BACKGROUND
plants are constantly exposed to microbes in natural and agricultural ecosystems.
to be pathogenic, most microbes must access the plant’s internal tissues, either by penetrating the plant surface directly or by entering through wounds or natural openings such as stomata  <cit> .
furthermore, the pathogen must pierce through the cell wall, and often the host plasma membrane, to be infective.
plants wield typical basal and resistance  gene-mediated defense mechanisms and mount a defensive response to pathogen attack to delay or arrest potential pathogenic microorganism growth  <cit>  through pathogen-associated molecular pattern-triggered immunity  and effector-triggered immunity  systems.
current analysis of plant immunity has moved towards an integrated view of plant–pathogen interactions  <cit> .
using oligonucleotides, cdna microarrays and transcriptome analysis, many comprehensive analyses of stress-induced changes in gene expression in model plants with small genomes have been undertaken, and consequently many genes associated with pathogenic stress responses in plants are known.
however, numerous fundamental molecular aspects remain unknown, such as the unique and common signaling components of pti and eti, and the induced host components that effect resistance.
a global gene expression approach is useful for elucidating the molecular mechanisms of wheat–fungus interactions, particularly the application of next-generation sequencing to study important non-model host–pathogen systems, such as wheat rusts  <cit> .
stripe rust  and powdery mildew  are important fungal diseases of wheat  in many wheat-growing regions of the world.
the filamentous powdery mildew and rust fungi develop haustoria within the lumen of the host cell, which function to absorb nutrients.
a number of studies on wheat–rust interactions have been carried out using the affymetrix® genechip® wheat genome array   <cit>  and cdna-aflp analysis  <cit> .
powdery mildew infection has been studied in grapevine  <cit> , hordeum
<cit>  and arabidopsis
<cit>  with the affymetrix genechip wheat genome array, cdna-aflp, and cdna microarrays, respectively.
however, a complete gene expression profile for the response to the stripe rust and powdery mildew pathogens in the same wheat germplasm is still lacking.
compared with other plants with smaller genomes, the use of most genetic and molecular techniques to study genes involved in wheat–pst or wheat–bgt interactions has been limited, because hexaploid wheat has a large and complex genome and its transformation is difficult, and both fungi show sexual reproduction and irreversible deletion of genes dispensable for biotrophy  <cit> .
use of the affymetrix genechip wheat genome array is often restricted by the known gene sequences arrayed on the chip, with a limited number of expressed sequence tags  non-specific to different wheat materials, whereas cdna-aflp is universally applicable for any organism or interaction without prior sequence information required, although false positives may frequently be observed because of technical reasons.
in contrast, rna sequencing  is not dependent on pre-existing databases of expressed genes and, therefore, provides an unbiased view of gene expression profiles.
in the present study, using illumina deep sequencing, we undertook a transcriptome analysis of leaves from different plants of the same wheat line, n <dig>  subjected to both pst and bgt stress treatments.
the main objective was to identify co-regulated mrnas that show a change in expression pattern after inoculation with pst or bgt, and to identify mrnas specific to the fungal stress response.
this is the first study to compare two biotic stresses using a global expression profiling strategy in the same wheat genetic background.
through a comparison of different pathogen stress treatments with biological replicates, we reasoned that we could better identify both shared and stimulus-specific responses.
RESULTS
in the present study, cdna libraries were constructed from leaves inoculated with pst or bgt at  <dig>   <dig>   <dig> and 3 days post-inoculation  with three biological replicates, and then sequenced using the illumina hiseq™  <dig> platform.
after cleaning and checking the read quality, we obtained almost  <dig>  million 101 bp paired-end clean reads.
among the clean reads, 100% had quality scores at the cycle q <dig> level .
the data sets were deposited in the ncbi sequence read archive .
because of deficiencies in the reference genome sequence, these reads were de novo assembled using the trinity platform software, resulting in  <dig>  unigenes with n <dig> length of 743 bp, of which  <dig>  unigenes were annotated after blast searches of the genbank nr, swissprot, kegg, cog and go databases.
the length of  <dig>  unigenes was more than 1 kb and contained  <dig>  simple sequence repeat sites.
as an aid to examining gene expression level distributions, the reads per kilobase of exon model per million of aligned reads  values were calculated as normalized expression estimates for each gene model in each sample.
also, correlation coefficients were calculated based on the log-transformed rpkm values after eliminating genes with a zero count in any of the three replicates.
the correlation coefficient values ranged from  <dig>  to  <dig>  , indicating there was a strong correlation between replicates.
a generalized linear model was applied based on a negative binomial distribution and an overall test was conducted to determine which genes varied in expression among any of the seven treatment groups, where a treatment group was defined by a strain-by-induction condition combination .
setting fold change ≥ <dig> and the false discovery rate  at  <dig> % using the method of benjamini and hochberg  <cit> , statistical analysis with deseq identified  <dig>  genes as differentially expressed among the six treatment groups compared with non-inoculated leaves as the control.
of these genes, the length of  <dig>  genes exceeded 1 kb.
table  <dig> lists details of the differentially expressed genes  and annotation numbers that were detected at the different time points in response to the fungal stress treatments.table  <dig>
statistical table of differentially expressed genes number and annotated degs
num
up
down
nr
swissprot
go
kegg
cog
note: p represents powdery mildew e <dig> inoculation condition; s represents stripe rust pathogen cyr  <dig> inoculation; ck represents the samples without pathogen stress as control.
the number of  <dig>   <dig> and 3: n <dig> infected at  <dig>   <dig> and  <dig> dpi, respectively.
to evaluate the reliability of our rna-seq and de novo assembled results, quantitative real-time pcr  was performed on eight selected genes of interest using rna samples as a fourth replication.
these genes were selected to represent a wide range of expression levels and patterns under fungal infection.
six gene expression patterns in response to bgt stress showed strong agreement and were highly correlated in the rna-seq and qpcr analyses .
additionally, six previously studied full-length genes were aligned with the present assembled unigene database and showed identities of up to  <dig> %.
more robust response to bgt- than pst-induced stress in bread wheat
although transcript levels do not necessarily reflect the amount of final active protein product, for simplicity, increased transcript abundance is often referred to as induced gene expression.
genes that were differentially expressed between non-inoculated and inoculated leaves were determined with rna-seq.
cluster analysis showed that the expression profiles of degs varied significantly in response to bgt and pst in the n <dig> genotype .figure  <dig>
hierarchical clustering of differentially-expressed genes  in resistant bread wheat accession n <dig> to
bgt
and
pst
infection.
the signal ratios are shown in a red-green color scale, where red represents up-regulation and green represents down-regulation.
each column  represents the mean expression value of the rna-seq obtained from three biological replicates and each row represents a differentially-expressed genes.
ck: resistant wheat n <dig> exempt from pathogen stress as control; s <dig>  s <dig> and s3: n <dig> infected with stripe rust pathogen cyr  <dig> at  <dig>   <dig> and  <dig> dpi, respectively; p <dig>  p <dig> and p3: n <dig> infected with powdery mildew pathogen e <dig> at  <dig>   <dig> and  <dig> dpi, respectively.
for the bgt test, using inoculated leaf samples, expression of  <dig>  degs was detected, of which  <dig>  genes exceeded 1 kb.
as shown in table  <dig> and , comparison of inoculated and non-inoculated leaves showed that  <dig> ,  <dig>  and  <dig>  genes were differentially expressed at  <dig>   <dig>  and  <dig> dpi, respectively.
of these genes,  <dig>  and  <dig>  were up- and down-regulated at  <dig> dpi,  <dig>  and  <dig>  at  <dig> dpi, and  <dig>  and  <dig>  at  <dig> dpi, respectively.
in the pst cyr  <dig> inoculation treatment,  <dig>  degs showed significantly lower expression than that in the bgt e <dig> stress treatment.
of these genes,  <dig>  ,  <dig> , and  <dig>   were differentially expressed at  <dig>   <dig>  and  <dig> dpi, respectively.
the number of up-regulated genes at  <dig> dpi was almost double those at  <dig> and  <dig> dpi after infection by bgt, whereas the number at  <dig> dpi was almost three-times higher than those at  <dig> and  <dig> dpi after pst infection.
this substantiated the contention that  <dig> dpi is the most important time point for wheat to respond to fungal attack by expression of race-specific resistance genes.
to further test the variation in response to pst and bgt inoculation, we compared gene expression in response to pst cyr  <dig> versus bgt e <dig> inoculation with fold change ≥ <dig> and fdr at  <dig> %.
