <dig> hours post inoculation  with p. sojae <cit> .
recently, zhang et al conducted a proteomics study in which  <dig> differentially expressed proteins were identified in soybean after infection with p. sojae <cit> .
among these,  <dig> were affected during the incompatible interaction, while the other  <dig> were altered during the compatible interaction.
these studies have contributed to our understanding of the interaction between soybean and p. sojae.
nevertheless, what mechanisms underlie compatible and incompatible interactions, how molecular responses mediated by a variety of rps genes/alleles resemble or differ from one another, as well as the nature of these similarity or difference, remain largely unknown.
access to the soybean genome sequence of williams  <dig>   <cit> , and the advent of high-throughput rna sequencing  by next-generation sequencing technology, have allowed researchers to take a novel look at the molecular interaction between soybean and p. sojae.
kim et al reported the first application of rna-seq for profiling gene expression in soybean in response to pathogen attack  <cit> .
in this study, the transcriptomes of two near isogenic lines , one resistant and one susceptible to bacterial leaf pustule, were analyzed  <dig>   <dig>  and  <dig> hpi and a total of  <dig>  differentially expressed genes , including a set of defense response genes, were identified.
nils are pure breeding lines that are developed by backcrossing a donor line with the recurrent parent for at least five generations to achieve introgression of a desired trait.
as such, they share more than 98% genetic identity with the recurrent parent, except for the region where the desired gene is located.
in order to determine the molecular mechanisms responsible for rps-mediated defense and to understand molecular basis for diverse responses to the pathogen, we analyzed the transcriptomes of  <dig> nils, each with a unique rps gene/allele, along with the susceptible recurrent parent williams, pre- and post-inoculation with a race  <dig> isolate of p. sojae .
RESULTS
symptom development in  <dig> nils and the recurrent susceptible parent inoculated with p. sojae
the  <dig> soybean nils carrying rps1-a, rps1-b, rps1-c, rps1-k, rps3-a, rps3-b, rps3-c, rps <dig>  rps <dig>  or rps <dig>  within the williams background, provided a unique resource for investigating the common and specific defense responses mediated by individual rps genes against p. sojae .
phenotypic reactions of the  <dig> nils and williams to p. sojae  <dig> days post-inoculation  with the pathogen were evaluated under greenhouse conditions.
in each of three experimental replications, approximately half the number of seedlings from each line was inoculated with race  <dig> of p. sojae.
the remaining seedlings were “mock” inoculated in the same manner without the pathogen.
in each experimental replicate, williams was susceptible  to p. sojae, while all nils were resistant .
nils containing rps1-a, rps1-b, rps1-c, rps1-k and rps <dig> displayed 100% survival when challenged with the pathogen.
in contrast, nils containing rps3-a, rps3-b, rps3-c, rps <dig> and rps <dig> showed a slight variation in the proportion of surviving seedlings across the three replicates, which may be attributed to minor differences in environmental conditions.
despite this variation, the percentage of surviving seedlings in each replicate was equal to or greater than 75%, which is generally used as a criterion for defining a pure line resistance  <cit> .
all mock-inoculated seedlings were asymptomatic of prr .
transcriptional changes in  <dig> nils and the recurrent susceptible parent in response to p. sojae
rnas, representing the entire transcriptomes of p. sojae inoculated and mock-inoculated seedlings from each of the  <dig> nils and williams  <dig> hpi with the pathogen, were sequenced and  <dig>  to  <dig>  million raw reads were generated for individual samples .
trimming adaptor sequences and elimination of low quality reads and reads shorter than 101 bp, resulted in  <dig>  to  <dig>  million clean reads for each sample.
among these,  <dig> % to  <dig> % per sample were uniquely mapped to the soybean reference genome v <dig>  .
a
b
c
areads with a quality score less than  <dig> and length less than  <dig> nucleotides were excluded.
breads uniquely mapped to g. max genome assembly  using tophat  <dig>  software  <cit> .
cpercentage of uniquely mapped reads out of clean reads.
based on the  <dig>  high-confidence gene models annotated in the soybean reference genome  <cit> , the relative change in the abundance of reads mapped to the same genes for inoculated and mock-treated samples, and the criteria for defining degs pre- and post-inoculation, a total of  <dig>  non-redundant gene models were identified as degs in at least one of the  <dig> soybean lines.
the numbers of degs identified between williams and the  <dig> nils or among the  <dig> nils vary greatly.
for example,  <dig>  to  <dig>  degs were found in individual nils containing rps1-a, rps1-b, rps1-c, or rps1-k, while  <dig>  to  <dig>  degs were detected in individual nils containing rps3-a, rps3-b, rps3-c, rps <dig>  rps <dig> or rps <dig> .
among the  <dig> nils,  <dig>  to  <dig>  degs were up-regulated, while  <dig> to  <dig>  degs were down-regulated in response to inoculation with the pathogen .
in comparison,  <dig>  up-regulated and  <dig>  down-regulated degs were identified in williams upon inoculation with p. sojae.
to validate degs profiled by rna-seq analysis, six detected degs per soybean line  were randomly chosen for quantitative rt-pcr  analysis with the same rna samples as used for rna-seq.
subsequently, the patterns of differential expression of these genes detected by rna-seq and qrt-pcr were compared.
as shown in additional file  <dig>  significant correlations between the patterns of degs detected by the two approaches were observed for each set of genes chosen from  <dig> individual lines , ranging from  <dig>  to  <dig> ), indicating the reliability of degs identified by rna-seq .
