BACKGROUND
fusarium head blight  caused e.g.
by f. graminearum schwabe  petch) is one of the most destructive diseases of wheat  worldwide, causing significant reductions in grain yield and quality.
the most efficient strategy to control fhb in wheat is the use of resistant cultivars
<cit> .
however, in hexaploid wheat the resistance to fhb is highly complex.
since  <dig>  over  <dig> qtl have been reported, whereas only a few qtl were found to be stable in different genetic backgrounds and useful for breeding.
the most stable qtl were obtained from the chinese wheat varieties sumai  <dig> and wangshuibai
<cit> .
however, poor agronomic performance and the frequent occurrence of genetic linkage drag make them less suitable donors of resistant genes
<cit> .
moreover, the genetic and molecular basis of the quantitative fhb resistance is still poorly understood.
recent studies on the mode of fusarium spike colonisation have revealed that the pathogens use a specific arsenal of virulence factors which are essential in nearly all phases of the disease making them interesting targets for novel resistance strategies.
trichothecene toxins, such as deoxynivalenol , and hydrolytic enzymes, such as subtilisin-like and trypsin-like proteases, are two virulence factors that were found to occur during almost the entire course of disease
<cit> .
don was found to be produced in the fungal infection structures already during the initial penetration of floret tissues
<cit> .
the reason for this early secretion remains unknown, because the initial infection is symptomless and indistinguishable between susceptible and resistant wheat cultivars in all respects
<cit> ; even the trichothecene-deficient fusarium mutants do not show any restrictions regarding their infectious ability
<cit> .
however, already in the second infection phase, don production gains relevance.
it is supposed that the general capacity to prevent protein synthesis makes the toxin an important suppressor of early plant defences
<cit> .
for that purpose, don seems to enable the fungal hyphae to break through the spike rachis node which is the central bottle-neck for both, the initial spread from infected florets into the spike rachis and the reverse direction from the rachis into uninoculated spikelets
<cit> .
during the rachis colonization when hyphae grow vertically
<cit> , the toxin may inhibit the onset of various cell wall reinforcement processes in the vicinity of invading hyphae
<cit> .
at the same time, fungal proteases are likely to participate in the suppression of plant defences by degrading pathogenesis-related  proteins or defence-signalling compounds according to their property to cause proteolytic protein digestion
<cit> .
in the spikes of the resistant landrace wangshuibai the down-regulation of different housekeeping proteins was reported already  <dig> to 24 h after f. graminearum inoculation as a consequence of the secretion of fungal hydrolytic enzymes and toxins
<cit> .
the intercellular spread through the spike rachis is accompanied by lateral hyphae growth to infect uninoculated spikelets.
this secondary colonisation is essentially associated with the secretion of don and proteases which initiate and facilitate necrotrophic intracellular nutrition.
the phase is characterized by dramatic changes in the interaction between pathogen and host concerning the respective transcriptomes, secretomes and metabolomes
<cit> , and is often described as switching point from fungal biotrophy to necrotrophy
<cit> .
increased don levels were observed 26
<cit>  to 96 h
<cit>  after infection .
in addition, between  <dig> and  <dig> hai f. graminearum transcripts were found to encode especially degrading enzymes such as proteases
<cit> .
these accumulations were typically linked to increased levels of systemic fungal development and collapsed host cells
<cit> .
both virulence factors are probably essential for the penetration and mortification of host cells, as fusarium pathogens use cell wall digestion to enter living host cells
<cit>  and don, in particular, is known to activate plant programmed cell death
<cit> .
in summary, don and proteases have a significant impact on cell wall digestion, protein matrix reduction and damage to starch granules, typically seen in fusarium-infected wheat kernels rendering grain yield unsuitable and unsafe for food, feed or malting purposes
<cit> .
in order to characterise the transcriptional changes in the resistant cv.
dream compared with the susceptible cv.
lynx, we performed gene expression profiling using the genechip® wheat genome array.
genechip expression data obtained  <dig> and 72 h after inoculation with f. graminearum or, respectively, mock have revealed indications for the presence of two main defence mechanisms in cv.
dream, reflecting a biphasic strategy against fhb disease.
one mechanism comprised jasmonate- and ethylene-mediated defence reactions directed against fungal growth and sporulation, while the second mechanism was specifically directed towards fungal mycotoxins and proteases.
quantitative real-time pcr  time-course study was applied to analyse the expressions of seven selected anti-virulence gene candidates in the cultivar pairs dream/lynx and sumai  <dig> /florence-aurore .
observed similarities between the resistant cultivars dream and sumai  <dig> in terms of fhb-responsive up-regulated genes from both described defence mechanisms will be reported.
RESULTS
identification of fhb-responsive genes in the resistant wheat cultivar dream
transcript abundances in the f. graminearum -inoculated and mock-inoculated wheat cultivars dream  and lynx  were measured and compared using the affymetrix genechip® wheat genome array.
the general disease progression was examined on single-floret inoculated samples that were collected  <dig> and  <dig> hours after inoculation .
all measurements were performed with three biological replicates.
for each timepoint the four genechip datasets were compared to identify differentially expressed genes involved in the different aspects of the inoculation response.
table
<dig> lists all comparisons with the respective numbers of differentially expressed genes.
* differentially expressed genes originated from the three replicates of genechip dataset ‘lynx 72 h after mock inoculation’ were not recorded due to low quality of the microarrays.
a gene set enrichment analysis
<cit>  of the comparisons was conducted to identify relevant functional classes associated to incompatible cv.
dream-f. graminearum interactions.
table
<dig> provides an overview of the nine gene ontology  terms that were enriched in those genes found to be significantly up-regulated in the resistant cv.
dream at  <dig> and 72 h after fusarium inoculation compared with the fusarium inoculated susceptible cv.
lynx.
all terms were found to be associated to the disease as the respective represented gene products were neither enriched in the analogous cultivar comparison after mock inoculation nor in the comparison ‘cv.
lynx fusarium inoculated versus cv.
lynx mock inoculated’.
no go terms were enriched in the significantly down-regulated genes at  <dig> hai while  <dig> enriched terms were observed at the later timepoint .
for enrichments at  <dig> hai associations to the infection with f. graminearum were restricted and, thus, were only possible if a go term was also enriched also at  <dig> hai.
1) significance levels for up-regulated go terms: fdr value  at <  <dig>  ; nom p-value  at <  <dig>  .
1) significance levels for down-regulated go terms: fdr value  at <  <dig>  ; nom p-value  at <  <dig>  .
the gsea provided insides into defence mechanisms that were induced during incompatible interactions .
at  <dig> hai an exclusive enrichment was observed for the terms ‘lipoxygenase activity’ , ‘oxylipin biosynthetic process’  and ‘lipid biosynthetic process’  including genes, such as lipoxygenases, involved in the plant oxylipin metabolism.
additionally, lipoxygenases genes were also frequent in the term ‘response to wounding’ .
putative cysteine-rich proteins, such as thionins, were detected in the go term ‘pathogenesis’ .
phyto-oxylipins comprising antimicrobial peptides and defence-signalling molecules such as jasmonates, together with cysteine-rich pathogenesis-related  genes indicate an induced antifungal defence mechanism
<cit> .
plant serine-protease inhibitors were enriched in the go terms ‘serine-type endopeptidase inhibitor activity’ go: <dig> and ‘peptidase activity’ go: <dig>  and represented the second class of genes enriched in the term ‘response to wounding’ .
serine-protease inhibitors as well as genes encoding serine-proteases identified by the term ‘serine-type carboxypeptidase activity’  were enriched at both timepoints.
these enriched terms represent an induced defence mechanism against pathogen-released proteases which as virulence factors are secreted to modify host proteins
<cit> .
on the basis of testing, this defence mechanism as well as the antifungal defence mechanism were found to be central and therefore, will be discussed in more detail later on.
finally, genes encoding for peptidyl-prolyl cis-trans isomerase  proteins  belonging to the immunophilin superfamily were found to be accumulated.
ppiase proteins have general functions in protein folding and protein degradation, and several proteins have shown antifungal properties, similar to pr-genes
<cit> .
in the down-regulated genes  three go terms were noticeable with regard to the pathogen presence, while associations to the disease were rather unclear for the other terms.
the detected go terms ‘chitin catabolic process’  and ‘chitinase activity’  demonstrate the down-regulation of genes which typically facilitate the breakdown of fungal cell walls
<cit> .
chitinase genes have shown to exhibit an enhanced resistance against f. graminearum, in barley
<cit>  while in the grains of emmer wheat , a progenitor of bread wheat , a similar down-regulation of chitinase genes was observed and discussed as a direct impact of f. graminearum signals
<cit> .
finally, the term ‘mycelium development’  comprises 10 f. graminearum genes, belonging to a set of  <dig> fusarium genes which were previously found to be present on the affymetrix wheat genechip®
<cit> .
as these genes are putatively associated to the progression of the fungal mycelium, their enrichment amongst down-regulated genes might reflect traces of an impaired fungal growth in the resistant dream cultivar.
a comparison was performed between the ‘cv.
dream fusarium inoculated versus cv.
lynx fusarium inoculated’ expression data and the analogous expression data from the mock inoculation , in order to address expression changes in the resistant cv.
dream associated with the fungal attack.
at  <dig> hai, the genes differentially expressed in cv.