additional file 1: figure s <dig> shows that  <dig>  genes were differentially expressed in response to bgt infection in comparison with pst infection, of which  <dig>  and  <dig> genes were up- and down-regulated at  <dig> dpi,  <dig>  and  <dig>  at  <dig> dpi, and  <dig>  and  <dig>  at  <dig> dpi, respectively.
this result indicated that the wheat resistance response to bgt inoculation triggered more robust alteration in gene expression than that observed in response to pst inoculation.
gene characterization of stimulus-specific responses to pstor bgt
to facilitate a closer comparison of pst-induced genes with bgt-induced genes, a pairwise comparison was conducted after degs were annotated using kegg classifications and go assignments.
all resistance-specific genes were analyzed using mas to identify the metabolic pathways in which they function.
the go enrichment results are shown in additional file 1: tables s <dig> , s <dig> , and s <dig> .
when the fdr-corrected p-value was set at  <dig> , seven significant enriched kegg pathways for bgt-induced stress and four for pst-induced stress versus the non-stressed control were identified.
two kegg pathways overlapped between the two infection treatments , namely ubiquinone and other terpenoid-quinone biosynthesis and photosynthesis-antenna proteins.
as there were insufficient pathway annotations in kegg, the pathways were retrieved using the degs comparing pst infection versus bgt infection directly at the specific time points and are listed in additional file 1: table s <dig>  taken together,  <dig> kegg pathways were identified.table  <dig>
significant kegg pathway of stimulus-specific responses on
pst
or
bgt
note: all kegg pathways were retrieved from mas <dig>  molecule annotation system.
some plant-specific pathways were computed repeat by extracting genes from comparing pst with bgt stress.
the values of correct-p were given and those less than  <dig>  were marked with asterisks.
some enriched kegg pathways with the high enrichment factor were noted and the factor was given in brackets.
the indistinctive pathway that was listed  had p-value < <dig>  for analysis degs from pst vs bgt.
phenylalanine metabolism, phenylpropanoid biosynthesis, alpha-linolenic acid metabolism, flavonoid biosynthesis, and phenylalanine, tyrosine and tryptophan biosynthesis showed specific significant differential enrichment in wheat in response to bgt-induced stress, whereas carotenoid biosynthesis and carbon fixation in photosynthetic organisms showed the opposite differential performance in response to pst-induced stress .
the analysis of kegg pathways showed that  <dig> specific pathways responded to bgt infection and four specific pathways responded to pst infection, although the p values were not significant for those present in the kegg database .
under pst-induced stress, no degs were detected in the mismatch repair, non-homologous end-joining, nucleotide excision repair, dna replication, and rna polymerase pathways, which are all involved in genetic information processing.
moreover, abcb <dig>  in abc transporters, mitochondrial trans-2-enoyl-coa reductase  in fatty acid elongation, lipoyl  transferase  in lipoic acid metabolism, biotin synthase  in biotin metabolism, glutamate decarboxylase, gamma-glutamyl transpeptidase and cysteamine dioxygenase in taurine and hypotaurine metabolism, and n-glycan biosynthesis were specifically enriched in response to bgt inoculation.
the number of dysregulation enzymes reaches to eight in n-glycan biosynthesis especially.
in addition, seven distinct kegg pathways were retrieved with mas <dig>  after comparative analysis of genes differentially expressed in response to pst- and bgt-induced stress, namely nitrogen metabolism, diterpenoid biosynthesis, stilbenoid, diarylheptanoid and gingerol biosynthesis, valine, leucine and isoleucine degradation, photosynthesis, benzoxazinoid biosynthesis, and ribosome pathway.
genes encoding components of conserved protein complexes, such as the proteasome and ribosome, are coexpressed in other organisms and show very similar expression profiles  <cit> .
surprisingly, half of the enzymes  showed dysregulated expression in the ribosome pathway in response to bgt, whereas  <dig> dysregulated enzymes were detected in response to pst-induced stress .
this result indicated that the ribosome pathway is one of the most complex and important pathways regulating genes antagonistic to fungal infection, and that further differential internal remodeling in response to different fungal signals is required.
this analysis showed that the nine functional pathways with p-values < <dig>  after fdr correction were significantly associated with resistance to fungal infection, whereas the stimulus-specific responses were more robust and  <dig> pathways included degs in comparison of the pst and bgt treatments at  <dig>   <dig>  and  <dig> dpi .
these results further confirmed the differences in gene regulation and pathways involved in wheat–pst interaction from those involved in wheat–bgt interaction.
identification of degs shared by pstand bgtinfection responses
from aforementioned information, a considerable number of degs would have overlapped between the bgt and pst inoculation treatments.
those genes that were significantly regulated in the defense response to both fungi were identified from among the  <dig>  overlapping degs with annotated information and are listed in table  <dig>  the overlapping degs at each time point are shown in a venn diagram .
these genes were annotated into  <dig> go biological processes with p-values < <dig>  and considerably dysregulated gene numbers.
protein tetramerization, oxidation-reduction process, xenobiotic metabolic process, response to hypoxia, response to abiotic stimulus, starch biosynthetic process, and cholesterol biosynthetic process were not only significantly enriched but also showed a higher number of dysregulated genes than expected  .
the number of dysregulated genes in the categories of regulation of t cell-mediated cytotoxicity, ketone body biosynthetic process, and ergosterol biosynthetic process was doubled that expected , although there seemed to be a relatively smaller number of degs in these three pathways than in the other above-mentioned ones.
the go pedigree analysis indicated that the third main go hierarchy of biological processes in which the shared genes may participate consisted of response to single-organism stimuli and metabolic process.table  <dig>
go characterization of overlapped degs responding to
pst
and
bgt
stress
inspired by similar expression patterns hinting at analogous gene functions,  <dig>  genes induced in response to both bgt- and pst-induced stress were screened with a model-based clustering algorithm to identify distinct gene expression profiles among the identified degs and to choose a total of eight clusters, to maintain as few tight clusters as possible while including most of the distinct expression patterns .
each gray line in figure  <dig> represents the expression pattern for an individual gene, and the single black line indicates the average behavior for induced genes in each cluster.
surprisingly, these overlapping genes showed different expression patterns and complex changes in response to pst infection.
this finding illustrated that these genes may perform different functions in response to different stimuli, and implied that wheat triggered distinct genes and regulatory networks to antagonize bgt and pst infection.figure  <dig>
clustering.
the expression patterns of  <dig> overlapped de genes for  <dig> clusters.
the horizontal axis indicates each time point and pathogen stress induction condition.
the number  <dig>   <dig> and  <dig> mean that n <dig> was infected at  <dig>   <dig> and  <dig> dpi, respectively.
p represents powdery mildew e <dig> inoculation condition; s represents stripe rust pathogen cyr  <dig> inoculation.
the vertical axis indicates the log <dig> fold change calculated between each condition and non-inoculation treatment.
each gray line symbolizes the expression pattern of one gene, and the bold back line illustrates the average expression pattern of all genes in each cluster under bgt or pst stress.
our results for the overlapping degs revealed changes in approximately 15% of the transcriptome, with most changes classified as stimulus-specific.
this view illustrates the “fluid” nature of the transcriptome and the challenge faced in understanding the complexity of any given stress response.
key fungal defense-related genes and pathways
transcript profiling is capable of revealing pathways of gene expression involved in a defense response, therefore detailed gene-by-gene analysis should be informative because the temporary kegg pathway and go annotations were incomplete.
a total of  <dig>  degs were identified, and the range of deg expression ratios was - <dig>  to  <dig>  for pst stress-induced genes and - <dig>  to  <dig>  for bgt stress-induced genes.
to identify the degs showing the greatest changes in transcript levels, the top 1% of up- and down-regulated genes in the bgt and pst treatments at each time point  were analyzed.
comparison of data for n <dig> pst- and bgt-inoculated plants with mock-inoculated data revealed gene expression changes that included basal defense transcripts and transcripts specific to the establishment of a biotrophic interaction with fungi.
twelve of the  <dig> top differentially expressed transcripts significant for treatment were induced by pst inoculation at  <dig> dpi, whereas  <dig> were repressed ,  <dig> were down-regulated and three were induced by pst infection at  <dig> dpi, while eight were repressed and five were up-regulated in response to pst infection at  <dig> dpi.
ninety-five genes were ranked in the top 1% of genes dysregulated by bgt inoculation, of which  <dig> were induced and four were repressed at  <dig> dpi,  <dig> were up-regulated and  <dig> were down-regulated at  <dig> dpi, and  <dig> were up-regulated and  <dig> were down-regulated at  <dig> dpi.