characterization of genes associated with incompatible, compatible, and basal interactions
in an attempt to decipher the molecular basis of resistance and susceptibility to p. sojae, we applied a comparative transcriptomics approach to identify degs specifically associated with a host response.
three major groups of genes were classified: 1) incompatible interaction genes , are degs identified in nils and associated with resistance; 2) compatible interaction genes  are degs identified in williams and specifically associated with the susceptible response; and 3) basal interaction genes , which are degs shared by all nils and williams .
a total of  <dig>  non-redundant iigs,  <dig> cigs, and  <dig> bigs were identified .
the number of up-regulated and down-regulated iigs range from  <dig> to  <dig>  and from  <dig> to  <dig> , respectively, among the  <dig> nils.
of the  <dig>  cigs,  <dig> were up-regulated and  <dig> were down-regulated; and of the  <dig> bigs,  <dig> were up-regulated and  <dig> were down-regulated .
clusters of rps-mediated iigs
to understand how transcriptomic changes mediated by different rps genes/alleles overlap, we performed hierarchical cluster analysis using pvclust, an r package for assessing the uncertainty in hierarchical clustering  <cit> , with log <dig> fold change  of the  <dig>  iigs identified in at least one of the ten nils.
the nils were grouped into three clusters, clusters i, ii, and iii .
cluster i  consisted of the nil containing rps1-a only.
cluster ii  was composed of those nils containing rps1-b, rps1-c and rps1-k, while cluster iii  included those nils containing rps3-a, rps3-b, rps3-c, rps <dig>  rps <dig>  and rps <dig>  this clustering of iigs was further supported by the hierarchical cluster structure of the ten nils revealed by heatmap analysis  <cit>  .
in addition, the numbers of iigs identified for each individual nil, were unevenly distributed among different clusters .
for example,  <dig>   <dig> - <dig> , and 556- <dig> iigs were found in c-i, c-ii, and c-iii, respectively.
the numbers of iigs in nils containing different alleles at a same gene locus also varied .
the iigs among the three clusters were analyzed by both broad range comparison, where all the genes within a cluster were counted, and narrow range comparison, where only those genes shared by the nils within a cluster were counted.
broad range comparison showed that  <dig> up-regulated and  <dig> down-regulated iigs were shared by the three clusters, whereas  <dig>   <dig>  and  <dig>  iigs were unique to nils in clusters i, ii, and iii, respectively .
in contrast, narrow range comparison indicated that only  <dig> up-regulated and  <dig> down-regulated iigs were shared among the three clusters .
the annotation of these  <dig> iigs is listed in table  <dig>
putative functions of degs based on gene ontology  analysis
to understand the functional components involved in molecular responses to p. sojae, we annotated the putative products encoded by iigs, cigs, and bigs based on go analysis .
these degs were grouped into seven categories: 1) response to biotic or abiotic stress; 2) biological regulation; 3) growth and development; 4) transport; 5) metabolism; 6) miscellaneous; and 7) unclassified.
for iigs, the majority of annotated genes fell into the category of “response to biotic stress and abiotc stress” .
this pattern was also observed for cigs and bigs .
of the  <dig> iigs shared by the  <dig> nils,  <dig>   <dig>  and  <dig>  were found to be related to “response to biotic or abiotic stress”, “biological regulation”, and “metabolism”, respectively .
the remaining four shared genes could not be classified into any functional category.
putative transcription factors  involved in molecular responses to p. sojae
it is documented that tfs play important roles in plant defense and stress responses.
as such, we annotated and analyzed putative tfs in the sets of degs detected in this study based on a soybean tf database that is composed of  <dig>  predicted tfs  <cit> .
of the  <dig>  non-redundant iigs,  <dig> up-regulated and  <dig> down-regulated genes were annotated as putative tfs.
the up-regulated and down-regulated iig tfs range from  <dig> to  <dig> and from  <dig> to  <dig>  respectively, among the  <dig> nils .
the number of iig tfs also varied among the three clusters, with  <dig>   <dig>  and  <dig> in c-i, c-ii, and c-iii, respectively.
the most abundant ones along these iig tfs  were of the wrky family, accounting for  <dig> % of all iig tfs identified in c-i,  <dig> - <dig> % in c-ii, and  <dig> - <dig> % in c-iii .
only three up-regulated iig tfs  and one down-regulated tf  were found to be shared by all the  <dig> nils .
of the  <dig>  cigs identified in williams,  <dig> up-regulated and  <dig> down-regulated genes were found to be putative tfs .
the largest proportion of up-regulated cig tfs belonged to the myb/hd-like family , followed by the ap2-erebp family .
in contrast, members of the ap2-erebp family represented the largest number  of down-regulated cig tfs, followed by myb/hd-like  and bhlh .
none of these  <dig> up-regulated cig tfs belonged to wrky family.
of the  <dig> non-redundant bigs shared by all the  <dig> soybean lines,  <dig> up-regulated and  <dig> down-regulated ones were found to be tfs .
among the up-regulated big tfs, the most abundant ones are of the wrky family , followed by the myb/hd-like family .
knowledge-based comparative analysis of rps gene-mediated defense signaling pathways
the degs showing homologies to previously identified protein genes responsible for pathogen recognition and defense, or defense-related phytohormone signaling genes were further analyzed.
overall, these degs exhibited diverse patterns of distribution among the three nil clusters .
for example, of the  <dig> up-regulated iigs homologous to previously reported defense/stress signaling genes,  <dig> were found only in nils within cluster iii.
in contrast, only one  of the two rboh b gene homologs was found to be up-regulated in all  <dig> nils.