dream could be separated into genes that were differentially expressed to higher levels or were only present after pathogen attack , after both treatments , and only after control treatment .
the genes only present in response to fhb were categorised as ‘fhb-responsive genes’.
especially, up-regulated transcripts are likely to represent defences, such as trigger mechanisms or direct antimicrobial activities.
genes with similar expression profiles after both treatments were categorised as ‘genotype-specific genes’ because they were differentially expressed to lower levels or absent in the cv.
lynx spike samples.
up-regulated genes were hereafter discussed as members of a basal defence if their induction has been demonstrated in previous related resistance studies.
finally, a comparison of the genes differentially expressed in cv.
dream at  <dig> and 72 h after fusarium inoculation was performed to separate expression changes which have been maintained  from those that were exclusive for one of the two timepoints .
genes that are only differentially expressed at  <dig> hai  were categorised as ‘ <dig> hai-specific genes’.
a mapping into one of the two categories ‘fhb-responsive genes’ and ‘genotype-specific genes’ could not be done, due to the low quality of the microarrays obtained from mock treated cv.
lynx samples at this timepoint .
the revised dataset contained a total of  <dig>  differentially expressed genes after the transcripts likely to represent general cellular processes in cv.
dream were removed.
in total  <dig> of those genes could be assigned to  <dig> different defence-related classes.
the gene classes and assignments were made based on the gsea results and on information obtained from fhb-related literature.
table
<dig> gives an overview of the respective number of individual genes which have been assigned to the three categories and the  <dig> defence-related gene classes.
detailed information on genes that were identified as putative defence-related is provided in additional files
<dig>
<dig> and
<dig>  the gsea results were contributing to the formation of the functional classes:  ‘jasmonic acid  and ethylene  related genes’;  ‘cysteine-rich antimicrobial peptides ’ including serine-protease inhibitors;  ‘jasmonate-regulated proteins ’ comprising a set of strictly fhb inducible genes;  ‘gdsl-lipases’; and  ‘proteolysis’ including serine proteases.
based on literature, genes with different direct or indirect antifungal properties were added to the following classes:  ‘peroxidases’;  ‘genes related to cell wall defence’, for example pgip1s , xylanase inhibitors and glucan endo- <dig> -beta-glucosidase precursors, and  ‘secondary metabolism/detoxification’.
the remaining gene classes ‘transcription or signalling genes’, ‘miscellaneous defence related genes’ and ‘hormone metabolism’ have been made for the convenience of discussion.
1)genes differentially expressed only at  <dig> hai were not mapped into one of the two categories ‘fhb-responsive genes’ and ‘genotype-specific genes’, because of restrictions that originated in low quality of microarrays obtained from mock treated cv.
lynx samples at this timepoint .
2)numbers of unique genes are listed that were significantly differentially expressed genes .
detailed information on the genes assigned to the different defence-related gene classes is provided in additional files  <dig>   <dig> and  <dig>
indications for a jasmonate-dependent enhancement of fhb resistance in wheat
indications for the presence of a ja signalling were found in the cv.
dream transcriptome after fhb infection by using gsea testing.
the go terms ‘lipoxygenase activity’ , ‘oxylipin biosynthetic process’  and ‘lipid biosynthetic process’  associated to the oxylipin metabolism were exclusively enriched in the early  <dig> hai gene expression data  indicating that the chloroplastic 13-lox-branch was induced upon fhb infection.
hormone-like compounds such as ja and methyl jasmonate , as well as 13-hpl-derived c <dig> aldehydes, are characteristic products of this pathway.
some oxylipins generated by the 13-lox pathway, for example thaumatin-like proteins and phytoalexins, exhibit antimicrobial activities by impairing fungal mycelial growth and spore germination
<cit> .
other oxylipins, such as ja and meja are well known to serve important roles in plant defence-signalling by mediating the induction of the expression of some pr-genes
<cit> .
moreover, as 13-lox oxylipins are substantially produced from cuticle- or cell membrane-associated fatty acids released during the fungal degradation of plant cell walls, they also act as elicitors involved in pathogen recognition
<cit> .
threeputative lox genes  were fhb-responsive induced at  <dig> hai .
the transcript ta.
<dig> .a1_at was found to be a homologue of the maize gene zmlox <dig>  which is a novel chloroplast localized lox gene described as uniquely regulated by phytohormones and pathogen infection
<cit>  .
the two transcripts ta.
<dig> .a1_x_at and taaffx.
<dig> .s1_s_at showed significant similarity to the barley gene hordeum vulgare methyljasmonate-inducible lipoxygenase  <dig>  .
therefore, both transcripts might encode for one or two putative methyljasmonate -inducible chloroplastic 13-lox genes.
it was shown that jasmonates regulate their synthesis via positive feedback control by inducing the transcription of biosynthesis genes such as lox <dig> <cit> .
it is remarkable that both transcripts were also already induced 24 h after f. graminearum inoculation in the resistant spring wheat cv.
sumai 3
<cit> .
a)published sources: 1) <cit> ; 2) <cit> ; 3) <cit> ; 4) <cit> ; 5) <cit> ; 6) <cit> ; 7) <cit> ; 8) <cit> ; 9) <cit> ; 10) <cit> ; 11) <cit> .
b)for blastn analyses the threshold for a significant homology  was set to an e-value ≤ 1e- <dig>  identity scale >70% according to
<cit> .
five lox genes were up-regulated after both treatments and, in contrast to the solely fhb dependent induced lox genes, three of them were also expressed at  <dig> hai .
here, except for the transcript ta.
<dig> .s1_x_at, none of the genes could be assigned to a ja-mediated defence based on sequence similarities to published genes .
ta.
<dig> .s1_x_at, however, a homologue of a barley gene lox <dig> involved in different stress responses
<cit>  , was also shown to be active in cv.
sumai  <dig> upon f. graminearum infection
<cit> .
in summary, putative functions regarding defence response mediation were assigned to genes showing fhb-associated expression alterations.
here, all genes were found to be jasmonate and pathogen inducible or were previously identified as being fhb-responsive in cv.
sumai 3
<cit> .
this is remarkable as the cultivars dream and sumai  <dig> represent entirely different  origins and resistance levels.
additionally, ja and et defence-signalling pathways were found to be essentially involved in the high level fhb resistance of wheat cv.
wangshuibai in a recent study and were supposed to mediate the early basal defences at  <dig> to 24 h after f. graminearum infection
<cit> .
however, the contribution of a salicylic acid  signalling towards fhb resistance reported in that study was neither observed in our study nor reported for the cv.
sumai 3
<cit> .
on the other hand, a continual ja production can be involved in pathogen defence as well
<cit> .
indications for ja-inducible as well as for a continual pr-gene expression were indeed observed in the cv.
dream and both might contribute to the present fhb resistance .
a jasmonate-responsive and non-specific antifungal defence contributes to fhb resistance
the enrichment of genes belonging to the 13-lox pathway indicates a systemic accumulation of endogenous jasmonates in the resistant cv.
dream as a result of f. graminearum infections.
it is known that members of the jasmonate family, whose levels increase on pathogen infection, activate a specific set of genes encoding antimicrobial peptides
<cit> .
several cysteine-rich amps were found to be up-regulated in fhb infected cv.
dream spikes, which are possible targets of such resistance-related ja signalling, when the two points in time were investigated.
the set of identified cysteine-rich amps comprises lipid transfer proteins , thionins , and defensins .
lipid transfer proteins  were the most frequently expressed class of amps.
three genes were up-regulated independent of the treatment, while two transcripts were up-regulated exclusively 72 h after fhb inoculation.
compared to the other identified cysteine-rich amps, most of the ltp genes have shown relatively high fold changes that remained constant at both timepoints .
blastn analyses proved that all present ltp genes encode for putative non-specific lipid-transfer proteins .
direct antifungal activities and a broad resistance spectrum against biotrophic and necrotrophic fungal pathogens have been reported for various crop species and tissues, notably with nsltps
<cit> .
the observed antifungal activities also include different fusarium pathogens, such as f. graminearum  and f. solani , as well as f. culmorum and f. oxysporum
<cit> .
thereby, nsltp proteins were found to strongly inhibit the growth of fungal mycelia as well as the germination of fungal spores, including the conidiospores of f. graminearum <cit> .
wheat ns-ltps are generally supposed to play a role in an enhanced non-specific defence response regulated by different hormonal signals, including jasmonates.
in particular, constitutively expressed genes are supposed to contribute to non-host resistance
<cit> .
a synergistic activity of nsltp genes with thionins  against f. solani and f. graminearum was shown in studies on barley, maize and wheat
<cit> .
in fact, two transcript sequences  homologous to the wheat thionin gene thi <dig>  <cit>  were differentially expressed in the cv.
dream after both treatments, but not in the cv.
lynx .
thionins have a general antimicrobial activity against early conidial germination
<cit> .
in addition, a highly inducible expression was observed in the case of the arabidopsis thionin thi <dig>  after both fungal infections as well as meja treatment leading to an enhanced resistance to f. oxysporum <cit> .
peptidase inhibitors of the defensin family  make up the third class of continual up-regulated amps , represented by homologues of the wheat gene tad <dig> <cit>   and the defensin precursor prpi- <dig> from durum wheat
<cit>   .
while the antimicrobial activity of defensins requires typically complex synergistic interactions with other amps
<cit> , their promoters are potentially interesting candidates for the targeted and tissue-specific expression of pr- and r-genes, particularly for the protection against f. graminearum in cereal grains
<cit> .
an induction by jasmonates was reported for most of the defensin genes and some of the putative antifungal defensins are reported to be markers for the presence of ja- and et-dependent defence-signalling pathways
<cit> .
indeed, indications for an active et signalling were found in the fhb-attacked cv.
dream transcriptome as well .
the majority of up-regulated cysteine-rich amps in cv.
dream have shown expression values that were independent of the treatment, but were lower or absent in the susceptible cv.
lynx .
it is likely that the majority of these peptides act synergistically in a generalized non-specific defence providing a basal protection.
amps transcribed at a constant level are known key components of an immediate defence against invading pathogens
<cit> , and many proteins that are pathogen-inducible, for example, in leaves were found to be constitutively present in storage tissues, such as seed
<cit> .
moreover, it is generally assumed that genes involved in the quantitative fhb resistance of adapted european wheat cultivars represent such a defence mechanism
<cit> .
nonetheless, amps can also be part of an induced plant defence
<cit> .
in fhb-treated cv.
dream spikes only nsltp genes were up-regulated in response to the disease .
among these ta.