as shown in additional file 1: table s <dig>  defense-related transcripts accounted for  <dig> %  of the dysregulated transcripts, while  <dig>  were involved in signal transduction,  <dig>  in oxidation-reduction,  <dig>  in biological regulation,  <dig>  in metabolic processes,  <dig>  in protein/carbohydrate transport,  <dig> in development,  <dig>  in the tricarboxylic acid cycle,  <dig> in protein modification, and  <dig>  were of unknown/unclear function.
comparing the top 1% transcriptomes after pst and bgt inoculation, most gene expression patterns showed opposing patterns in the two treatments , especially at the same time point.
those genes induced by bgt infection were usually repressed in the same genotype by pst infection, and vice versa.
for example, this pattern was shown by l-type lectin-domain containing receptor kinase ix , hemoglobin  <dig> , luminal-binding protein , delta-cadinene synthase isozyme xc <dig> , and hypothetical protein mtr_7g <dig> .
plants sharing the microbe gene fragments antagonistic to fungal infection
blast searches of the nr and swissport databases showed that  <dig>  annotated unigenes matched genes from aegilops tauschii , triticum urartu , hordeum vulgare , brachypodium distachyon , oryza sativa , and marssonina brunnea , while  <dig> unigenes  were related to t. aestivum in the current databases.
interestingly,  <dig> unigenes matched genes from puccinia graminis , of which  <dig> were expressed differentially in the pst treatment, while  <dig> unigenes were similar to apple stem pitting virus  genes and  <dig> were induced in the bgt treatment.
a blast search of the wheat genome with microbial sequences showed that some partial microbial genetic fragments were detected in the wheat genome.
for example, a 115 bp fragment of t16_unigene_bmk.
<dig>  was mapped to chromosomes 7b and 7d with identity of 100% and an e value of  <dig> e- <dig>  and a 227 bp fragment of t16_unigene_bmk.
<dig>  was mapped to chromosome 1dl with an e value of  <dig> e- <dig>
ten pairs of gene-specific primers were designed based on de novo assembled suspected microbe sequences with the primer  <dig>  software and used in pcrs with genomic dna extracted from non-infected wheat n <dig> leaves as the template.
the pcr products showed clear bands, and sequencing confirmed that the two fragments t16_unigene_bmk.
<dig> and t17_unigene_bmk.
<dig>  which were  <dig> and 280 bp in length, respectively, were amplified from genomic dna of the resistant wheat germplasm n <dig> .
however, most of the checked unigenes could not be verified by sequencing of the pcr products amplified from the genomic dna, although clear bands were observed in  <dig> % agarose gels.
this result indicated that most of the unigenes were transcribed from fungi, but some were homologous to wheat genes.
taken together, we inferred that the wheat line shared some genetic material with the microbes, which may be exploited by a virulent fungal pathogen to induce a response directed towards the wrong pathogen.
high diversity of rgas
because of the interest in wheat disease-resistance genes and plant–fungus interactions, the matched disease-resistance-related unigenes were clustered and those unigenes involved in plant–pathogen interaction kegg pathways were compared between the pst and bgt treatments.
in this transcriptome, we detected  <dig>  unigenes that matched disease-resistance proteins with blastx, including  <dig> unigenes with a nb-arc conserved domain and  <dig> containing a nb-lrr domain.
seventy-eight genes were regulated in response to bgt infection of which  <dig> were induced, whereas  <dig> unigenes were disturbed by pst inoculation of which only two were induced.
five regulated unigenes were shared by the two fungal stress responses and matched rpm <dig> and rga <dig>  this finding indicated that rgas are highly diverse.
in addition, kegg enrichment analysis showed that  <dig> differentially expressed unigenes were associated with plant–fungus interaction pathways and these genes dominated  <dig> crucial proteins or joints in the bgt stress response, whereas nine differentially expressed unigenes that regulate seven points were detected in the pst stress response.
comparison of the change in expression pattern of these enzymes at  <dig>   <dig>  and  <dig> dpi indicated that  <dig> dpi was the most important time point because nearly all regulated genes were detected at this stage .
for resistance to pst infection, wheat manipulated flagellin-sensitive  <dig>  and calcium-dependent protein kinase  to trigger the hr response, and induced nonhost  <dig>  at  <dig> dpi.
the respiratory burst oxidase homolog  and heat shock protein  were down-regulated.
however, the expression levels of fls <dig> relative gene was severely decreased compared with that in non-inoculated leaves, whereas nho <dig> expression returned to normal at  <dig> dpi.
perhaps to compensate for the loss, the expression level of hsp <dig> was induced and attained a level similar to that of cdpk, whereas rboh expression was restored to normal.
at  <dig> dpi, fls <dig>  cdpk, and hsp <dig> genes maintained their respective expression levels observed at  <dig> dpi at the cost of calmodulin/calmodulin-like  repression.figure  <dig>
differences in disturbed genes matched with enzymes in plant-pathogen interaction pathway between
pst
and
bgt
infection at  <dig> dpi.
green means that the degs encoding corresponding enzyme were up regulated in contrasting with non-inoculation; red represent down regulation and blue indicated that de unigenes are mixed expressed.
the diagram of network was cited from kegg website.
in response to bgt inoculation, wheat redeployed most pathways to trigger the hr response at  <dig> dpi, including pti, eti and nho <dig>  pti was activated through the reactive oxygen species  and nitric oxide  signaling pathway mediated by cdpk, rboh and cam/cml, fls <dig>  mkk1/ <dig> , and wrky25/ <dig> involved a mapk cascade.
eti was triggered via rin <dig>  rps <dig>  rar <dig> , sgt <dig> , and hsp <dig>  in addition, min <dig>  and jaz  were induced to activate protein-mediated proteolysis and induce two chitin prr proteins, chitin elicitor receptor kinase  <dig>  and chitin elicitor-binding protein , but started to repress cyclic nucleotide-gated ion channel  proteins at  <dig> dpi.
however, most gene induction had ceased at  <dig> dpi except for six unigenes that matched cam/cml, mkk1/ <dig>  hsp <dig>  nho <dig> and min <dig>  and three enzymes were repressed, namely rboh, jaz and myc <dig> as cngcs.
nho <dig> and min <dig> continued to be expressed, although the cam/cml gene and chitin prr protein cebip were significantly down-regulated at  <dig> dpi.
figure  <dig> shows that wheat accession n <dig> employed multi-layered mechanisms to detect and combat pathogens, including preformed physical barriers and physiological and biochemical responses, upon recognition of pathogen-derived elicitors.
discussion
in the present study, we performed triplicate deep transcriptome surveys in leaves of the same wheat line inoculated with bgt and pst.
using high-throughput rna sequencing technology , we compared in detail the transcriptional differences and overlap between bgt- and pst-induced stress, and produced currently the most robust and reliable data for investigation of the response of wheat to fungal pathogen attack.
identification of differentially expressed genes and comparison with previously reported fungal transcriptome data
stripe rust and powdery mildew are among the most devastating diseases of wheat in cool regions.
several transcriptome and gene expression analyses of wheat following inoculation with these pathogens have been reported, such as the yr <dig>  yr <dig> high-temperature, adult-plant resistance line versus pst using the affymetrix genechip wheat genome array  <cit> , ‘shaanmai 139’ versus cyr  <dig> and ‘shuiyuan 11’ against cyr  <dig> using suppression subtractive hybridization and cdna-aflp approaches  <cit> .
similarly, several reports on wheat–bgt interaction have been published.
for example, mirnas and long non-coding rna regulating the response to bgt infection were analyzed using solexa high-throughput sequencing  <cit> , and gene expression profiling of wheat in response to bgt infection  <cit> .
without the requirement for known gene sequences, high-throughput rna sequencing provides more powerful data for observation of global gene expression profiles in different physiological processes in response to fungal attack than those contained in previous reports.
although we selected the top 1% dysregulated genes as representative,  <dig> unigenes were listed in additional file 1: table s <dig> owing to the thousands of genes observed in this study.
the fungal-induced biological function genes could be divided into nine groups, which covered those reported in previous studies.
defense-related transcripts comprised the biggest group followed by oxidation-reduction, and signal transduction ranked as the third-largest group.
the other groups consisted of metabolic, biological regulation, protein/carbohydrate transport, tricarboxylic acid cycle, development, and protein modification, as well as a tenth group categorized as “unknown/unclear”.
the results confirmed the expectation, based on observation of resistance-specific transcripts significantly induced at 1– <dig> dpi, that the top 1% genes were representative of the overall transcriptome.