it is notable that the majority of the putative degs involved in defense-related phytohormone signaling pathways showed distinct or opposite patterns of variation in gene expression between williams and nils, particularly the nils within the cluster iii .
previous studies indicated that the resistance to p. sojae in soybean was mediated by sa signaling  <cit> , with the npr <dig> as the key component of sa-mediated signaling  <cit> .
in this study, two npr1-like iigs  were identified .
however, these two genes differ from the two npr1-like genes reported by sandhu et al  <cit> .
in the later study, they studied soybean leaves four dpi with p. sojae race  <dig> while, in the present study, we studied stems  <dig> hpi with p. sojae race  <dig>  as such, these two studies are less comparable.
as an antagonist of sa, the ja pathway is repressed by jaz proteins in arabidopsis <cit> .
several jaz homologs were identified as iig and/or cig, including homologs of jaz <dig>  jaz <dig>  jaz <dig> and jaz <dig> .
these homologs showed a consistent expression pattern that was up-regulated nils and down-regulated in williams.
in contrast to the jaz proteins, jar <dig>  appears to be required for the activation of the ja signaling pathway in arabidopsis <cit> .
we found that a soybean jar <dig> homolog  was up-regulated in williams upon inoculation with the p. sojae.
these observations suggest that the ja signaling pathway may have played opposite roles in phytohormone signaling between incompatible and compatible interactions.
the et signaling pathway is generally considered to work cooperatively with ja in response to necrotrophic pathogens  <cit> .
in arabidopsis, et receptors etr <dig> <cit>  and ein <dig> <cit>  were both negative regulators of et signaling, and an ubiquitin/proteasome pathway for the degradation of ein <dig> was mediated by an f-box protein, ebf <dig>  to negatively regulate et responses  <cit> .
we found that one homolog of etr <dig> and two homologs of ein <dig> were down-regulated in nils, while another homolog of etr <dig> was up-regulated in williams.
in addition to etr <dig> and ein <dig>  we identified five ebf <dig> homologs that were down-regulated in all nils and up-regulated in williams , suggesting that the et signaling response to the pathogen was repressed during compatible interactions but activated during incompatible interactions.
in addition to the three major phytohormones described above, br have also been found to enhance disease resistance against viral and bacterial pathogens in tobacco and rice  <cit> .
bak <dig> is a crucial component of br signaling, and can interact with lrr-rlk  genes such as fls <dig> to promote their function in plant defense responses  <cit> .
as shown in figure  <dig>  three bak <dig> homologs were identified as iigs and all were up-regulated upon inoculation with the pathogen, suggesting a positive role of br signaling in defense response to p. sojae.
discussion
the nature of distinction in molecular immune response among nils
one of the most striking observations in this study was the distinction in defense response to inoculation with p. sojae among the ten resistant nils as revealed by comparative transcriptomic analysis.
this was reflected not only by the level of variation in the number of iigs  detected in individual nils, but also by the relative small numbers of up-regulated  and down-regulated  iigs shared by all nils .
in general, different rps genes recognize distinct avr genes in a p. sojae population  <cit> , but the avr determinants of different rps genes in a same isolate of the pathogen  may not be distinct.
one hypothesis that may explain the observed variations of degs among different nils may be differential timing and robustness in defense responses and signaling mediated by different rps genes/alleles  <cit> .
although the timing of gene expression mediated by different r genes in response to the same pathogen has not been characterized in plants, dramatic and rapid changes in gene expression after inoculation with, or following infection by a pathogen, have been observed  <cit> .
a recent study demonstrated that ~5% of degs were shared by soybean lines resistant to bacterial leaf pustule at  <dig> and  <dig> hpi with xanthomonas axonopodis <cit> .
in this study, the majority  of degs showed less than an eight-fold difference in expression levels upon inoculation with the pathogen when given the same cutoff time for each of the  <dig> lines.
such a low level of expressional changes and uniformed cutoff may have affected the detectability of shared degs among different lines.
in addition, the nils remain 1-2% genomic difference  from each other, which may have affected expression patterns of a small proportion of genes.
furthermore, inoculated regions of seedling stems, near which layers of cells respond to the pathogen, vary in size among different plants and nils.
such a variation may affect effective identification of degs.
therefore, the numbers of degs, and particularly iigs specific to each nil could be over-estimated.
nevertheless, the degs in each line were detected pre-and post-inoculation with the same pathogen, and thus the influence of genomic difference surrounding rps genes on our pipeline for detection of degs triggered by the pathogen may be minimal.
it is documented that resistance genes recognize pathogens either directly or indirectly by guarding a protein or using a decoy  <cit> .
in arabidopsis, rin <dig> is ‘guarded’ by resistance genes rpm <dig> and rps <dig>  which trigger the immune response  <cit> .
in soybean, two of four rin4- homologous genes appear to function as a heteromeric complex in mediating rpg1-b and rpm1-derived resistance to pseudomonas syringae, and silencing gmrin4a or gmrin4b in rpg1-b plants enhances basal resistance to virulent strains of p. syringae and the oomycete p. sojae <cit> .
in our study, a single rin <dig> homolog was found to be up-regulated in c-iii nils, but not in nils within c-i and c-ii.
if this rin <dig> homolog did encode the “guard” protein that was recognized by the rps genes in the c-iii nils, then the relatively low numbers of iigs identified among nils in c-i and c-ii, as compared with the c-iii nils, may be explained by the distinct “recognition” processes needed for triggering the immune response, which may result in variations in speed, timing, and magnitude of molecular responses to the pathogen  <cit> .