<dig> .s1_a_at  seems to be an interesting resistance candidate, as the gene combines a general high antifungal property with considerable fold change expression ratios at both timepoints .
moreover, the putative defensin gene prpi- <dig>  might be a relevant finding as well due to its possible utilization in a resistant strategy aiming at over-expressions of pathogen-inducible promoters to directly target the infection sites or the most vulnerable tissues
<cit> .
such an approach becomes even more interesting with the recent observation that the biotrophic life form of f. graminearum persists in all colonized tissues
<cit> .
living host cells form a zone surrounding the most advancing hyphae and could be targets for such an approach as they allow a continuous supply with antifungals onto the intercellular hyphal tips.
identification of fhb-responsive jasmonate-regulated proteins
taking into account the observations on the dream and sumai  <dig> cultivars it can be hypothesised that a fhb-responsive ja signalling is active from  <dig> to  <dig> hai covering the presumed phase of the general biotrophic fungal growth, since the switching point to increased necrotrophic nutrition was timed around  <dig> hai
<cit> .
an interesting conformance was observed with the fhb-responsive expressions of genes that encode for jasmonate-regulated proteins , belonging to the subfamily of mannose-specific jacalin-like lectin containing proteins  during that period
<cit> .
three mjrl genes, taaffx.
<dig> .s1_at, ta.
<dig> .s1_at and ta.
<dig> .s1_at, were up-regulated in cv.
dream at  <dig> hai.
the first two transcripts are prominent due to the considerable fold change expression ratios of  <dig>  and  <dig>    and their up-regulation exclusively in the fhb treated spikes of cv.
dream.
as many mjrl genes are described as strictly inducible defence proteins
<cit> , taaffx.
<dig> .s1_at and ta.
<dig> .s1_at might be involved in the fhb defence.
blast analysis showed that all detected putative mjrl genes belong to the mjrp- <dig> protein subfamily.
in general, mjrp- <dig> genes are specifically induced by ja via transcriptional activation and were initially identified in jasmonate-treated barley leaves.
the first mjrp- <dig> gene analysed in detail was the bgaf  gene from maize
<cit> .
the sequence of the transcript taaffx.
<dig> .s1_at shows similarities to another maize bgaf gene
<cit>  as well as to the wheat gene ta-ja <dig> .
although detailed knowledge on the defence function of mjrp- <dig> proteins is still to be gained, a broad resistance spectrum has already been observed.
one prominent example is the ta-ja <dig> gene that encodes a modular bgaf-related protein with a proven broad-spectrum resistance to infections by bacterial, fungal and viral pathogens in transgenic tobacco plants
<cit> .
all currently known mjrp- <dig> genes come from poaceae and share important traits separating them from other mjrls, for example their exclusive, tissue-specific induction via jasmonates and their single-copy status.
however, notably due to their strict tissue-specific expressions, mjrp- <dig> genes are not supposed to be orthologous, although the proteins share numerous common features
<cit> .
an mjrp- <dig> gene expressed in spike tissues has not been reported so far.
for this reason and due to its fhb-responsive high level induction, a separate study should reveal whether the taaffx.
<dig> .s1_at gene represents a new spike-specific member of the mjrp- <dig> family.
in addition to ta-ja <dig>  the poaceae jrp- <dig> family comprises three other wheat genes: ver <dig> , wci- <dig>  and hfr- <dig> .
in the present work, the wheat chemically-induced gene wci- <dig> and the vernalisation-related gene ver <dig> were up-regulated in cv.
dream upon f. graminearum infection.
wci- <dig>  was characterised as a plant disease resistant response gene that is induced by benzothiadiazole
<cit> .
bth is a functional analogue to sa which was not successful in reducing the fhb-disease caused by f. graminearum <cit> .
on the other hand, an up-regulation of wci- <dig> upon meja application has been reported
<cit> , and the wci- <dig> orthologous pea gene dir <dig> was found to be involved in the resistance to different fusarium pathogens
<cit> .
due to these contradictory observations further examinations are required to clarify the role of wci- <dig> in fhb resistance.
the up-regulation of the vernalisation-related gene ver <dig>  upon f. graminearum infection is interesting.
indeed, due to the proven specific induction by meja, ver <dig> was initially proposed to be involved in a jasmonate mediated plant defence response.
however, an induction of expression upon f. culmorum infection could not be confirmed and a native ver <dig> induction has so far only been observed in young wheat seedlings during the vernalisation process
<cit> .
thus, whether the untypical expression of ver <dig> in wheat kernels is associated with fhb resistance, or rather is a side effect caused by jasmonate-signalling remains unanswered at this point.
an increased ethylene  production contributes to wheat fhb resistance
ethylene  plays an important role in plant growth and development but it is also known to be involved in the regulation of primary resistance responses
<cit> .
indications for an increased et-metabolism in cv.
dream spikes following fhb infection are provided by several up-regulated putative 1-aminocyclopropane-1-carboxylate  oxidases and gdsl-like lipases genes.
the acc oxidase, also called the et-forming enzyme, catalyses, together with the enzyme acc synthase, the last biosynthetic step to convert acc into et.
both enzymes are known to be rate-limiting components in the et biosynthetic pathway
<cit> .
a total of  <dig> acc oxidase genes were either up-regulated or down-regulated in the cv.
dream, mainly in a constitutive manner .
in fact, the expression of individual acc oxidase genes is generally frequent and differentially regulated at all times due to developmental changes as well as abiotic and biotic stress factors
<cit> .
the occurrence of several gdsl-like lipase genes in the cv.
dream assay further indicates an elevated et-signalling.
gdsl-like lipases were mainly differentially expressed upon both treatments .
among the characterised gdsl-like lipases, the genes glip <dig> and glip <dig> of arabidopsis are known to play an important role in plant immunity by eliciting local as well as systemic resistance against necrotrophic and hemibiotrophic pathogens.
moreover, gdsl-like lipase transcription was exclusively enhanced by et, but not by sa or ja
<cit> .
however, none of cv.
dream gdsl-like lipases has shown a sequence homology to the reported resistance candidates from arabidopsis.
it is generally accepted that the plant defence against necrotrophic pathogens is usually regulated by ja and et while sa plays a major role in the defences against biotrophic pathogens
<cit> .
a possible involvement of et in fhb resistance has also been demonstrated for sumai  <dig>  based on an up-regulated acc oxidase gene.
furthermore, an increased resistance against fhb was observed for the susceptible cultivar y <dig> after spraying spikelets with ja as well as et before and after fungal infection
<cit> .
different studies in arabidopsis <cit>  and tobacco
<cit>  have shown that et and, in particular, the over-expression of certain acc oxidase genes can extend the symptomless biotrophic phase during hemibiotrophic fungal infections.
in addition, it was found that et can reduce cell death caused by the fungal toxin fumonisin b <dig> which is produced by several cereal-attacking fusarium species
<cit> .
indications for fhb-responsive suppression of fungal virulence factors
in addition to the presence of ja- and et-mediated general antifungal defences, a second line of defence was found to be based on a fhb-responsive and targeted suppression of relevant fusarium virulence factors, such as proteases and mycotoxins.
this defence mechanism was assembled from genes encoding protease inhibitor  proteins  and different genes which are proposed to be associated with the detoxification of pathogen-derived mycotoxins.
both, fusarium proteases and mycotoxins take on relevant roles in the fungal pathogenesis and were found to be secreted in nearly all phases of the fungal wheat spike colonisation
<cit> .
wheat-derived protease inhibitor genes in fhb disease resistance
in the fhb-treated cv.
dream transcriptome, serine pi proteins of the subtilisin-like protease  superfamily were significant enriched at both timepoints , represented by the go terms ‘serine-type endopeptidase inhibitor activity’  and ‘peptidase activity’ .
pi proteins generally feature a high substrate specificity and therefore, it is likely that those genes encode for proteins that specifically bind and impair secreted fusarium sl proteases
<cit> .
proteases generally cause the proteolytic digestion of proteins via the hydrolysation of peptide bonds.
fusarium subtilisin-like  and trypsin-like  proteases are released in infected wheat kernels mainly to disrupt host cell membranes during necrotrophic intracellular nutrition.
consequently, defence-related interactions between plant pi proteins and subtilisin-like and trypsin-like proteases of f. graminearum and f. culmorum have already been proven in the grains of barley
<cit>  and ancient emmer wheat
<cit> .
in total, five serine-protease inhibitors were differentially up-regulated in cv.
dream .
two transcripts were functionally annotated to the bowman-birk inhibitor  family based on sequence homologies to the wrsi <dig> gene .
wrsi <dig> was described as a salt-responsive gene with a suggested role in regulating plant growth
<cit> .
among the remaining transcripts, ta.