the mass sequence data obtained by deep sequencing of the wheat–fungus interactions provides a robust platform for future functional and molecular research than previously published data.
meta-analysis of the kegg metabolism pathway
in classical genetics, the pathogen-infected host plants and host responses usually showed monogenic control  <cit> .
however, it is probable that thousands of genes can be detected in the early stages of infection, which causes induction of key resistance genes.
this means that priming or core control genes must trigger other defense-related and downstream genes.
accordingly, the analysis of pathway enrichment and metabolomics will open up the study of important host–pathogen systems, and may be helpful to identify the important factors that regulate such pathways.
in contrast with previous transcript analyses, we focused mainly on enriched pathways and biological processes in more detail, using meta-analysis of transcripts associated with race-specific pathogen resistance in addition to isolation of early response genes.
when responding to pathogen attack, the host plant activates a network of pathways in an endeavor to impair pathogen invasion and to escape damage, which usually includes thousands of genes through a synergistic effect.
in addition, bread wheat is hexaploid; as a consequence, characterization of multiple gene copies having mutual or compensatory interactions, such as those encoding the aforementioned disease resistance protein.
in this study, we identified seven highly enriched kegg pathways in response to bgt infection and four in response to pst infection, for example phenylalanine metabolism , phenylalanine, tyrosine and tryptophan biosynthesis , and carbon fixation in photosynthetic organisms .
a meta-analysis is advantageous in helping to narrow the field of potential resistance gene analogues and in the search for the critical genes or triggers, and to construct an integrated metabolism network as shown in figure  <dig>  the final products of the ko <dig> and ko <dig> pathways  are capsaicin and curcumin diglucoside.
capsaicin and curcumin have antibacterial and anticancer properties and reduce inflammatory reactions in mammals  <cit> ; capsaicin also activates serine/threonine kinase and amp-activated kinase , causing increased apoptosis  <cit> .
this may imply that capsaicin and curcumin are similarly employed and processed as antifungal agents in plants, although further physiological and biochemical investigations are needed to test this hypothesis.
in addition, this network of pathways will accelerate identification of upstream regulators and key transcription factors.figure  <dig>
overview of a hypothetical metabolism network model constructed with enriched degs kegg pathways according to kegg annotation.
aforementioned significant enriched kee pathways were marked with solid frame, indistinctive kegg pathways marked with dotted frame.
arrows indicated the fluid direction of metabolic products.
one component of this network is photosynthesis, and followed by photosynthesis-antenna proteins pathway, which has been reported to modulate plant defense responses induced by pathogen infection and by abiotic signals such as light, circadian rhythm, and temperature  <cit> .
in the present study,  <dig> enzymes that were dysregulated are participants in the light-harvesting chlorophyll protein complex  belonging to the photosynthesis-antenna proteins pathway which was significantly enriched in both bgt and pst treatments.
modulation acts on specific key components of plant resistance, indicating that intricate integration of biotic and abiotic signals occurs.
relationship between transcriptome divergence and differences in resistance
although both bgt and pst are fungi, the number of genes induced by bgt infection was  <dig>  times higher than the number induced by pst infection at  <dig> dpi, while the ratio was more than  <dig> times higher at  <dig> and  <dig> dpi.
genes that show highly correlated levels and temporal patterns of expression are often involved in similar functions or cellular processes  <cit> .
therefore, we clustered  <dig>  overlapping degs to assess possible crosstalk between transcripts.
unexpectedly, those genes whose expression pattern was similar under bgt attack showed differing temporal patterns of expression under pst infection.
this finding indicated that these genes have multiple or different functional roles in response to different biotic stresses.
considering that the wheat germplasm n <dig> harbored two pathogen resistance loci, namely a stripe rust resistance gene on chromosome 1b and a powdery mildew resistance gene on chromosome 5b  <cit> , we hypothesized that this discrepancy results from different loci acting as a trigger or elicitor.
most importantly, this study has identified transcripts associated with multi-gene resistance in wheat n <dig>  which will be useful in future functional studies to identify relevant disease-responsive genes.
this result also dispels the current belief that similar mechanisms are activated in response to bgt and pst infection.
microbe genes were shared with the wheat genome
the ability to detect and mount a defense response against a potential pathogen has been paramount to the evolutionary and developmental success of plants.
apart from natural selection, an alternative model of evolution involving symbiotic relationships has been proposed and supported by experimental evidence  <cit> .
selection may act mutually and may drive parasite evolution and host–parasite co-evolution  <cit> .
likewise, the evolution of plants has also been shaped by molecular interactions with epiphytic, symbiotic, and pathogenic microbes  <cit> .
rna–rna recombination is considered to be one of the strongest forces shaping the genomes of plant rna viruses  <cit> .
profiting from gene sequences of rust fungi  <cit> , the present transcriptome analysis found that the microbe genes or genomic hallmarks, including genes of aspv and pgt origin, were shared by wheat and could be activated to respond to fungal attack.
based on present knowledge, a reasonable expectation is that wheat expresses the microbe-activated genes to outcompete or overcome hostile rna as huang suggested  <cit> .
paradoxically, molecular evidence proves that only partial unigenes are regulated by fungi, and the remarkable abundance of microbe-derivative genes in infected tissues cannot be an accurate reflection of resistance responses.
a satisfactory explanation for the uneven spatial distribution of microbe genes in different fungus-infected tissues will require fine-tuned analyses taking into account the influence of fungal factors.
nevertheless, our results raise the possibility of genetic communication between plants and microbial pathogens, and further substantiates the previous hypothesis that recombination has occurred not only between viruses but even with host rnas  <cit> .
these features may represent tradeoff advantages of increased genetic variation independent of sexual recombination for biotrophy.
hence, it may provide a notable example of dollo’s law.
this may explain why powdery mildews, stripe rust and possibly other biotrophic parasites became obligate pathogens.
key functional genes in plant-pathogen interaction
plants have evolved innate immune systems that recognize the presence of potential pathogens and initiate effective defense responses.
accumulation of resistance genes has been reported in the pti and eti pathways  <cit> .
there are, however, some notable highlights or differences in resistance mechanisms against pst and bgt in wheat.
first, previous observations indicated that cngcs, which are members of the superfamily of ion channels with six transmembrane domains, are involved in diverse physiological functions and have an iq domain  <cit> , such as atcngc <dig> and atcngc <dig> responding to pathogen attack  <cit> .
in the present study, the transcriptome analysis indicated that cngc <dig> was repressed in wheat from  <dig> to 3 days after infection with the avirulent bgt e <dig>  whereas transcription of  <dig> genes similar to cngc <dig>   <dig>   <dig>   <dig>   <dig>   <dig>  and  <dig> were not disturbed in the wheat–pathogen interaction process.
in addition, cam/cml was markedly down-regulated after fungal inoculation, especially at  <dig> dpi.
although much evidence indicates that deregulation of cam/cml gene expression or loss of cam/cml function strongly affects immune responses  <cit> , this change in resistance phenotype was not observed in n <dig>  this finding indicated that bread wheat synthetically used multiple pathways to perceive and fight fungal infection in addition to the nos signal pathway.
inspired by the function of dnd <dig>  a cyclic nucleotide-gated ion channel reported to accelerate cell death  <cit> , we hypothesize that cngc <dig> may play a similar role to resist attack by the powdery mildew pathogen because of the perfect ‘no death phenotype’ resistance of n <dig> to bgt e <dig>
second, plant cdpks are a key regulator of innate immune responses to pathogen-associated molecular pattern stimulation  <cit> , and a mutual activation circuit consisting of cpk <dig> and nadph oxidase rboh facilitates rapid signal propagation in the plant  <cit> .
in the current study, we detected one cdpk gene in wheat that was positively persistent and four-fold regulated at all time points  in response to pst attack, whereas three cdpks were induced only at  <dig> dpi.
intriguingly, we found that the rboh was selectively repressed or induced in the early stages of fungal attack, which provides a chance to identify in vivo substrates to unravel cdpk functions and correlate cdpks with their corresponding substrates to establish their biological significance.
the leucine-rich repeat receptor kinases fls <dig> contribute to resistance against bacterial infection in arabidopsis thaliana
<cit> .
the third notable difference in resistance mechanisms against pst and bgt infection in wheat was that the similar serine/threonine-protein kinase fls <dig> genes were implicated in protecting wheat seedlings from infection by the two fungi.
however, no flg <dig> or homolog was detected among the unigenes.
our results indicated that the elicitation of this basal immune response is an effective strategy for protecting the plant from both bacterial and fungal pathogens in wheat as well as arabidopsis.
we speculate that in wheat an alternative elicitor must interact with fls <dig> to contribute to plant resistance.