do patterns in transcriptomics distinguish rps genes from alleles?
an allele is defined as an alternative form of the same gene that is located at a specific position on a specific chromosome.
in general, different alleles of the same gene are determined by genetic tests for allelism.
however, due to the complexity of phenotypes, such as variation in the proportion of surviving plants of a pure nil that contains an rps gene, as observed in this study, such a test may not be effective in determining whether the resistance carried by two individual lines is controlled by different alleles or different genes.
moreover, many nbs-lrr gene models predicted in the williams <dig> genome are clustered in a small number of genomic regions  <cit> , which makes it more difficult to distinguish genes resistant to the same pathogen by classic genetic analysis, especially if they are physically adjacent or closely linked to each other.
the rps genes/alleles in the  <dig> nils have been previously mapped to three genomic regions: rps1-a, rps1-b, rps1-c, and rps1-k on chromosome  <dig>  <cit> , rps3-a, rps3-b and rps3-c on chromosome  <dig>  <cit>  and rps <dig>  rps <dig> and rps <dig> on chromosome  <dig>  <cit> .
however, because limited numbers of molecular markers were available and used when these genes/alleles were identified, the genetic distances between genes or alleles mapped in the same chromosomal regions have not been determined.
actually, the alleles at the rps <dig> and rps <dig> loci were simply designated based on their disease reaction to a set of p. sojae races.
as these three regions are highly enriched with nbs-lrr genes, according to the soybean reference genome, it is possible that some designated alleles of the same gene may be located at different loci.
as different alleles of the same gene are generally more identical to each other than to other genes, people are inclined to speculate that the defense response mediated by different alleles of a same gene may be more similar than those mediated by different genes.
at first glance, this hypothesis seems to be supported by the observation that the nils with rps3-a and rps3-b exhibited similar patterns of transcriptional changes upon inoculation with p. sojae, as did the nils with rps1-b and rps1-c .
however, the pattern of transcriptional changes mediated by rps3-c was found to be most similar to those mediated by rps <dig>  rps <dig>  and rps <dig>  thus, the patterns of transcriptomic changes do not appear to predict genetic similarity and difference among these rps genes and alleles, although we could not rule out the possibility that rps3-c and rps1-a are non-allelic to the rps <dig> and rps <dig> loci, respectively.
an integrated picture of molecular responses to p. sojae
based on analysis of putative defense-related genes identified in this study and the patterns of their expressions upon inoculation with the pathogen, we propose a hypothesis that four major phytohormone signaling pathways are involved in defense responses .
during incompatible interaction, the rps protein recognizes the avirulence effector of p. sojae, initiating signaling transduction that involves the sa, ja, et and br pathways.
the sa, et, and br pathways are activated, whereas the ja pathway is suppressed.
although the specific functions of individual genes encoding tfs belonging to the wrky family, as well as those genes for reactive oxygen species  and map kinase  cascades remain to be elucidated, it is apparent that some of the genes belonging to these families play pivotal roles in defense signaling transduction.
during compatible interaction, the avirulence effector of p. sojae induces a set of downstream responses that lead to disease development .
the signatures of the susceptible responses would include up-regulation of the ja pathway, suppression of et pathway and with no significant changes in sa and br pathways.
it seems possible that the defense responses might be delayed in the compatible interaction due to insufficient activation of the expression of genes related to ros and phytoalexin biosynthesis, as well as mapk signaling.
together, these data gained from a unique set of soybean nils provide a comprehensive picture regarding the molecular mechanisms underlying incompatible and compatible interaction between soybean and p. sojae, which shed light on the nature and timing of molecular responses mediated by individual rps genes.
CONCLUSIONS
comparative transcriptomic analysis of ten resistant nils and susceptible recurrent line williams with and without inoculation allowed us to identify degs associated with defense responses to p. sojae that were unique in each nil and common among all these nils, and thus depicted functional “fingerprints” of individual rps-mediated resistance.
further analysis revealed multiple branches of putative et, ja, ros, and mapk regulatory networks underlying the defense responses.
such responses exhibited dramatic variations among the soybean nils containing distinct rps genes/alleles.
we propose different timing of individual rps-mediated defense signaling to the same pathogen may be largely responsible for such variations.