<dig> .s1_x_at and ta.
<dig> .s1_x_at were up-regulated in response to fhb at  <dig> hai, while ta.
<dig> .s1_at was regulated solely at  <dig> hai.
the ta.
<dig> .s1_at gene was selected for qpcr analysis because of its relativelyhigh and fhb-responsive fold change  at  <dig> hai .
a possible expression at  <dig> h after fusarium inoculation was not reliably determined due to missing expression data from two of the three biological replicates.
the ta.
<dig> .s1_at expression was measured with an inoculation time-courses of two cultivar pairs: first, the winter cultivars dream and lynx described as moderately resistant and susceptible ; second, the spring cultivars sumai  <dig> and florence-aurore described as resistant and susceptible .
sumai  <dig> and florence-aurore, in particular, were found to represent the extremes of spring wheat responses to fusarium spread
<cit> .
upon comparing the fhb-responsive transcript induction levels in the cultivars dream and lynx, a generally higher induction over control and cv.
lynx samples was observed for cv.
dream for the period between  <dig> to  <dig> hai .
as a matter of fact, >4-fold inductions were only obtained at  <dig> and  <dig> hai.
however, even the 2-fold inductions of earlier and later timepoints were considered relevant due to the strictly suppressed expression in the susceptible genotype .
in the fhb-treated spike tissues of sumai  <dig>  > <dig> and >300-fold inductions were already observed between  <dig> to  <dig> hai and a third peak of >200-fold was found at  <dig> hai .
no gene expression was verifiable in spike samples of cultivars sumai  <dig> and florence-aurore at  <dig> hai which was the last timepoint .
in the first instance, the relative induction peak at  <dig> hai in cv.
dream is consistent with previous observations that endogenous wheat serine-protease inhibitor proteins are not induced until 72 h after fusarium inoculation
<cit> .
in fact, in the period between  <dig> and  <dig> hai, during which the necrotrophic nutrition becomes predominant, f. graminearum transcripts were found to dominantly encode degrading enzymes such as proteases, lipases and nucleases
<cit> .
a transcript accumulation was even observed in the susceptible genotypes lynx  and florence-aurore  particularly in this period, however, to a lower extent than in the respective resistant counterparts.
the physiological responses of pis are furthermore triggered by negative feedback mechanisms
<cit> .
therefore, the remarkable suppression of ta.
<dig> .s <dig> in sumai  <dig> during this crucial time might be a consequence of the already high transcript abundance and the subsequent induction at  <dig> hai is assumed to be stimulated by further secreted fungal proteases .
the early high level activity of ta.
<dig> .s <dig> until the timepoint  <dig> hai in cv.
sumai  <dig>  is consistent with previous data from sumai  <dig> gene expression studies, demonstrating the fhb-responsive expression of several pis at already  <dig> hai
<cit> .
in this period, an exclusive induction of the tested serine-protease inhibitor was also observed for the moderately resistant cv.
dream .
consequently, the early expression of wheat pi genes could be an immediate reaction to early levels of secreted fusarium tl and sl proteases which have been reported for different compatible interactions, amongst others between f. graminearum and barley as well as wheat.
here, the activity of fungal proteases has been attested already at  <dig> to  <dig> hai, long before a corresponding expression could be observed in kernels
<cit> .
in fact, beside their harmful roles during the phase of a necrotrophic intracellular nutrition, fungal proteases were found to be secreted already during the earlier intercellular colonisation of spike rachis, probably to suppress certain plant defence reactions by degrading pr-proteins
<cit> .
in this sense, the serine-protease inhibitor ta.
<dig> .s1_at seems to be an interesting resistance candidate as transcript accumulations were present during the early and the later phases of fungal spike colonisation.
however, this potential still needs to be confirmed in a further study.
nevertheless, pis are discussed as candidates for an improved resistance strategy against grain infecting fungal pathogens
<cit>  and our results from qpcr and transcriptome analyses do not contradict these considerations.
analysis of the detoxification mechanisms in wheat concerning fhb resistance
fusarium proteases and mycotoxins act in a kind of strategic cooperation during spike and kernel colonisation by featuring complementary roles during the host defence suppression and the intracellular colonisation of spikelets.
from an economic perspective, fusarium species causing fhb belong to the most important trichothecene producers and don is a predominant trichothecene toxin produced by these species
<cit> .
silencing the fusarium tri <dig> gene down-regulates more than  <dig> genes involved in the mycotoxin production and results in a reduction of don production and pathogenicity
<cit> .
meanwhile, several different plant genes are known to be up-regulated at the transcriptional level in response to either don treatment or don production which are thus likely to be involved in the don-resistance
<cit> .
to analyze the expected impact of a specific mycotoxin defence on the general fhb resistance of cv.
dream, a literature-to-transcriptome approach was used.
known toxin resistance-related genes from wheat and barley were checked for homologous genes on the wheat array and their respective expression profiles in the cultivars dream and lynx.
a diverse set of  <dig> wheat genes could be identified as possible members of a general detoxification mechanism.
those genes are listed in table
<dig>  including the respective literature sources.
within this set,  <dig> genes originate from a study of trichothecene-induced gene expression in barley
<cit> .
screening the expression patterns of those  <dig> genes in the cv.
dream vs. cv.
lynx microarray data revealed for all genes similar expression patterns.
they were exclusively expressed or induced in fusarium treated samples collected 72 h after infection.
moreover, they were also up-regulated in both genotypes and, in addition, they were up-regulated in both genotypes and the level of up-regulation was higher in susceptible cv.
lynx in all cases.
however, expression differences between both genotypes never reached a level of statistical significance.
finally, they were not expressed in mock control samples at all, although this observation was not reliable in the case of cv.
lynx samples collected at  <dig> hai due to the above mentioned restrictions.
to analyse the observed congruities in more detail and to test whether or not the expression in the susceptible cv.
lynx is just a temporary phenomenon, a selection of six genes representing different functional categories was forwarded to qpcr analysis using the above mentioned inoculation time-courses of the cultivar pairs dream vs. lynx and sumai  <dig> vs. florence-aurore.
a)class association as obtained from respective literature sources.
b)gene annotations were updated by blastn analysis in the ncbi database, otherwise published annotations were considered.
c)published sources: 1) <cit> ; 2) <cit> ; 3) <cit> ; 4) <cit> ; 5) <cit> ; 6) <cit> ; 7) <cit> ; 8) <cit> ; 9) <cit> ; 10) <cit> ; 11) <cit> ; 12) <cit> .
d)predicted mrp gene sequence contains est cluster: ca <dig>  bj <dig> and bj303163
<cit> .
the analysed genes associated with don detoxification are taugt <dig>  and a homologue of the barley udp-glucosyltransferase gene hvugt <dig>  genes that are supposed to be involved in the resistance to don accumulation are tapdr <dig>  and tamdr <dig> .
as representatives of the functional categories “defence-related” and “general” a further putative serine-protease gene and a 12-oxophytodienoate reductase gene were included .
the qpcr data for the winter wheat cultivars dream vs. lynx  showed similar expression patterns for all tested genes as did the microarray experiments.
consequently, a temporary and higher induction peak was found for lynx at  <dig> hai compared to dream.
on the other hand, the transcripts of all tested genes peaked at  <dig> hai in the cv.
dream samples, while lynx revealed suppressed or consistent inductions.
in addition, a >4-fold induction was already observed before  <dig> hai for most of the cv.
dream alleles and the expressions were showing a general and increasing trend towards the peak at  <dig> hai .
such a maximum induction at  <dig> hai has likewise been observed for the don resistance candidate gene pdr <dig> -like in infected spikes of the chinese landrace wangshuibai
<cit>  which represents one of the most important genetic resources for fhb and don resistance
<cit> .
like the analysed genes tapdr <dig> and tamdr <dig> , pdr5-like is like a plasma membrane abc transporter which co-segregates with the don resistance qtl qfhs.ndus-6bs from cv.
wangshuibai
<cit> .
in the cultivar pair sumai  <dig> vs. florence-aurore the fusarium-induced expression levels obtained for the udp-glucosyltransferase  and abc transporter genes were showing typical curve characteristics in cv.
sumai  <dig> samples; starting with a low level induction at  <dig> hai, followed by a consistent increase up to the peak at  <dig> or  <dig> hai and showing a continuous downtrend thereafter .
in contrast, considerable inductions for the susceptible cv.
florence-aurore did not appear until  <dig> to  <dig> hai .
interestingly, both ugt genes show induction peaks at  <dig> hai in cv.
sumai  <dig> while both abc transporter genes peak at  <dig> hai.
deviating induction patterns were observed for the representatives of the functional categories “defence-related” and “general” .
for all tested genes no expressions were measured from samples collected at  <dig> hai .