CONCLUSIONS
the findings presented here provide evidence to clarify the hypothesis that wheat antagonizes stripe rust and powdery mildew infection with similar molecular mechanisms and gene transcripts because both pathogens are biotrophic fungi.
in contrast, we found that a disease-resistant wheat line triggers various defense mechanisms to strengthen disease resistance, and that expression patterns of the same defense-associated genes were altered in adaptation to different pathogens.
transcripts of the infection-induced wheat genes were more abundant in bgt-infected leaves than in pst-infected leaves at an early stage following inoculation, especially of genes in plant–pathogen interaction pathways.
our experiment showed that the wheat line shared some microbial genetic materials with the pathogens, which may be exploited to resist infection by a virulent fungal pathogen.
our study provides new insights into the underlying mechanisms related to modulation and regulation of various biochemical pathways in response to fungal infection.
moreover, we provide a powerful platform for further exploration of disease-resistance genes, gene function, molecular research on wheat–fungus interactions, and marker development for classical genetics.

 plants are constantly exposed to microbes in natural and agricultural ecosystems. to be pathogenic, most microbes must access the plant’s internal tissues, either by penetrating the plant surface directly or by entering through wounds or natural openings such as stomata. furthermore, the pathogen must pierce through the cell wall, and often the host plasma membrane, to be infective. plants wield typical basal and resistance gene-mediated defense mechanisms and mount a defensive response to pathogen attack to delay or arrest potential pathogenic microorganism growth through pathogen-associated molecular pattern-triggered immunity and effector-triggered immunity systems. current analysis of plant immunity has moved towards an integrated view of plant–pathogen interactions. using oligonucleotides, cdna microarrays and transcriptome analysis, many comprehensive analyses of stress-induced changes in gene expression in model plants with small genomes have been undertaken, and consequently many genes associated with pathogenic stress responses in plants are known. however, numerous fundamental molecular aspects remain unknown, such as the unique and common signaling components of pti and eti, and the induced host components that effect resistance. a global gene expression approach is useful for elucidating the molecular mechanisms of wheat–fungus interactions, particularly the application of next-generation sequencing to study important non-model host–pathogen systems, such as wheat rusts. stripe rust and powdery mildew are important fungal diseases of wheat in many wheat-growing regions of the world. the filamentous powdery mildew and rust fungi develop haustoria within the lumen of the host cell, which function to absorb nutrients. a number of studies on wheat–rust interactions have been carried out using the affymetrix® genechip® wheat genome array and cdna-aflp analysis. powdery mildew infection has been studied in grapevine, hordeum and arabidopsis with the affymetrix genechip wheat genome array, cdna-aflp, and cdna microarrays, respectively. however, a complete gene expression profile for the response to the stripe rust and powdery mildew pathogens in the same wheat germplasm is still lacking. compared with other plants with smaller genomes, the use of most genetic and molecular techniques to study genes involved in wheat–pst or wheat–bgt interactions has been limited, because hexaploid wheat has a large and complex genome and its transformation is difficult, and both fungi show sexual reproduction and irreversible deletion of genes dispensable for biotrophy. use of the affymetrix genechip wheat genome array is often restricted by the known gene sequences arrayed on the chip, with a limited number of expressed sequence tags non-specific to different wheat materials, whereas cdna-aflp is universally applicable for any organism or interaction without prior sequence information required, although false positives may frequently be observed because of technical reasons. in contrast, rna sequencing is not dependent on pre-existing databases of expressed genes and, therefore, provides an unbiased view of gene expression profiles. in the present study, using illumina deep sequencing, we undertook a transcriptome analysis of leaves from different plants of the same wheat line, n subjected to both pst and bgt stress treatments. the main objective was to identify co-regulated mrnas that show a change in expression pattern after inoculation with pst or bgt, and to identify mrnas specific to the fungal stress response. this is the first study to compare two biotic stresses using a global expression profiling strategy in the same wheat genetic background. through a comparison of different pathogen stress treatments with biological replicates, we reasoned that we could better identify both shared and stimulus-specific responses.  in the present study, cdna libraries were constructed from leaves inoculated with pst or bgt at and 3 days post-inoculation with three biological replicates, and then sequenced using the illumina hiseq™ platform. after cleaning and checking the read quality, we obtained almost million 101 bp paired-end clean reads. among the clean reads, 100% had quality scores at the cycle q level. the data sets were deposited in the ncbi sequence read archive. because of deficiencies in the reference genome sequence, these reads were de novo assembled using the trinity platform software, resulting in unigenes with n length of 743 bp, of which unigenes were annotated after blast searches of the genbank nr, swissprot, kegg, cog and go databases. the length of unigenes was more than 1 kb and contained simple sequence repeat sites. as an aid to examining gene expression level distributions, the reads per kilobase of exon model per million of aligned reads values were calculated as normalized expression estimates for each gene model in each sample. also, correlation coefficients were calculated based on the log-transformed rpkm values after eliminating genes with a zero count in any of the three replicates. the correlation coefficient values ranged from to, indicating there was a strong correlation between replicates. a generalized linear model was applied based on a negative binomial distribution and an overall test was conducted to determine which genes varied in expression among any of the seven treatment groups, where a treatment group was defined by a strain-by-induction condition combination. setting fold change ≥ and the false discovery rate at % using the method of benjamini and hochberg, statistical analysis with deseq identified genes as differentially expressed among the six treatment groups compared with non-inoculated leaves as the control. of these genes, the length of genes exceeded 1 kb. table  lists details of the differentially expressed genes and annotation numbers that were detected at the different time points in response to the fungal stress treatments.table statistical table of differentially expressed genes number and annotated degs num up down nr swissprot go kegg cog note: p represents powdery mildew e inoculation condition; s represents stripe rust pathogen cyr inoculation; ck represents the samples without pathogen stress as control. the number of and 3: n infected at and dpi, respectively. to evaluate the reliability of our rna-seq and de novo assembled results, quantitative real-time pcr was performed on eight selected genes of interest using rna samples as a fourth replication. these genes were selected to represent a wide range of expression levels and patterns under fungal infection. six gene expression patterns in response to bgt stress showed strong agreement and were highly correlated in the rna-seq and qpcr analyses. additionally, six previously studied full-length genes were aligned with the present assembled unigene database and showed identities of up to %. more robust response to bgt- than pst-induced stress in bread wheat although transcript levels do not necessarily reflect the amount of final active protein product, for simplicity, increased transcript abundance is often referred to as induced gene expression. genes that were differentially expressed between non-inoculated and inoculated leaves were determined with rna-seq. cluster analysis showed that the expression profiles of degs varied significantly in response to bgt and pst in the n genotype.figure hierarchical clustering of differentially-expressed genes in resistant bread wheat accession n to bgt and pst infection. the signal ratios are shown in a red-green color scale, where red represents up-regulation and green represents down-regulation. each column represents the mean expression value of the rna-seq obtained from three biological replicates and each row represents a differentially-expressed genes. ck: resistant wheat n exempt from pathogen stress as control; s s and s3: n infected with stripe rust pathogen cyr at and dpi, respectively; p p and p3: n infected with powdery mildew pathogen e at and dpi, respectively. for the bgt test, using inoculated leaf samples, expression of degs was detected, of which genes exceeded 1 kb. as shown in table  and, comparison of inoculated and non-inoculated leaves showed that, and genes were differentially expressed at and dpi, respectively. of these genes, and were up- and down-regulated at dpi, and at dpi, and and at dpi, respectively. in the pst cyr inoculation treatment, degs showed significantly lower expression than that in the bgt e stress treatment. of these genes,,, and were differentially expressed at and dpi, respectively. the number of up-regulated genes at dpi was almost double those at and dpi after infection by bgt, whereas the number at dpi was almost three-times higher than those at and dpi after pst infection. this substantiated the contention that dpi is the most important time point for wheat to respond to fungal attack by expression of race-specific resistance genes. to further test the variation in response to pst and bgt inoculation, we compared gene expression in response to pst cyr versus bgt e inoculation with fold change ≥ and fdr at %. additional file 1: figure s shows that genes were differentially expressed in response to bgt infection in comparison with pst infection, of which and genes were up- and down-regulated at dpi, and at dpi, and and at dpi, respectively. this result indicated that the wheat resistance response to bgt inoculation triggered more robust alteration in gene expression than that observed in response to pst inoculation. gene characterization of stimulus-specific responses to pstor bgt to facilitate a closer