 phytophthora root and stem rot is one of the most devastating diseases of soybean, causing nearly $ million in annual yield losses in the u.s. alone. prr is caused by the soil-borne, hemibiotrophic pathogen phytophthora sojae, and is most effectively controlled by rps genes. the resistance conferred by an rps gene is race specific, and the interaction between an rps gene and the corresponding avirulence gene in p. sojae, follows the gene-for-gene model. currently, rps genes/alleles from soybean and avr genes from p. sojae have been identified. like most resistance genes, the rps gene family encodes, or is predicted to encode nucleotide binding leucine rich repeat -type proteins, which are able to recognize the avr effector proteins of pathogens directly or indirectly to induce the appropriate defense response. the first evidence of a direct interaction between an r and avr protein was reported in the flax-melampsora lini pathosystem. indirect interactions, however, are more common. in these cases, the r protein appears to require the existence of a ‘guard protein’ or ‘decoy’ in order to recognize an avr protein. a classical example of this type of interaction is the rpm1-interacting protein, which is ‘guarded’ by rpm and rps proteins in arabidopsis. the recognition of pathogen effectors triggers an innate immunity response that is mediated by two distinct types of nb-lrr proteins, the toll-interleukin- receptor -nb-lrr proteins and the coiled-coil -nb-lrr proteins. the former requires eds, a central regulator of effector-triggered immunity, which functions together with pad. in contrast, the latter nb-lrr proteins are independent of eds but require ndr. the interaction among these intracellular proteins results in a regulation network of phytohormone signaling, which is mainly mediated by salicylic acid for biotrophic and hemibiotrophic pathogens, and jasmonic acid and ethylene for necrotrophic pathogens. in addition to sa, ja and et, other phytohormones such as brassinosteroids, abscisic acid, auxin, gibberellins and cytokinin also contribute to plant immune responses, with complex crosstalk between one another. it is suggested that the resistance to p. sojae conferred by rps genes is mediated by the sa signaling pathway, with the induction of sa-mediated systemic acquired resistance occurring several days post infection via expression of the gene npr. during this process, two putative regulators of the chromosome condensation gene family are down-regulated during the incompatible interaction. besides sa, exogenous treatment of 1-aminocyclopropane-1-carboxylic acid has been shown to enhance resistance while applications of ga and aba induce susceptibility, highlighting the complexity of phytohormone signaling pathways in response to attack by p. sojae. a microarray study of transcriptomes from one susceptible and two partially resistant soybean genotypes indicated that 97-99% of all detectable genes experienced transcriptional modulation five dpi in response to infection by p. sojae. however, the majority of these differences were less than two fold. another microarray study of transcriptomic changes in soybean revealed a peak in most defense related genes at hours post inoculation with p. sojae. recently, zhang et al conducted a proteomics study in which differentially expressed proteins were identified in soybean after infection with p. sojae. among these, were affected during the incompatible interaction, while the other were altered during the compatible interaction. these studies have contributed to our understanding of the interaction between soybean and p. sojae. nevertheless, what mechanisms underlie compatible and incompatible interactions, how molecular responses mediated by a variety of rps genes/alleles resemble or differ from one another, as well as the nature of these similarity or difference, remain largely unknown. access to the soybean genome sequence of williams, and the advent of high-throughput rna sequencing by next-generation sequencing technology, have allowed researchers to take a novel look at the molecular interaction between soybean and p. sojae. kim et al reported the first application of rna-seq for profiling gene expression in soybean in response to pathogen attack. in this study, the transcriptomes of two near isogenic lines, one resistant and one susceptible to bacterial leaf pustule, were analyzed and hpi and a total of differentially expressed genes, including a set of defense response genes, were identified. nils are pure breeding lines that are developed by backcrossing a donor line with the recurrent parent for at least five generations to achieve introgression of a desired trait. as such, they share more than 98% genetic identity with the recurrent parent, except for the region where the desired gene is located. in order to determine the molecular mechanisms responsible for rps-mediated defense and to understand molecular basis for diverse responses to the pathogen, we analyzed the transcriptomes of nils, each with a unique rps gene/allele, along with the susceptible recurrent parent williams, pre- and post-inoculation with a race isolate of p. sojae.  symptom development in nils and the recurrent susceptible parent inoculated with p. sojae the soybean nils carrying rps1-a, rps1-b, rps1-c, rps1-k, rps3-a, rps3-b, rps3-c, rps rps or rps within the williams background, provided a unique resource for investigating the common and specific defense responses mediated by individual rps genes against p. sojae. phenotypic reactions of the nils and williams to p. sojae days post-inoculation with the pathogen were evaluated under greenhouse conditions. in each of three experimental replications, approximately half the number of seedlings from each line was inoculated with race of p. sojae. the remaining seedlings were “mock” inoculated in the same manner without the pathogen. in each experimental replicate, williams was susceptible to p. sojae, while all nils were resistant. nils containing rps1-a, rps1-b, rps1-c, rps1-k and rps displayed 100% survival when challenged with the pathogen. in contrast, nils containing rps3-a, rps3-b, rps3-c, rps and rps showed a slight variation in the proportion of surviving seedlings across the three replicates, which may be attributed to minor differences in environmental conditions. despite this variation, the percentage of surviving seedlings in each replicate was equal to or greater than 75%, which is generally used as a criterion for defining a pure line resistance. all mock-inoculated seedlings were asymptomatic of prr. transcriptional changes in nils and the recurrent susceptible parent in response to p. sojae rnas, representing the entire transcriptomes of p. sojae inoculated and mock-inoculated seedlings from each of the nils and williams hpi with the pathogen, were sequenced and to million raw reads were generated for individual samples. trimming adaptor sequences and elimination of low quality reads and reads shorter than 101 bp, resulted in to million clean reads for each sample. among these, % to % per sample were uniquely mapped to the soybean reference genome v. a b c areads with a quality score less than and length less than nucleotides were excluded. breads uniquely mapped to g. max genome assembly using tophat