in summary, the expression profiles of genes related to detoxification in both resistant cultivars were following an early beginning steady-state model, providing transcript levels during the reported period of increased don accumulations between  <dig> and  <dig> hai
<cit> .
in contrast, both susceptible cultivars showed typically late and temporary inductions.
our observations for the expressions of ugt and abc transporter genes in cv.
sumai  <dig> are furthermore in accordance with expression patterns previously observed for the abc transporter gene tapdr <dig> <cit>  as well as the ugt gene taugt <dig> <cit>  in fhb-treated spike samples of cv.
wangshuibai.
the tapdr <dig> gene is a member of the atp-binding cassette  protein superfamily and has been identified in cv.
wangshuibai due to its strong up-regulation upon don treatment as well as f. graminearum inoculation.
after fungal infection, the relative amount of tapdr <dig> transcripts increased in wangshuibai at  <dig> hai
<cit> .
the function of tapdr <dig> in fhb resistance is proposed to be don-related because gene expressions were found to peak after  <dig> to 12 h of don-inoculation and declined slowly thereafter.
in addition, a late expression peak was observed for the susceptible cv.
alondra
<cit>  similar to our observations in the susceptible cv.
florence-aurore .
the general role of pdr transporters in the resistance to antifungal drugs was first characterized in yeast  and a particular function in don resistance was confirmed based on a yeast mutant carrying a knockout variant of the pdr <dig> transporter gene resulting in a non-natural hypersensitivity to don
<cit> .
the second analysed transporter gene tamdr <dig> was initially isolated from wheat root apices as being induced by aluminium toxicity
<cit> .
however, tamdr <dig> was up-regulation together with tapdr <dig> in cv.
wangshuibai and, thus, was supposed to be involved in don resistance as well
<cit> .
in fact, our time-course qpcr expression data were able to reveal that both genes show similar expression profiles upon fusarium infection in the resistant cultivars dream  and sumai  <dig> , respectively.
although genotype-specific differences were present, the observed similar expression patterns indicate a possible trichothecene-responsive up-regulation for tamdr <dig> as well.
using nullisomic-tetrasomic wheat lines, we have also located the tamdr <dig> allele on chromosome 5a where tapdr <dig> had already been placed before
<cit> .
these observations may reflect a common mechanism of transcriptional co-regulation for both genes.
in general, there is accumulating evidence that gene order in eukaryotic genomes is not completely random and that pathogen-responsive as well as other genes with similar expression levels tend to be clustered within the same genomic neighbourhoods
<cit> .
in fact, for tapdr <dig> it was discovered that the gene expression is not induced by ja, sa and abiotic stress factors but by decreasing concentrations of al3+ and free [ca2+.
this mode of regulation was also reported for the tamdr <dig> gene due to its general induction by al injury in wheat roots
<cit> .
both toxicities activate plant programmed cell death via an oxidative burst and both inhibit calcium channels of plasma membranes which causes a decrease of the intracellular second messenger [ca2+ <cit> .
in order to explain the commonalities we have found between both abc transporters in terms of induction, we suggest that don induces the expression of tapdr <dig> and tamdr <dig> indirectly via decreased levels of [ca2+.
whether tamdr <dig> thus has a similar relevance for the detoxification process as can be suggested for tapdr <dig>  still needs to be proven in a further study.
two ugt genes supposed to be involved in the don detoxification were analysed with qpcr.
quite a few of the plant ugts are related to disease resistance where they play important roles in the detoxification of exogenous compounds, for example fungal metabolites such as don
<cit> .
blastn analysis revealed the homology between the transcript ta.
<dig> .s1_at and the taugt <dig>  gene which had originally been cloned from cv.
wangshuibai
<cit> .
ta.
<dig> .s1_at has revealed a significant full length sequence homology to the barley ugt gene hvugt <dig> <cit>  .
both genes have displayed the respective characteristic qpcr expression profiles for cvs.
dream  and sumai  <dig>  as described above.
however, higher induction levels were observed for the putative hvugt <dig> gene when compared to taugt <dig>
at the first instance, the wheat gene taugt <dig> was the most interesting candidate since it was suggested to be an efficient candidate gene for improving don resistance
<cit> .
however, our expression data are in accordance with recent observations which have demonstrated that hvugt <dig> can protect yeast from don by converting it to don-3-glucoside while taugt <dig> was not able to convert don
<cit> .
in addition, with our observations in the cultivars dream and sumai  <dig>  hvugt <dig> has demonstrated relevant activities in a number of fhb-treated wheat cultivars as well as in barley, indicating that it be might of general relevance.
hvugt <dig>  and also taugt <dig>  were detected as don resistance candidates in don inoculated spikes of cv.
wangshuibai in a gene expression study using the affymetrix wheat gene-chip®
<cit> .
moreover, blastn analysis could demonstrate that hvugt <dig> has also been identified as don resistance related gene  in wheat dh-lines carrying the major fhb resistance qtl fhb <dig> from cv.
cm82036
<cit>  as well as in two related barley transcriptome studies
<cit> .
finally, the gene hvugt <dig> appears to be a remarkable candidate gene for fhb resistance.
it is considered relevant for a promising strategy to improve fhb resistance not only in wheat but also other cereal species.
as representative for the functional category “general”, the expressions of a putative wheat gene encoding for a 12-oxophytodienoate reductase was analysed .
ta.
<dig> .s1_at was functionally characterised by significant homology to the maize 12-oxo-phytodienoate reductase gene zmopr <dig> .
the homologous barley gene  was previously found to respond to pathogen-derived trichothecene accumulation
<cit> .
in addition to ta.
<dig> .s1_at, two more putative opr genes were identified as up-regulated in response to fhb: the gene zmopr <dig>  and the gene zmopr <dig> .
all three genes are putative wheat homologous of the opr i group members which preferentially catalyse the formation of the natural ja precursor 12-oxo-phytodienoic acid
<cit> .
in our qpcr analysis, the zmopr <dig> homologue ta.
<dig> .s1_at has shown a fhb-associated induction at  <dig> hai which was common for both the resistant genotypes .
this might indicate a rapid and transient up-regulation of ta.
<dig> .s1_at.
in fact, the genes zmopr <dig> and zmopr <dig> have demonstrated a transient induction upon fusarium verticillioides infection in maize
<cit> .
a similar rapid and transient up-regulation caused by a variety of environmental cues including hydrogen peroxide  was observed for the ta.
<dig> .s1_at homologous gene osopr <dig>  in rice
<cit> .
don is known to induce the transient accumulation of h2o <dig> as the most stable compound involved in oxidative burst
<cit> .
indeed, yeast studies indicate detoxifying functions for opri enzymes
<cit> .
indications for a complex crosstalk between fungal and plant proteases and their inhibitors during fhb defence
the putative wheat serine protease gene  belongs to the subtilisin-like protease family and was initially detected as a gene  that strictly responds to pathogen-derived trichothecene accumulation in barley
<cit>  .
in addition, serine proteases were found to be enriched in the cv.
dream transcriptome upon fhb treatment and were annotated to the go term ‘serine-type carboxypeptidase activity’  .
an early ta.
<dig> .a1_at expression was found for cv.
sumai  <dig>  here, exclusive and equal >2-fold inductions were present at  <dig>   <dig> and  <dig> hai .
at  <dig> hai, both resistant cultivars showed the highest induction level, in cv.
dream even with a peak of >60-fold, while at this timepoints no expressions were found in the susceptible cultivars .
an opposing effect was observed at  <dig> hai, when exclusive expression was observed for both susceptible wheat cultivars, while no expression was detectable in the resistant ones .
as proteolytic and protein-binding enzymes proteases feature important functions for the selective breakdown of regulatory proteins and several plant proteases have been linked to defence responses
<cit> .
although many questions remain unanswered concerning their mode of action, there is evidence that plant proteases, in particular subtilisin-like proteases, are involved in the crosstalk between pathogen and host.
in this context, a defence-counter-defence mechanism was observed between the plant-pathogen interaction tomato/phytophthora infestans, in which both, host and pathogen are supposed to release specific sets of proteases and protease inhibitors mutually impairing each other
<cit> .
moreover, such counter-defence mechanism is supported by the assumption of a strong co-evolution between proteases and protease inhibitors which are mutually released during a pathogen-host interaction
<cit> .
it is interesting in this context, that proteases as well as protease inhibitors were enriched in the transcriptome of the resistant cultivar dream upon f. graminearum infection .
regarding the role of the reported plant proteases involved in don resistance , we suggest that they do not act in response to a don accumulation but rather in response to a fusarium protease-rich environment as fusarium proteases appear together with mycotoxins during spike rachis and kernel colonisation.
in addition, a specific function within a detoxification mechanism has yet not been described for plant proteases.