comparison of pst-induced genes with bgt-induced genes, a pairwise comparison was conducted after degs were annotated using kegg classifications and go assignments. all resistance-specific genes were analyzed using mas to identify the metabolic pathways in which they function. the go enrichment results are shown in additional file 1: tables s, s, and s. when the fdr-corrected p-value was set at, seven significant enriched kegg pathways for bgt-induced stress and four for pst-induced stress versus the non-stressed control were identified. two kegg pathways overlapped between the two infection treatments, namely ubiquinone and other terpenoid-quinone biosynthesis and photosynthesis-antenna proteins. as there were insufficient pathway annotations in kegg, the pathways were retrieved using the degs comparing pst infection versus bgt infection directly at the specific time points and are listed in additional file 1: table s taken together, kegg pathways were identified.table significant kegg pathway of stimulus-specific responses on pst or bgt note: all kegg pathways were retrieved from mas molecule annotation system. some plant-specific pathways were computed repeat by extracting genes from comparing pst with bgt stress. the values of correct-p were given and those less than were marked with asterisks. some enriched kegg pathways with the high enrichment factor were noted and the factor was given in brackets. the indistinctive pathway that was listed had p-value < for analysis degs from pst vs bgt. phenylalanine metabolism, phenylpropanoid biosynthesis, alpha-linolenic acid metabolism, flavonoid biosynthesis, and phenylalanine, tyrosine and tryptophan biosynthesis showed specific significant differential enrichment in wheat in response to bgt-induced stress, whereas carotenoid biosynthesis and carbon fixation in photosynthetic organisms showed the opposite differential performance in response to pst-induced stress. the analysis of kegg pathways showed that specific pathways responded to bgt infection and four specific pathways responded to pst infection, although the p values were not significant for those present in the kegg database. under pst-induced stress, no degs were detected in the mismatch repair, non-homologous end-joining, nucleotide excision repair, dna replication, and rna polymerase pathways, which are all involved in genetic information processing. moreover, abcb in abc transporters, mitochondrial trans-2-enoyl-coa reductase in fatty acid elongation, lipoyl transferase in lipoic acid metabolism, biotin synthase in biotin metabolism, glutamate decarboxylase, gamma-glutamyl transpeptidase and cysteamine dioxygenase in taurine and hypotaurine metabolism, and n-glycan biosynthesis were specifically enriched in response to bgt inoculation. the number of dysregulation enzymes reaches to eight in n-glycan biosynthesis especially. in addition, seven distinct kegg pathways were retrieved with mas after comparative analysis of genes differentially expressed in response to pst- and bgt-induced stress, namely nitrogen metabolism, diterpenoid biosynthesis, stilbenoid, diarylheptanoid and gingerol biosynthesis, valine, leucine and isoleucine degradation, photosynthesis, benzoxazinoid biosynthesis, and ribosome pathway. genes encoding components of conserved protein complexes, such as the proteasome and ribosome, are coexpressed in other organisms and show very similar expression profiles. surprisingly, half of the enzymes showed dysregulated expression in the ribosome pathway in response to bgt, whereas dysregulated enzymes were detected in response to pst-induced stress. this result indicated that the ribosome pathway is one of the most complex and important pathways regulating genes antagonistic to fungal infection, and that further differential internal remodeling in response to different fungal signals is required. this analysis showed that the nine functional pathways with p-values < after fdr correction were significantly associated with resistance to fungal infection, whereas the stimulus-specific responses were more robust and pathways included degs in comparison of the pst and bgt treatments at and dpi. these results further confirmed the differences in gene regulation and pathways involved in wheat–pst interaction from those involved in wheat–bgt interaction. identification of degs shared by pstand bgtinfection responses from aforementioned information, a considerable number of degs would have overlapped between the bgt and pst inoculation treatments. those genes that were significantly regulated in the defense response to both fungi were identified from among the overlapping degs with annotated information and are listed in table  the overlapping degs at each time point are shown in a venn diagram. these genes were annotated into go biological processes with p-values < and considerably dysregulated gene numbers. protein tetramerization, oxidation-reduction process, xenobiotic metabolic process, response to hypoxia, response to abiotic stimulus, starch biosynthetic process, and cholesterol biosynthetic process were not only significantly enriched but also showed a higher number of dysregulated genes than expected. the number of dysregulated genes in the categories of regulation of t cell-mediated cytotoxicity, ketone body biosynthetic process, and ergosterol biosynthetic process was doubled that expected, although there seemed to be a relatively smaller number of degs in these three pathways than in the other above-mentioned ones. the go pedigree analysis indicated that the third main go hierarchy of biological processes in which the shared genes may participate consisted of response to single-organism stimuli and metabolic process.table go characterization of overlapped degs responding to pst and bgt stress inspired by similar expression patterns hinting at analogous gene functions, genes induced in response to both bgt- and pst-induced stress were screened with a model-based clustering algorithm to identify distinct gene expression profiles among the identified degs and to choose a total of eight clusters, to maintain as few tight clusters as possible while including most of the distinct expression patterns. each gray line in figure  represents the expression pattern for an individual gene, and the single black line indicates the average behavior for induced genes in each cluster. surprisingly, these overlapping genes showed different expression patterns and complex changes in response to pst infection. this finding illustrated that these genes may perform different functions in response to different stimuli, and implied that wheat triggered distinct genes and regulatory networks to antagonize bgt and pst infection.figure clustering. the expression patterns of overlapped de genes for clusters. the horizontal axis indicates each time point and pathogen stress induction condition. the number and mean that n was infected at and dpi, respectively. p represents powdery mildew e inoculation condition; s represents stripe rust pathogen cyr inoculation. the vertical axis indicates the log fold change calculated between each condition and non-inoculation treatment. each gray line symbolizes the expression pattern of one gene, and the bold back line illustrates the average expression pattern of all genes in each cluster under bgt or pst stress. our results for the overlapping degs revealed changes in approximately 15% of the transcriptome, with most changes classified as stimulus-specific. this view illustrates the “fluid” nature of the transcriptome and the challenge faced in understanding the complexity of any given stress response. key fungal defense-related genes and pathways transcript profiling is capable of revealing pathways of gene expression involved in a defense response, therefore detailed gene-by-gene analysis should be informative because the temporary kegg pathway and go annotations were incomplete. a total of degs were identified, and the range of deg expression ratios was - to for pst stress-induced genes and - to for bgt stress-induced genes. to identify the degs showing the greatest changes in transcript levels, the top 1% of up- and down-regulated genes in the bgt and pst treatments at each time point were analyzed. comparison of data for n pst- and bgt-inoculated plants with mock-inoculated data revealed gene expression changes that included basal defense transcripts and transcripts specific to the establishment of a biotrophic interaction with fungi. twelve of the top differentially expressed transcripts significant for treatment were induced by pst inoculation at dpi, whereas were repressed, were down-regulated and three were induced by pst infection at dpi, while eight were repressed and five were up-regulated in response to pst infection at dpi. ninety-five genes were ranked in the top 1% of genes dysregulated by bgt inoculation, of which were induced and four were repressed at dpi, were up-regulated and were down-regulated at dpi, and were up-regulated and were down-regulated at dpi. as shown in additional file 1: table s defense-related transcripts accounted for % of the dysregulated transcripts, while were involved in signal transduction, in oxidation-reduction, in biological regulation, in metabolic processes, in protein/carbohydrate transport, in development, in the tricarboxylic acid cycle, in protein modification, and were of unknown/unclear function. comparing the top 1% transcriptomes after pst and bgt inoculation, most gene expression patterns showed opposing patterns in the two treatments, especially at the same time point. those genes induced by bgt infection were usually repressed in the same genotype by pst infection, and vice versa. for example, this pattern was shown by l-type lectin-domain containing receptor kinase ix, hemoglobin, luminal-binding protein, delta-cadinene synthase isozyme xc, and hypothetical protein mtr_7g. plants sharing the microbe gene fragments antagonistic to fungal infection blast searches of the nr and swissport databases showed that annotated unigenes matched genes from aegilops tauschii, triticum urartu, hordeum vulgare, brachypodium distachyon, oryza sativa, and marssonina brunnea, while unigenes were related to t. aestivum in the current databases. interestingly, unigenes matched genes from puccinia graminis, of which were expressed differentially in the pst treatment, while unigenes were similar to apple stem pitting virus genes and were induced in the bgt treatment. a blast search of the wheat genome with microbial sequences showed that some partial microbial genetic fragments were detected in the wheat genome. for example, a 115 bp fragment of t16_unigene_bmk. was mapped to chromosomes 7b and 7d with identity of 100% and an e value of e- and a 227 bp fragment of t16_unigene_bmk. was mapped to chromosome 1dl with an e value of e- ten pairs of gene-specific primers