software. cpercentage of uniquely mapped reads out of clean reads. based on the high-confidence gene models annotated in the soybean reference genome, the relative change in the abundance of reads mapped to the same genes for inoculated and mock-treated samples, and the criteria for defining degs pre- and post-inoculation, a total of non-redundant gene models were identified as degs in at least one of the soybean lines. the numbers of degs identified between williams and the nils or among the nils vary greatly. for example, to degs were found in individual nils containing rps1-a, rps1-b, rps1-c, or rps1-k, while to degs were detected in individual nils containing rps3-a, rps3-b, rps3-c, rps rps or rps. among the nils, to degs were up-regulated, while to degs were down-regulated in response to inoculation with the pathogen. in comparison, up-regulated and down-regulated degs were identified in williams upon inoculation with p. sojae. to validate degs profiled by rna-seq analysis, six detected degs per soybean line were randomly chosen for quantitative rt-pcr analysis with the same rna samples as used for rna-seq. subsequently, the patterns of differential expression of these genes detected by rna-seq and qrt-pcr were compared. as shown in additional file significant correlations between the patterns of degs detected by the two approaches were observed for each set of genes chosen from individual lines, ranging from to ), indicating the reliability of degs identified by rna-seq. characterization of genes associated with incompatible, compatible, and basal interactions in an attempt to decipher the molecular basis of resistance and susceptibility to p. sojae, we applied a comparative transcriptomics approach to identify degs specifically associated with a host response. three major groups of genes were classified: 1) incompatible interaction genes, are degs identified in nils and associated with resistance; 2) compatible interaction genes are degs identified in williams and specifically associated with the susceptible response; and 3) basal interaction genes, which are degs shared by all nils and williams. a total of non-redundant iigs, cigs, and bigs were identified. the number of up-regulated and down-regulated iigs range from to and from to, respectively, among the nils. of the cigs, were up-regulated and were down-regulated; and of the bigs, were up-regulated and were down-regulated. clusters of rps-mediated iigs to understand how transcriptomic changes mediated by different rps genes/alleles overlap, we performed hierarchical cluster analysis using pvclust, an r package for assessing the uncertainty in hierarchical clustering, with log fold change of the iigs identified in at least one of the ten nils. the nils were grouped into three clusters, clusters i, ii, and iii. cluster i consisted of the nil containing rps1-a only. cluster ii was composed of those nils containing rps1-b, rps1-c and rps1-k, while cluster iii included those nils containing rps3-a, rps3-b, rps3-c, rps rps and rps this clustering of iigs was further supported by the hierarchical cluster structure of the ten nils revealed by heatmap analysis. in addition, the numbers of iigs identified for each individual nil, were unevenly distributed among different clusters. for example, -, and 556- iigs were found in c-i, c-ii, and c-iii, respectively. the numbers of iigs in nils containing different alleles at a same gene locus also varied. the iigs among the three clusters were analyzed by both broad range comparison, where all the genes within a cluster were counted, and narrow range comparison, where only those genes shared by the nils within a cluster were counted. broad range comparison showed that up-regulated and down-regulated iigs were shared by the three clusters, whereas and iigs were unique to nils in clusters i, ii, and iii, respectively. in contrast, narrow range comparison indicated that only up-regulated and down-regulated iigs were shared among the three clusters. the annotation of these iigs is listed in table  putative functions of degs based on gene ontology analysis to understand the functional components involved in molecular responses to p. sojae, we annotated the putative products encoded by iigs, cigs, and bigs based on go analysis. these degs were grouped into seven categories: 1) response to biotic or abiotic stress; 2) biological regulation; 3) growth and development; 4) transport; 5) metabolism; 6) miscellaneous; and 7) unclassified. for iigs, the majority of annotated genes fell into the category of “response to biotic stress and abiotc stress”. this pattern was also observed for cigs and bigs. of the iigs shared by the nils, and were found to be related to “response to biotic or abiotic stress”, “biological regulation”, and “metabolism”, respectively. the remaining four shared genes could not be classified into any functional category. putative transcription factors involved in molecular responses to p. sojae it is documented that tfs play important roles in plant defense and stress responses. as such, we annotated and analyzed putative tfs in the sets of degs detected in this study based on a soybean tf database that is composed of predicted tfs. of the non-redundant iigs, up-regulated and down-regulated genes were annotated as putative tfs. the up-regulated and down-regulated iig tfs range from to and from to respectively, among the nils. the number of iig tfs also varied among the three clusters, with and in c-i, c-ii, and c-iii, respectively. the most abundant ones along these iig tfs were of the wrky family, accounting for % of all iig tfs identified in c-i, - % in c-ii, and - % in c-iii. only three up-regulated iig tfs and one down-regulated tf were found to be shared by all the nils. of the cigs identified in williams, up-regulated and down-regulated genes were found to be putative tfs. the largest proportion of up-regulated cig tfs belonged to the myb/hd-like family, followed by the ap2-erebp family. in contrast, members of the ap2-erebp family represented the largest number of down-regulated cig tfs, followed by myb/hd-like and bhlh. none of these up-regulated cig tfs belonged to wrky family. of the non-redundant bigs shared by all the soybean lines, up-regulated and down-regulated ones were found to be tfs. among the up-regulated big tfs, the most abundant ones are of the wrky family, followed by the myb/hd-like family. knowledge-based comparative analysis of rps gene-mediated defense signaling pathways the degs showing homologies to previously identified protein genes responsible for pathogen recognition and defense, or defense-related phytohormone signaling genes were further analyzed. overall, these degs exhibited diverse patterns of distribution among the three nil clusters. for example, of the up-regulated iigs homologous to previously reported defense/stress signaling genes, were found only in nils within cluster iii. in contrast, only one of the two rboh b gene homologs was found to be up-regulated in all nils. it is notable that the majority of the putative degs involved in defense-related phytohormone signaling pathways showed distinct or opposite patterns of variation in gene expression between williams and nils, particularly the nils within the cluster iii. previous studies indicated that the