CONCLUSIONS
our transcriptome study provides evidence for the existence of a biphasic defence reaction against fhb in wheat.
jasmonate and ethylene regulated non-specific antifungal protections are supplemented by host gene networks associated to the accumulation of f. graminearum-derived trichothecenes and subtilisin-like proteases.
using a literature-to-transcriptome approach,  <dig> genes described as related to don resistance were identified due to analogies in their microarray expression profiles which hence, may belong to a detoxification pathway that is active in different resistant wheat cultivars as well as in barley.
our qpcr expression analyses of seven wheat genes associated with the suppression of fungal virulence factors have demonstrated similar fhb-responsive inductions in the cultivars dream and sumai  <dig>  moreover, an earlier first induction and a steady-state level of expression were found to be associated with fhb resistance, while fhb-responsive gene expression in susceptible cultivars was typically late and temporary.
these results will help not only to understand changes in overall gene expression in wheat during fusarium infection, but will also help to identify potential targets for development of disease control strategies.
in fact, genes interesting for further investigations in this direction were identified in both wheat defence mechanisms.
these are, nsltp , defensin  and mjrp  genes as well as the pdr-transporter gene tamdr <dig> , the ugt gene hvugt <dig>  and the putative serine-protease inhibitor gene ta.
<dig> .s1_at.
the last three genes have shown regulations in response to fhb in the cultivars dream and sumai  <dig>  in general, the identification of resistance candidate genes that are commonly active in different resistant wheat and barley cultivars is an important result with regard to the development of novel strategies against fhb severity and grain toxin contamination.

 fusarium head blight caused e.g. by f. graminearum schwabe petch) is one of the most destructive diseases of wheat worldwide, causing significant reductions in grain yield and quality. the most efficient strategy to control fhb in wheat is the use of resistant cultivars. however, in hexaploid wheat the resistance to fhb is highly complex. since over qtl have been reported, whereas only a few qtl were found to be stable in different genetic backgrounds and useful for breeding. the most stable qtl were obtained from the chinese wheat varieties sumai and wangshuibai. however, poor agronomic performance and the frequent occurrence of genetic linkage drag make them less suitable donors of resistant genes. moreover, the genetic and molecular basis of the quantitative fhb resistance is still poorly understood. recent studies on the mode of fusarium spike colonisation have revealed that the pathogens use a specific arsenal of virulence factors which are essential in nearly all phases of the disease making them interesting targets for novel resistance strategies. trichothecene toxins, such as deoxynivalenol, and hydrolytic enzymes, such as subtilisin-like and trypsin-like proteases, are two virulence factors that were found to occur during almost the entire course of disease. don was found to be produced in the fungal infection structures already during the initial penetration of floret tissues. the reason for this early secretion remains unknown, because the initial infection is symptomless and indistinguishable between susceptible and resistant wheat cultivars in all respects; even the trichothecene-deficient fusarium mutants do not show any restrictions regarding their infectious ability. however, already in the second infection phase, don production gains relevance. it is supposed that the general capacity to prevent protein synthesis makes the toxin an important suppressor of early plant defences. for that purpose, don seems to enable the fungal hyphae to break through the spike rachis node which is the central bottle-neck for both, the initial spread from infected florets into the spike rachis and the reverse direction from the rachis into uninoculated spikelets. during the rachis colonization when hyphae grow vertically, the toxin may inhibit the onset of various cell wall reinforcement processes in the vicinity of invading hyphae. at the same time, fungal proteases are likely to participate in the suppression of plant defences by degrading pathogenesis-related proteins or defence-signalling compounds according to their property to cause proteolytic protein digestion. in the spikes of the resistant landrace wangshuibai the down-regulation of different housekeeping proteins was reported already to 24 h after f. graminearum inoculation as a consequence of the secretion of fungal hydrolytic enzymes and toxins. the intercellular spread through the spike rachis is accompanied by lateral hyphae growth to infect uninoculated spikelets. this secondary colonisation is essentially associated with the secretion of don and proteases which initiate and facilitate necrotrophic intracellular nutrition. the phase is characterized by dramatic changes in the interaction between pathogen and host concerning the respective transcriptomes, secretomes and metabolomes, and is often described as switching point from fungal biotrophy to necrotrophy. increased don levels were observed 26 to 96 h after infection. in addition, between and hai f. graminearum transcripts were found to encode especially degrading enzymes such as proteases. these accumulations were typically linked to increased levels of systemic fungal development and collapsed host cells. both virulence factors are probably essential for the penetration and mortification of host cells, as fusarium pathogens use cell wall digestion to enter living host cells and don, in particular, is known to activate plant programmed cell death. in summary, don and proteases have a significant impact on cell wall digestion, protein matrix reduction and damage to starch granules, typically seen in fusarium-infected wheat kernels rendering grain yield unsuitable and unsafe for food, feed or malting purposes. in order to characterise the transcriptional changes in the resistant cv. dream compared with the susceptible cv. lynx, we performed gene expression profiling using the genechip® wheat genome array. genechip expression data obtained and 72 h after inoculation with f. graminearum or, respectively, mock have revealed indications for the presence of two main defence mechanisms in cv. dream, reflecting a biphasic strategy against fhb disease. one mechanism comprised jasmonate- and ethylene-mediated defence reactions directed against fungal growth and sporulation, while the second mechanism was specifically directed towards fungal mycotoxins and proteases. quantitative real-time pcr time-course study was applied to analyse the expressions of seven selected anti-virulence gene candidates in the cultivar pairs dream/lynx and sumai /florence-aurore. observed similarities between the resistant cultivars dream and sumai in terms of fhb-responsive up-regulated genes from both described defence mechanisms will be reported.  identification of fhb-responsive genes in the resistant wheat cultivar dream transcript abundances in the f. graminearum -inoculated and mock-inoculated wheat cultivars dream and lynx were measured and compared using the affymetrix genechip® wheat genome array. the general disease progression was examined on single-floret inoculated samples that were collected and hours after inoculation. all measurements were performed with three biological replicates. for each timepoint the four genechip datasets were compared to identify differentially expressed genes involved in the different aspects of the inoculation response. table lists all comparisons with the respective numbers of differentially expressed genes. * differentially expressed genes originated from the three replicates of genechip dataset ‘lynx 72 h after mock inoculation’ were not recorded due to low quality of the microarrays. a gene set enrichment analysis of the comparisons was conducted to identify relevant functional classes associated to incompatible cv. dream-f. graminearum interactions. table provides an overview of the nine gene ontology terms that were enriched in those genes found to be significantly up-regulated in the resistant cv. dream at and 72 h after fusarium inoculation compared with the fusarium inoculated susceptible cv. lynx. all terms were found to be associated to the disease as the respective represented gene products were neither enriched in the analogous cultivar comparison after mock inoculation nor in the comparison ‘cv. lynx fusarium inoculated versus cv. lynx mock inoculated’. no go terms were enriched in the significantly down-regulated genes at hai while enriched terms were observed at the later timepoint. for enrichments at hai associations to the infection with f. graminearum were restricted and, thus, were only possible if a go term was also enriched also at hai. 1) significance levels for up-regulated go terms: fdr value at < ; nom p-value at < . 1) significance levels for down-regulated go terms: fdr value at < ; nom p-value at < . the gsea provided insides into defence mechanisms that were induced during incompatible interactions. at hai an exclusive enrichment was observed for the terms ‘lipoxygenase activity’, ‘oxylipin biosynthetic process’ and ‘lipid biosynthetic process’ including genes, such as lipoxygenases, involved in the plant oxylipin metabolism. additionally, lipoxygenases genes were also frequent in the term ‘response to wounding’. putative cysteine-rich proteins, such as thionins, were detected in the go term ‘pathogenesis’. phyto-oxylipins comprising antimicrobial peptides and defence-signalling molecules such as jasmonates, together with cysteine-rich pathogenesis-related genes indicate an induced antifungal defence mechanism. plant serine-protease inhibitors were enriched in the go terms ‘serine-type endopeptidase inhibitor activity’ go: and ‘peptidase activity’ go: and represented the second class of genes enriched in the term ‘response to wounding’. serine-protease inhibitors as well as genes encoding serine-proteases identified by the term ‘serine-type carboxypeptidase activity’ were enriched at both timepoints. these enriched terms represent an induced defence mechanism against pathogen-released proteases which as virulence factors are secreted to modify host proteins. on the basis of testing, this defence mechanism as well as the antifungal defence mechanism were found to be central and therefore, will be discussed in more detail later on. finally, genes encoding for peptidyl-prolyl cis-trans isomerase proteins belonging to the immunophilin superfamily were found to be accumulated. ppiase proteins have general functions in protein folding and protein degradation, and several proteins have shown antifungal properties, similar to pr-genes. in the down-regulated genes three go terms were noticeable with regard to the pathogen presence, while associations to the disease were rather unclear for the other terms. the detected go terms ‘chitin catabolic process’ and ‘chitinase activity’ demonstrate the down-regulation of genes which typically facilitate the breakdown of fungal cell walls. chitinase genes have shown to exhibit an enhanced resistance against f. graminearum, in barley while in the grains of emmer wheat, a progenitor of bread wheat, a similar down-regulation of chitinase genes was observed and discussed as a direct impact of f. graminearum signals. finally, the term ‘mycelium development’ comprises 10 f. graminearum genes, belonging to a set of fusarium genes which were previously found to be present on the