were designed based on de novo assembled suspected microbe sequences with the primer software and used in pcrs with genomic dna extracted from non-infected wheat n leaves as the template. the pcr products showed clear bands, and sequencing confirmed that the two fragments t16_unigene_bmk. and t17_unigene_bmk. which were and 280 bp in length, respectively, were amplified from genomic dna of the resistant wheat germplasm n. however, most of the checked unigenes could not be verified by sequencing of the pcr products amplified from the genomic dna, although clear bands were observed in % agarose gels. this result indicated that most of the unigenes were transcribed from fungi, but some were homologous to wheat genes. taken together, we inferred that the wheat line shared some genetic material with the microbes, which may be exploited by a virulent fungal pathogen to induce a response directed towards the wrong pathogen. high diversity of rgas because of the interest in wheat disease-resistance genes and plant–fungus interactions, the matched disease-resistance-related unigenes were clustered and those unigenes involved in plant–pathogen interaction kegg pathways were compared between the pst and bgt treatments. in this transcriptome, we detected unigenes that matched disease-resistance proteins with blastx, including unigenes with a nb-arc conserved domain and containing a nb-lrr domain. seventy-eight genes were regulated in response to bgt infection of which were induced, whereas unigenes were disturbed by pst inoculation of which only two were induced. five regulated unigenes were shared by the two fungal stress responses and matched rpm and rga this finding indicated that rgas are highly diverse. in addition, kegg enrichment analysis showed that differentially expressed unigenes were associated with plant–fungus interaction pathways and these genes dominated crucial proteins or joints in the bgt stress response, whereas nine differentially expressed unigenes that regulate seven points were detected in the pst stress response. comparison of the change in expression pattern of these enzymes at and dpi indicated that dpi was the most important time point because nearly all regulated genes were detected at this stage. for resistance to pst infection, wheat manipulated flagellin-sensitive and calcium-dependent protein kinase to trigger the hr response, and induced nonhost at dpi. the respiratory burst oxidase homolog and heat shock protein were down-regulated. however, the expression levels of fls relative gene was severely decreased compared with that in non-inoculated leaves, whereas nho expression returned to normal at dpi. perhaps to compensate for the loss, the expression level of hsp was induced and attained a level similar to that of cdpk, whereas rboh expression was restored to normal. at dpi, fls cdpk, and hsp genes maintained their respective expression levels observed at dpi at the cost of calmodulin/calmodulin-like repression.figure differences in disturbed genes matched with enzymes in plant-pathogen interaction pathway between pst and bgt infection at dpi. green means that the degs encoding corresponding enzyme were up regulated in contrasting with non-inoculation; red represent down regulation and blue indicated that de unigenes are mixed expressed. the diagram of network was cited from kegg website. in response to bgt inoculation, wheat redeployed most pathways to trigger the hr response at dpi, including pti, eti and nho pti was activated through the reactive oxygen species and nitric oxide signaling pathway mediated by cdpk, rboh and cam/cml, fls mkk1/, and wrky25/ involved a mapk cascade. eti was triggered via rin rps rar, sgt, and hsp in addition, min and jaz were induced to activate protein-mediated proteolysis and induce two chitin prr proteins, chitin elicitor receptor kinase and chitin elicitor-binding protein, but started to repress cyclic nucleotide-gated ion channel proteins at dpi. however, most gene induction had ceased at dpi except for six unigenes that matched cam/cml, mkk1/ hsp nho and min and three enzymes were repressed, namely rboh, jaz and myc as cngcs. nho and min continued to be expressed, although the cam/cml gene and chitin prr protein cebip were significantly down-regulated at dpi. figure  shows that wheat accession n employed multi-layered mechanisms to detect and combat pathogens, including preformed physical barriers and physiological and biochemical responses, upon recognition of pathogen-derived elicitors. discussion in the present study, we performed triplicate deep transcriptome surveys in leaves of the same wheat line inoculated with bgt and pst. using high-throughput rna sequencing technology, we compared in detail the transcriptional differences and overlap between bgt- and pst-induced stress, and produced currently the most robust and reliable data for investigation of the response of wheat to fungal pathogen attack. identification of differentially expressed genes and comparison with previously reported fungal transcriptome data stripe rust and powdery mildew are among the most devastating diseases of wheat in cool regions. several transcriptome and gene expression analyses of wheat following inoculation with these pathogens have been reported, such as the yr yr high-temperature, adult-plant resistance line versus pst using the affymetrix genechip wheat genome array, ‘shaanmai 139’ versus cyr and ‘shuiyuan 11’ against cyr using suppression subtractive hybridization and cdna-aflp approaches. similarly, several reports on wheat–bgt interaction have been published. for example, mirnas and long non-coding rna regulating the response to bgt infection were analyzed using solexa high-throughput sequencing, and gene expression profiling of wheat in response to bgt infection. without the requirement for known gene sequences, high-throughput rna sequencing provides more powerful data for observation of global gene expression profiles in different physiological processes in response to fungal attack than those contained in previous reports. although we selected the top 1% dysregulated genes as representative, unigenes were listed in additional file 1: table s owing to the thousands of genes observed in this study. the fungal-induced biological function genes could be divided into nine groups, which covered those reported in previous studies. defense-related transcripts comprised the biggest group followed by oxidation-reduction, and signal transduction ranked as the third-largest group. the other groups consisted of metabolic, biological regulation, protein/carbohydrate transport, tricarboxylic acid cycle, development, and protein modification, as well as a tenth group categorized as “unknown/unclear”. the results confirmed the expectation, based on observation of resistance-specific transcripts significantly induced at 1– dpi, that the top 1% genes were representative of the overall transcriptome. the mass sequence data obtained by deep sequencing of the wheat–fungus interactions provides a robust platform for future functional and molecular research than previously published data. meta-analysis of the kegg metabolism pathway in classical genetics, the pathogen-infected host plants and host responses usually showed monogenic control. however, it is probable that thousands of genes can be detected in the early stages of infection, which causes induction of key resistance genes. this means that priming or core control genes must trigger other defense-related and downstream genes. accordingly, the analysis of pathway enrichment and metabolomics will open up the study of important host–pathogen systems, and may be helpful to identify the important factors that regulate such pathways. in contrast with previous transcript analyses, we focused mainly on enriched pathways and biological processes in more detail, using meta-analysis of transcripts associated with race-specific pathogen resistance in addition to isolation of early response genes. when responding to pathogen attack, the host plant activates a network of pathways in an endeavor to impair pathogen invasion and to escape damage, which usually includes thousands of genes through a synergistic effect. in addition, bread wheat is hexaploid; as a consequence, characterization of multiple gene copies having mutual or compensatory interactions, such as those encoding the aforementioned disease resistance protein. in this study, we identified seven highly enriched kegg pathways in response to bgt infection and four in response to pst infection, for example phenylalanine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, and carbon fixation in photosynthetic organisms. a meta-analysis is advantageous in helping to narrow the field of potential resistance gene analogues and in the search for the critical genes or triggers, and to construct an integrated metabolism network as shown in figure  the final products of the ko and ko pathways are capsaicin and curcumin diglucoside. capsaicin and curcumin have antibacterial and anticancer properties and reduce inflammatory reactions in mammals; capsaicin also activates serine/threonine kinase and amp-activated kinase, causing increased apoptosis. this may imply that capsaicin and curcumin are similarly employed and processed as antifungal agents in plants, although further physiological and biochemical investigations are needed to test this hypothesis. in addition, this network of pathways will accelerate identification of upstream regulators and key transcription factors.figure overview of a hypothetical metabolism network model constructed with enriched degs kegg pathways according to kegg annotation. aforementioned significant enriched kee pathways were marked with solid frame, indistinctive kegg pathways marked with dotted frame. arrows indicated the fluid direction of metabolic products. one component of this network is photosynthesis, and followed by photosynthesis-antenna proteins pathway, which has been reported to modulate plant defense responses induced by pathogen infection and by abiotic signals such as light, circadian rhythm, and temperature. in the present study, enzymes that were dysregulated are participants in the light-harvesting chlorophyll protein complex belonging to the photosynthesis-antenna proteins pathway which was significantly enriched in both bgt and pst treatments. modulation acts on specific key components of plant resistance, indicating that intricate integration of biotic and abiotic signals occurs. relationship between transcriptome divergence and differences in resistance although both bgt and pst are fungi, the number of genes induced by bgt infection was times higher than the number induced by pst infection at dpi, while the ratio was more than times higher at and dpi. genes that show highly correlated levels and temporal patterns of expression are often