resistance to p. sojae in soybean was mediated by sa signaling, with the npr as the key component of sa-mediated signaling. in this study, two npr1-like iigs were identified. however, these two genes differ from the two npr1-like genes reported by sandhu et al. in the later study, they studied soybean leaves four dpi with p. sojae race while, in the present study, we studied stems hpi with p. sojae race as such, these two studies are less comparable. as an antagonist of sa, the ja pathway is repressed by jaz proteins in arabidopsis. several jaz homologs were identified as iig and/or cig, including homologs of jaz jaz jaz and jaz. these homologs showed a consistent expression pattern that was up-regulated nils and down-regulated in williams. in contrast to the jaz proteins, jar appears to be required for the activation of the ja signaling pathway in arabidopsis. we found that a soybean jar homolog was up-regulated in williams upon inoculation with the p. sojae. these observations suggest that the ja signaling pathway may have played opposite roles in phytohormone signaling between incompatible and compatible interactions. the et signaling pathway is generally considered to work cooperatively with ja in response to necrotrophic pathogens. in arabidopsis, et receptors etr and ein were both negative regulators of et signaling, and an ubiquitin/proteasome pathway for the degradation of ein was mediated by an f-box protein, ebf to negatively regulate et responses. we found that one homolog of etr and two homologs of ein were down-regulated in nils, while another homolog of etr was up-regulated in williams. in addition to etr and ein we identified five ebf homologs that were down-regulated in all nils and up-regulated in williams, suggesting that the et signaling response to the pathogen was repressed during compatible interactions but activated during incompatible interactions. in addition to the three major phytohormones described above, br have also been found to enhance disease resistance against viral and bacterial pathogens in tobacco and rice. bak is a crucial component of br signaling, and can interact with lrr-rlk genes such as fls to promote their function in plant defense responses. as shown in figure  three bak homologs were identified as iigs and all were up-regulated upon inoculation with the pathogen, suggesting a positive role of br signaling in defense response to p. sojae. discussion the nature of distinction in molecular immune response among nils one of the most striking observations in this study was the distinction in defense response to inoculation with p. sojae among the ten resistant nils as revealed by comparative transcriptomic analysis. this was reflected not only by the level of variation in the number of iigs detected in individual nils, but also by the relative small numbers of up-regulated and down-regulated iigs shared by all nils. in general, different rps genes recognize distinct avr genes in a p. sojae population, but the avr determinants of different rps genes in a same isolate of the pathogen may not be distinct. one hypothesis that may explain the observed variations of degs among different nils may be differential timing and robustness in defense responses and signaling mediated by different rps genes/alleles. although the timing of gene expression mediated by different r genes in response to the same pathogen has not been characterized in plants, dramatic and rapid changes in gene expression after inoculation with, or following infection by a pathogen, have been observed. a recent study demonstrated that ~5% of degs were shared by soybean lines resistant to bacterial leaf pustule at and hpi with xanthomonas axonopodis. in this study, the majority of degs showed less than an eight-fold difference in expression levels upon inoculation with the pathogen when given the same cutoff time for each of the lines. such a low level of expressional changes and uniformed cutoff may have affected the detectability of shared degs among different lines. in addition, the nils remain 1-2% genomic difference from each other, which may have affected expression patterns of a small proportion of genes. furthermore, inoculated regions of seedling stems, near which layers of cells respond to the pathogen, vary in size among different plants and nils. such a variation may affect effective identification of degs. therefore, the numbers of degs, and particularly iigs specific to each nil could be over-estimated. nevertheless, the degs in each line were detected pre-and post-inoculation with the same pathogen, and thus the influence of genomic difference surrounding rps genes on our pipeline for detection of degs triggered by the pathogen may be minimal. it is documented that resistance genes recognize pathogens either directly or indirectly by guarding a protein or using a decoy. in arabidopsis, rin is ‘guarded’ by resistance genes rpm and rps which trigger the immune response. in soybean, two of four rin4- homologous genes appear to function as a heteromeric complex in mediating rpg1-b and rpm1-derived resistance to pseudomonas syringae, and silencing gmrin4a or gmrin4b in rpg1-b plants enhances basal resistance to virulent strains of p. syringae and the oomycete p. sojae. in our study, a single rin homolog was found to be up-regulated in c-iii nils, but not in nils within c-i and c-ii. if this rin homolog did encode the “guard” protein that was recognized by the rps genes in the c-iii nils, then the relatively low numbers of iigs identified among nils in c-i and c-ii, as compared with the c-iii nils, may be explained by the distinct “recognition” processes needed for triggering the immune response, which may result in variations in speed, timing, and magnitude of molecular responses to the pathogen. do patterns in transcriptomics distinguish rps genes from alleles? an allele is defined as an alternative form of the same gene that is located at a specific position on a specific chromosome. in general, different alleles of the same gene are determined by genetic tests for allelism. however, due to the complexity of phenotypes, such as variation in the proportion of surviving plants of a pure nil that contains an rps gene, as observed in this study, such a test may not be effective in determining whether the resistance carried by two individual lines is controlled by different alleles or different genes. moreover, many nbs-lrr gene models predicted in the williams genome are clustered in a small number of genomic regions, which makes it more difficult to distinguish genes resistant to the same pathogen by classic genetic analysis, especially if they are physically adjacent or closely linked to each other. the rps genes/alleles in the nils have been previously mapped to three genomic regions: rps1-a, rps1-b, rps1-c, and rps1-k on chromosome, rps3-a, rps3-b and rps3-c on chromosome and rps rps and rps on chromosome. however, because limited numbers of molecular markers were available and used when these genes/alleles were identified, the genetic distances between genes or alleles mapped in the same chromosomal regions have not been determined. actually, the alleles at the rps and rps loci were simply designated based on their disease reaction to a set of p. sojae races. as these three regions are