affymetrix wheat genechip®. as these genes are putatively associated to the progression of the fungal mycelium, their enrichment amongst down-regulated genes might reflect traces of an impaired fungal growth in the resistant dream cultivar. a comparison was performed between the ‘cv. dream fusarium inoculated versus cv. lynx fusarium inoculated’ expression data and the analogous expression data from the mock inoculation, in order to address expression changes in the resistant cv. dream associated with the fungal attack. at hai, the genes differentially expressed in cv. dream could be separated into genes that were differentially expressed to higher levels or were only present after pathogen attack, after both treatments, and only after control treatment. the genes only present in response to fhb were categorised as ‘fhb-responsive genes’. especially, up-regulated transcripts are likely to represent defences, such as trigger mechanisms or direct antimicrobial activities. genes with similar expression profiles after both treatments were categorised as ‘genotype-specific genes’ because they were differentially expressed to lower levels or absent in the cv. lynx spike samples. up-regulated genes were hereafter discussed as members of a basal defence if their induction has been demonstrated in previous related resistance studies. finally, a comparison of the genes differentially expressed in cv. dream at and 72 h after fusarium inoculation was performed to separate expression changes which have been maintained from those that were exclusive for one of the two timepoints. genes that are only differentially expressed at hai were categorised as ‘ hai-specific genes’. a mapping into one of the two categories ‘fhb-responsive genes’ and ‘genotype-specific genes’ could not be done, due to the low quality of the microarrays obtained from mock treated cv. lynx samples at this timepoint. the revised dataset contained a total of differentially expressed genes after the transcripts likely to represent general cellular processes in cv. dream were removed. in total of those genes could be assigned to different defence-related classes. the gene classes and assignments were made based on the gsea results and on information obtained from fhb-related literature. table gives an overview of the respective number of individual genes which have been assigned to the three categories and the defence-related gene classes. detailed information on genes that were identified as putative defence-related is provided in additional files and the gsea results were contributing to the formation of the functional classes: ‘jasmonic acid and ethylene related genes’; ‘cysteine-rich antimicrobial peptides ’ including serine-protease inhibitors; ‘jasmonate-regulated proteins ’ comprising a set of strictly fhb inducible genes; ‘gdsl-lipases’; and ‘proteolysis’ including serine proteases. based on literature, genes with different direct or indirect antifungal properties were added to the following classes: ‘peroxidases’; ‘genes related to cell wall defence’, for example pgip1s, xylanase inhibitors and glucan endo- -beta-glucosidase precursors, and ‘secondary metabolism/detoxification’. the remaining gene classes ‘transcription or signalling genes’, ‘miscellaneous defence related genes’ and ‘hormone metabolism’ have been made for the convenience of discussion. 1)genes differentially expressed only at hai were not mapped into one of the two categories ‘fhb-responsive genes’ and ‘genotype-specific genes’, because of restrictions that originated in low quality of microarrays obtained from mock treated cv. lynx samples at this timepoint. 2)numbers of unique genes are listed that were significantly differentially expressed genes. detailed information on the genes assigned to the different defence-related gene classes is provided in additional files and indications for a jasmonate-dependent enhancement of fhb resistance in wheat indications for the presence of a ja signalling were found in the cv. dream transcriptome after fhb infection by using gsea testing. the go terms ‘lipoxygenase activity’, ‘oxylipin biosynthetic process’ and ‘lipid biosynthetic process’ associated to the oxylipin metabolism were exclusively enriched in the early hai gene expression data indicating that the chloroplastic 13-lox-branch was induced upon fhb infection. hormone-like compounds such as ja and methyl jasmonate, as well as 13-hpl-derived c aldehydes, are characteristic products of this pathway. some oxylipins generated by the 13-lox pathway, for example thaumatin-like proteins and phytoalexins, exhibit antimicrobial activities by impairing fungal mycelial growth and spore germination. other oxylipins, such as ja and meja are well known to serve important roles in plant defence-signalling by mediating the induction of the expression of some pr-genes. moreover, as 13-lox oxylipins are substantially produced from cuticle- or cell membrane-associated fatty acids released during the fungal degradation of plant cell walls, they also act as elicitors involved in pathogen recognition. threeputative lox genes were fhb-responsive induced at hai. the transcript ta..a1_at was found to be a homologue of the maize gene zmlox which is a novel chloroplast localized lox gene described as uniquely regulated by phytohormones and pathogen infection. the two transcripts ta..a1_x_at and taaffx..s1_s_at showed significant similarity to the barley gene hordeum vulgare methyljasmonate-inducible lipoxygenase. therefore, both transcripts might encode for one or two putative methyljasmonate -inducible chloroplastic 13-lox genes. it was shown that jasmonates regulate their synthesis via positive feedback control by inducing the transcription of biosynthesis genes such as lox. it is remarkable that both transcripts were also already induced 24 h after f. graminearum inoculation in the resistant spring wheat cv. sumai 3. a)published sources: 1); 2); 3); 4); 5); 6); 7); 8); 9); 10); 11). b)for blastn analyses the threshold for a significant homology was set to an e-value ≤ 1e- identity scale >70% according to. five lox genes were up-regulated after both treatments and, in contrast to the solely fhb dependent induced lox genes, three of them were also expressed at hai. here, except for the transcript ta..s1_x_at, none of the genes could be assigned to a ja-mediated defence based on sequence similarities to published genes. ta..s1_x_at, however, a homologue of a barley gene lox involved in different stress responses, was also shown to be active in cv. sumai upon f. graminearum infection. in summary, putative functions regarding defence response mediation were assigned to genes showing fhb-associated expression alterations. here, all genes were found to be jasmonate and pathogen inducible or were previously identified as being fhb-responsive in cv. sumai 3. this is remarkable as the cultivars dream and sumai represent entirely different origins and resistance levels. additionally, ja and et defence-signalling pathways were found to be essentially involved in the high level fhb resistance of wheat cv. wangshuibai in a recent study and were supposed to mediate the early basal defences at to 24 h after f. graminearum infection. however, the contribution of a salicylic acid signalling towards fhb resistance reported in that study was neither observed in our study nor reported for the cv. sumai 3. on the other hand, a continual ja production can be involved in pathogen defence as well. indications for ja-inducible as well as for a continual pr-gene expression were indeed observed in the cv. dream and both might contribute to the present fhb resistance. a jasmonate-responsive and non-specific antifungal defence contributes to fhb resistance the enrichment of genes belonging to the 13-lox pathway indicates a systemic accumulation of endogenous jasmonates in the resistant cv. dream as a result of f. graminearum infections. it is known that members of the jasmonate family, whose levels increase on pathogen infection, activate a specific set of genes encoding antimicrobial peptides. several cysteine-rich amps were found to be up-regulated in fhb infected cv. dream spikes, which are possible targets of such resistance-related ja signalling, when the two points in time were investigated. the set of identified cysteine-rich amps comprises lipid transfer proteins, thionins, and defensins. lipid transfer proteins were the most frequently expressed class of amps. three genes were up-regulated independent of the treatment, while two transcripts were up-regulated exclusively 72 h after fhb inoculation. compared to the other identified cysteine-rich amps, most of the ltp genes have shown relatively high fold changes that remained constant at both timepoints. blastn analyses proved that all present ltp genes encode for putative non-specific lipid-transfer proteins. direct antifungal activities and a broad resistance spectrum against biotrophic and necrotrophic fungal pathogens have been reported for various crop species and tissues, notably with nsltps. the observed antifungal activities also include different fusarium pathogens, such as f. graminearum and f. solani, as well as f. culmorum and f. oxysporum. thereby, nsltp proteins were found to strongly inhibit the growth of fungal mycelia as well as the germination of fungal spores, including the conidiospores of f. graminearum. wheat ns-ltps are generally supposed to play a role in an enhanced non-specific defence response regulated by different hormonal signals, including jasmonates. in particular, constitutively expressed genes are supposed to contribute to non-host resistance. a synergistic activity of nsltp genes with thionins against f. solani and f. graminearum was shown in studies on barley, maize and wheat. in fact, two transcript sequences homologous to the wheat thionin gene thi were differentially expressed in the cv. dream after both treatments, but not in the cv. lynx. thionins have a general antimicrobial activity against early conidial germination. in addition, a highly inducible expression was observed in the case of the arabidopsis thionin thi after both fungal infections as well as meja treatment leading to an enhanced resistance to f. oxysporum. peptidase inhibitors of the defensin family make up the third class of continual up-regulated amps, represented by homologues of the wheat gene tad and the defensin precursor prpi- from durum wheat. while the antimicrobial activity of defensins requires typically complex synergistic interactions with other amps, their promoters are potentially interesting candidates for the targeted and tissue-specific expression of pr- and r-genes, particularly for the protection against f. graminearum in cereal grains. an induction by jasmonates was reported for most of the defensin genes and some of the putative antifungal defensins are reported to be markers for the presence of ja- and et-dependent defence-signalling pathways. indeed, indications for an active et signalling were found in the fhb-attacked cv. dream transcriptome as well. the majority of up-regulated cysteine-rich amps in cv. dream have shown expression values that were independent of the treatment, but were lower or absent in the susceptible cv. lynx. it is likely that the majority of these peptides act synergistically in a generalized non-specific defence providing a basal protection. amps transcribed at a constant level are known key components of an immediate defence against invading pathogens, and many proteins that are pathogen-inducible, for example, in leaves were found to be constitutively present in storage tissues, such as seed. moreover, it is generally assumed that genes involved in the quantitative fhb resistance of