involved in similar functions or cellular processes. therefore, we clustered overlapping degs to assess possible crosstalk between transcripts. unexpectedly, those genes whose expression pattern was similar under bgt attack showed differing temporal patterns of expression under pst infection. this finding indicated that these genes have multiple or different functional roles in response to different biotic stresses. considering that the wheat germplasm n harbored two pathogen resistance loci, namely a stripe rust resistance gene on chromosome 1b and a powdery mildew resistance gene on chromosome 5b, we hypothesized that this discrepancy results from different loci acting as a trigger or elicitor. most importantly, this study has identified transcripts associated with multi-gene resistance in wheat n which will be useful in future functional studies to identify relevant disease-responsive genes. this result also dispels the current belief that similar mechanisms are activated in response to bgt and pst infection. microbe genes were shared with the wheat genome the ability to detect and mount a defense response against a potential pathogen has been paramount to the evolutionary and developmental success of plants. apart from natural selection, an alternative model of evolution involving symbiotic relationships has been proposed and supported by experimental evidence. selection may act mutually and may drive parasite evolution and host–parasite co-evolution. likewise, the evolution of plants has also been shaped by molecular interactions with epiphytic, symbiotic, and pathogenic microbes. rna–rna recombination is considered to be one of the strongest forces shaping the genomes of plant rna viruses. profiting from gene sequences of rust fungi, the present transcriptome analysis found that the microbe genes or genomic hallmarks, including genes of aspv and pgt origin, were shared by wheat and could be activated to respond to fungal attack. based on present knowledge, a reasonable expectation is that wheat expresses the microbe-activated genes to outcompete or overcome hostile rna as huang suggested. paradoxically, molecular evidence proves that only partial unigenes are regulated by fungi, and the remarkable abundance of microbe-derivative genes in infected tissues cannot be an accurate reflection of resistance responses. a satisfactory explanation for the uneven spatial distribution of microbe genes in different fungus-infected tissues will require fine-tuned analyses taking into account the influence of fungal factors. nevertheless, our results raise the possibility of genetic communication between plants and microbial pathogens, and further substantiates the previous hypothesis that recombination has occurred not only between viruses but even with host rnas. these features may represent tradeoff advantages of increased genetic variation independent of sexual recombination for biotrophy. hence, it may provide a notable example of dollo’s law. this may explain why powdery mildews, stripe rust and possibly other biotrophic parasites became obligate pathogens. key functional genes in plant-pathogen interaction plants have evolved innate immune systems that recognize the presence of potential pathogens and initiate effective defense responses. accumulation of resistance genes has been reported in the pti and eti pathways. there are, however, some notable highlights or differences in resistance mechanisms against pst and bgt in wheat. first, previous observations indicated that cngcs, which are members of the superfamily of ion channels with six transmembrane domains, are involved in diverse physiological functions and have an iq domain, such as atcngc and atcngc responding to pathogen attack. in the present study, the transcriptome analysis indicated that cngc was repressed in wheat from to 3 days after infection with the avirulent bgt e whereas transcription of genes similar to cngc and were not disturbed in the wheat–pathogen interaction process. in addition, cam/cml was markedly down-regulated after fungal inoculation, especially at dpi. although much evidence indicates that deregulation of cam/cml gene expression or loss of cam/cml function strongly affects immune responses, this change in resistance phenotype was not observed in n this finding indicated that bread wheat synthetically used multiple pathways to perceive and fight fungal infection in addition to the nos signal pathway. inspired by the function of dnd a cyclic nucleotide-gated ion channel reported to accelerate cell death, we hypothesize that cngc may play a similar role to resist attack by the powdery mildew pathogen because of the perfect ‘no death phenotype’ resistance of n to bgt e second, plant cdpks are a key regulator of innate immune responses to pathogen-associated molecular pattern stimulation, and a mutual activation circuit consisting of cpk and nadph oxidase rboh facilitates rapid signal propagation in the plant. in the current study, we detected one cdpk gene in wheat that was positively persistent and four-fold regulated at all time points in response to pst attack, whereas three cdpks were induced only at dpi. intriguingly, we found that the rboh was selectively repressed or induced in the early stages of fungal attack, which provides a chance to identify in vivo substrates to unravel cdpk functions and correlate cdpks with their corresponding substrates to establish their biological significance. the leucine-rich repeat receptor kinases fls contribute to resistance against bacterial infection in arabidopsis thaliana. the third notable difference in resistance mechanisms against pst and bgt infection in wheat was that the similar serine/threonine-protein kinase fls genes were implicated in protecting wheat seedlings from infection by the two fungi. however, no flg or homolog was detected among the unigenes. our results indicated that the elicitation of this basal immune response is an effective strategy for protecting the plant from both bacterial and fungal pathogens in wheat as well as arabidopsis. we speculate that in wheat an alternative elicitor must interact with fls to contribute to plant resistance.  the findings presented here provide evidence to clarify the hypothesis that wheat antagonizes stripe rust and powdery mildew infection with similar molecular mechanisms and gene transcripts because both pathogens are biotrophic fungi. in contrast, we found that a disease-resistant wheat line triggers various defense mechanisms to strengthen disease resistance, and that expression patterns of the same defense-associated genes were altered in adaptation to different pathogens. transcripts of the infection-induced wheat genes were more abundant in bgt-infected leaves than in pst-infected leaves at an early stage following inoculation, especially of genes in plant–pathogen interaction pathways. our experiment showed that the wheat line shared some microbial genetic materials with the pathogens, which may be exploited to resist infection by a virulent fungal pathogen. our study provides new insights into the underlying mechanisms related to modulation and regulation of various biochemical pathways in response to fungal infection. moreover, we provide a powerful platform for further exploration of disease-resistance genes, gene function, molecular research on wheat–fungus interactions, and marker development for classical genetics. 
stripe rust and powdery mildew are important diseases of wheat worldwide. bread wheatstripe rustpowdery mildewrna-seqgene expressionissue-copyright-statement© the author 2014 the mass sequence data for wheat–fungus interaction generated in this study provides a powerful platform for future functional and molecular research on wheat–fungus interactions. the main objective of our study was to identify co-regulated mrnas that show a change in expression pattern after inoculation with pst or bgt, and to identify mrnas specific to the fungal stress response. infection by bgt triggered a more robust alteration in gene expression in n compared with the response to pst infection. similar mechanisms and gene transcripts are assumed to be involved in the host defense response because both pathogens are biotrophic fungi. the transcriptome of the hexaploid wheat line n inoculated with the chinese pst race cyr was compared with that of the same line inoculated with bgt race e at and 3 days post-inoculation. an array of overlapping gene clusters with distinctive expression patterns provided insight into the regulatory differences in the responses to bgt and pst infection. genomic analysis revealed that the wheat genome shared some microbial genetic fragments, which were specifically induced in response to bgt and pst infection. in the plant–pathogen interaction pathway, n activated a higher number of genes and pathways in response to bgt infection than in response to pst invasion.
 stripe rust and powdery mildew are important diseases of wheat worldwide. similar mechanisms and gene transcripts are assumed to be involved in the host defense response because both pathogens are biotrophic fungi. the main objective of our study was to identify co-regulated mrnas that show a change in expression pattern after inoculation with pst or bgt, and to identify mrnas specific to the fungal stress response.  the transcriptome of the hexaploid wheat line n inoculated with the chinese pst race cyr was compared with that of the same line inoculated with bgt race e at and 3 days post-inoculation. infection by pst and bgt affected transcription of % of all t. aestivum genes. infection by bgt triggered a more robust alteration in gene expression in n compared with the response to pst infection. an array of overlapping gene clusters with distinctive expression patterns provided insight into the regulatory differences in the responses to bgt and pst infection. the differentially expressed genes were grouped into seven enriched kyoto encyclopedia of genes and genomes pathways in bgt-infected leaves and four pathways in pst-infected leaves, while only two pathways overlapped. in the plant–pathogen interaction pathway, n activated a higher number of genes and pathways in response to bgt infection than in response to pst invasion. genomic analysis revealed that the wheat genome shared some microbial genetic fragments, which were specifically induced in response to bgt and pst infection.  taken together, our findings indicate that the responses of wheat n to infection by bgt and pst shows differences in the pathways and genes activated. the mass sequence data for wheat–fungus interaction generated in this study provides a powerful platform for future functional and molecular research on wheat–fungus interactions. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords bread wheatstripe rustpowdery mildewrna-seqgene expressionissue-copyright-statement© the author 2014 