highly enriched with nbs-lrr genes, according to the soybean reference genome, it is possible that some designated alleles of the same gene may be located at different loci. as different alleles of the same gene are generally more identical to each other than to other genes, people are inclined to speculate that the defense response mediated by different alleles of a same gene may be more similar than those mediated by different genes. at first glance, this hypothesis seems to be supported by the observation that the nils with rps3-a and rps3-b exhibited similar patterns of transcriptional changes upon inoculation with p. sojae, as did the nils with rps1-b and rps1-c. however, the pattern of transcriptional changes mediated by rps3-c was found to be most similar to those mediated by rps rps and rps thus, the patterns of transcriptomic changes do not appear to predict genetic similarity and difference among these rps genes and alleles, although we could not rule out the possibility that rps3-c and rps1-a are non-allelic to the rps and rps loci, respectively. an integrated picture of molecular responses to p. sojae based on analysis of putative defense-related genes identified in this study and the patterns of their expressions upon inoculation with the pathogen, we propose a hypothesis that four major phytohormone signaling pathways are involved in defense responses. during incompatible interaction, the rps protein recognizes the avirulence effector of p. sojae, initiating signaling transduction that involves the sa, ja, et and br pathways. the sa, et, and br pathways are activated, whereas the ja pathway is suppressed. although the specific functions of individual genes encoding tfs belonging to the wrky family, as well as those genes for reactive oxygen species and map kinase cascades remain to be elucidated, it is apparent that some of the genes belonging to these families play pivotal roles in defense signaling transduction. during compatible interaction, the avirulence effector of p. sojae induces a set of downstream responses that lead to disease development. the signatures of the susceptible responses would include up-regulation of the ja pathway, suppression of et pathway and with no significant changes in sa and br pathways. it seems possible that the defense responses might be delayed in the compatible interaction due to insufficient activation of the expression of genes related to ros and phytoalexin biosynthesis, as well as mapk signaling. together, these data gained from a unique set of soybean nils provide a comprehensive picture regarding the molecular mechanisms underlying incompatible and compatible interaction between soybean and p. sojae, which shed light on the nature and timing of molecular responses mediated by individual rps genes.  comparative transcriptomic analysis of ten resistant nils and susceptible recurrent line williams with and without inoculation allowed us to identify degs associated with defense responses to p. sojae that were unique in each nil and common among all these nils, and thus depicted functional “fingerprints” of individual rps-mediated resistance. further analysis revealed multiple branches of putative et, ja, ros, and mapk regulatory networks underlying the defense responses. such responses exhibited dramatic variations among the soybean nils containing distinct rps genes/alleles. we propose different timing of individual rps-mediated defense signaling to the same pathogen may be largely responsible for such variations. 
comparisons of the degs between the nils and williams identified incompatible interaction genes and compatible interaction genes. we thus sequenced, analyzed, and compared the transcriptomes of near isogenic lines, each with a unique rps gene/allele, and the susceptible parent williams, pre- and post-inoculation with the pathogen. we hypothesize that different timing and robustness in defense signaling to the same pathogen may be largely responsible for such variations. phytophthora root and stem rot of soybean, caused by phytophthora sojae, is controlled by rps genes. hierarchical cluster and heatmap analyses consistently grouped the nils into three clusters: cluster i, cluster ii and cluster iii, suggesting an overlap in rps-induced defense signaling among certain nils. however, little is known regarding the rps-induced molecular responses to p. sojae and how they actually overlap. of particular interest are dramatic variations of detected degs, putatively involved in ethylene -, jasmonic acid -, ros-, and mapk- signaling, among these soybean nils, implicating their important roles of these signaling in differentiating molecular defense responses. gene ontology analysis revealed associations between members of the wrky family and incompatible reactions and between a number of phytohormone signaling pathways and incompatible/compatible interactions. this study characterized genes and multiple branches of putative regulatory networks associated with resistance to p. sojae in ten soybean nils, and depicted functional “fingerprints” of individual rps-mediated resistance responses through comparative transcriptomic analysis. these associations appear to be distinguished according to the nil clusters. a total of differentially expressed genes were identified in williams versus to degs in individual nils upon inoculation with the pathogen.
 phytophthora root and stem rot of soybean, caused by phytophthora sojae, is controlled by rps genes. however, little is known regarding the rps-induced molecular responses to p. sojae and how they actually overlap. we thus sequenced, analyzed, and compared the transcriptomes of near isogenic lines, each with a unique rps gene/allele, and the susceptible parent williams, pre- and post-inoculation with the pathogen.  a total of differentially expressed genes were identified in williams versus to degs in individual nils upon inoculation with the pathogen. comparisons of the degs between the nils and williams identified incompatible interaction genes and compatible interaction genes. hierarchical cluster and heatmap analyses consistently grouped the nils into three clusters: cluster i, cluster ii and cluster iii, suggesting an overlap in rps-induced defense signaling among certain nils. gene ontology analysis revealed associations between members of the wrky family and incompatible reactions and between a number of phytohormone signaling pathways and incompatible/compatible interactions. these associations appear to be distinguished according to the nil clusters.  this study characterized genes and multiple branches of putative regulatory networks associated with resistance to p. sojae in ten soybean nils, and depicted functional “fingerprints” of individual rps-mediated resistance responses through comparative transcriptomic analysis. of particular interest are dramatic variations of detected degs, putatively involved in ethylene -, jasmonic acid -, ros-, and mapk- signaling, among these soybean nils, implicating their important roles of these signaling in differentiating molecular defense responses. we hypothesize that different timing and robustness in defense signaling to the same pathogen may be largely responsible for such variations. comparative transcriptomicsresistance to phytophthora sojaesoybean 