adapted european wheat cultivars represent such a defence mechanism. nonetheless, amps can also be part of an induced plant defence. in fhb-treated cv. dream spikes only nsltp genes were up-regulated in response to the disease. among these ta..s1_a_at seems to be an interesting resistance candidate, as the gene combines a general high antifungal property with considerable fold change expression ratios at both timepoints. moreover, the putative defensin gene prpi- might be a relevant finding as well due to its possible utilization in a resistant strategy aiming at over-expressions of pathogen-inducible promoters to directly target the infection sites or the most vulnerable tissues. such an approach becomes even more interesting with the recent observation that the biotrophic life form of f. graminearum persists in all colonized tissues. living host cells form a zone surrounding the most advancing hyphae and could be targets for such an approach as they allow a continuous supply with antifungals onto the intercellular hyphal tips. identification of fhb-responsive jasmonate-regulated proteins taking into account the observations on the dream and sumai cultivars it can be hypothesised that a fhb-responsive ja signalling is active from to hai covering the presumed phase of the general biotrophic fungal growth, since the switching point to increased necrotrophic nutrition was timed around hai. an interesting conformance was observed with the fhb-responsive expressions of genes that encode for jasmonate-regulated proteins, belonging to the subfamily of mannose-specific jacalin-like lectin containing proteins during that period. three mjrl genes, taaffx..s1_at, ta..s1_at and ta..s1_at, were up-regulated in cv. dream at hai. the first two transcripts are prominent due to the considerable fold change expression ratios of and and their up-regulation exclusively in the fhb treated spikes of cv. dream. as many mjrl genes are described as strictly inducible defence proteins, taaffx..s1_at and ta..s1_at might be involved in the fhb defence. blast analysis showed that all detected putative mjrl genes belong to the mjrp- protein subfamily. in general, mjrp- genes are specifically induced by ja via transcriptional activation and were initially identified in jasmonate-treated barley leaves. the first mjrp- gene analysed in detail was the bgaf gene from maize. the sequence of the transcript taaffx..s1_at shows similarities to another maize bgaf gene as well as to the wheat gene ta-ja. although detailed knowledge on the defence function of mjrp- proteins is still to be gained, a broad resistance spectrum has already been observed. one prominent example is the ta-ja gene that encodes a modular bgaf-related protein with a proven broad-spectrum resistance to infections by bacterial, fungal and viral pathogens in transgenic tobacco plants. all currently known mjrp- genes come from poaceae and share important traits separating them from other mjrls, for example their exclusive, tissue-specific induction via jasmonates and their single-copy status. however, notably due to their strict tissue-specific expressions, mjrp- genes are not supposed to be orthologous, although the proteins share numerous common features. an mjrp- gene expressed in spike tissues has not been reported so far. for this reason and due to its fhb-responsive high level induction, a separate study should reveal whether the taaffx..s1_at gene represents a new spike-specific member of the mjrp- family. in addition to ta-ja the poaceae jrp- family comprises three other wheat genes: ver, wci- and hfr-. in the present work, the wheat chemically-induced gene wci- and the vernalisation-related gene ver were up-regulated in cv. dream upon f. graminearum infection. wci- was characterised as a plant disease resistant response gene that is induced by benzothiadiazole. bth is a functional analogue to sa which was not successful in reducing the fhb-disease caused by f. graminearum. on the other hand, an up-regulation of wci- upon meja application has been reported, and the wci- orthologous pea gene dir was found to be involved in the resistance to different fusarium pathogens. due to these contradictory observations further examinations are required to clarify the role of wci- in fhb resistance. the up-regulation of the vernalisation-related gene ver upon f. graminearum infection is interesting. indeed, due to the proven specific induction by meja, ver was initially proposed to be involved in a jasmonate mediated plant defence response. however, an induction of expression upon f. culmorum infection could not be confirmed and a native ver induction has so far only been observed in young wheat seedlings during the vernalisation process. thus, whether the untypical expression of ver in wheat kernels is associated with fhb resistance, or rather is a side effect caused by jasmonate-signalling remains unanswered at this point. an increased ethylene production contributes to wheat fhb resistance ethylene plays an important role in plant growth and development but it is also known to be involved in the regulation of primary resistance responses. indications for an increased et-metabolism in cv. dream spikes following fhb infection are provided by several up-regulated putative 1-aminocyclopropane-1-carboxylate oxidases and gdsl-like lipases genes. the acc oxidase, also called the et-forming enzyme, catalyses, together with the enzyme acc synthase, the last biosynthetic step to convert acc into et. both enzymes are known to be rate-limiting components in the et biosynthetic pathway. a total of acc oxidase genes were either up-regulated or down-regulated in the cv. dream, mainly in a constitutive manner. in fact, the expression of individual acc oxidase genes is generally frequent and differentially regulated at all times due to developmental changes as well as abiotic and biotic stress factors. the occurrence of several gdsl-like lipase genes in the cv. dream assay further indicates an elevated et-signalling. gdsl-like lipases were mainly differentially expressed upon both treatments. among the characterised gdsl-like lipases, the genes glip and glip of arabidopsis are known to play an important role in plant immunity by eliciting local as well as systemic resistance against necrotrophic and hemibiotrophic pathogens. moreover, gdsl-like lipase transcription was exclusively enhanced by et, but not by sa or ja. however, none of cv. dream gdsl-like lipases has shown a sequence homology to the reported resistance candidates from arabidopsis. it is generally accepted that the plant defence against necrotrophic pathogens is usually regulated by ja and et while sa plays a major role in the defences against biotrophic pathogens. a possible involvement of et in fhb resistance has also been demonstrated for sumai based on an up-regulated acc oxidase gene. furthermore, an increased resistance against fhb was observed for the susceptible cultivar y after spraying spikelets with ja as well as et before and after fungal infection. different studies in arabidopsis and tobacco have shown that et and, in particular, the over-expression of certain acc oxidase genes can extend the symptomless biotrophic phase during hemibiotrophic fungal infections. in addition, it was found that et can reduce cell death caused by the fungal toxin fumonisin b which is produced by several cereal-attacking fusarium species. indications for fhb-responsive suppression of fungal virulence factors in addition to the presence of ja- and et-mediated general antifungal defences, a second line of defence was found to be based on a fhb-responsive and targeted suppression of relevant fusarium virulence factors, such as proteases and mycotoxins. this defence mechanism was assembled from genes encoding protease inhibitor proteins and different genes which are proposed to be associated with the detoxification of pathogen-derived mycotoxins. both, fusarium proteases and mycotoxins take on relevant roles in the fungal pathogenesis and were found to be secreted in nearly all phases of the fungal wheat spike colonisation. wheat-derived protease inhibitor genes in fhb disease resistance in the fhb-treated cv. dream transcriptome, serine pi proteins of the subtilisin-like protease superfamily were significant enriched at both timepoints, represented by the go terms ‘serine-type endopeptidase inhibitor activity’ and ‘peptidase activity’. pi proteins generally feature a high substrate specificity and therefore, it is likely that those genes encode for proteins that specifically bind and impair secreted fusarium sl proteases. proteases generally cause the proteolytic digestion of proteins via the hydrolysation of peptide bonds. fusarium subtilisin-like and trypsin-like proteases are released in infected wheat kernels mainly to disrupt host cell membranes during necrotrophic intracellular nutrition. consequently, defence-related interactions between plant pi proteins and subtilisin-like and trypsin-like proteases of f. graminearum and f. culmorum have already been proven in the grains of barley and ancient emmer wheat. in total, five serine-protease inhibitors were differentially up-regulated in cv. dream. two transcripts were functionally annotated to the bowman-birk inhibitor family based on sequence homologies to the wrsi gene. wrsi was described as a salt-responsive gene with a suggested role in regulating plant growth. among the remaining transcripts, ta..s1_x_at and ta..s1_x_at were up-regulated in response to fhb at hai, while ta..s1_at was regulated solely at hai. the ta..s1_at gene was selected for qpcr analysis because of its relativelyhigh and fhb-responsive fold change at hai. a possible expression at h after fusarium inoculation was not reliably determined due to missing expression data from two of the three biological replicates. the ta..s1_at expression was measured with an inoculation time-courses of two cultivar pairs: first, the winter cultivars dream and lynx described as moderately resistant and susceptible; second, the spring cultivars sumai and florence-aurore described as resistant and susceptible. sumai and florence-aurore, in particular, were found to represent the extremes of spring wheat responses to fusarium spread. upon comparing the fhb-responsive transcript induction levels in the cultivars dream and lynx, a generally higher induction over control and cv. lynx samples was observed for cv. dream for the period between to hai. as a matter of fact, >4-fold inductions were only obtained at and hai. however, even the 2-fold inductions of earlier and later timepoints were considered relevant due to the strictly suppressed expression in the susceptible genotype. in the fhb-treated spike tissues of sumai > and >300-fold inductions were already observed between to hai and a third peak of >200-fold was found at hai. no gene expression was verifiable in spike samples of cultivars sumai and florence-aurore at hai which was the last timepoint. in the first instance, the relative induction peak at hai in cv. dream is consistent with previous observations that endogenous wheat serine-protease inhibitor proteins are not induced until 72 h after fusarium inoculation. in fact, in the period between and hai, during which the necrotrophic nutrition becomes predominant, f. graminearum transcripts were found to dominantly encode degrading enzymes such as proteases, lipases and nucleases. a transcript accumulation was even observed in the susceptible genotypes lynx and florence-aurore particularly in this period, however, to a lower extent than in the respective resistant counterparts. the physiological responses of pis are furthermore triggered by negative feedback mechanisms. therefore, the remarkable suppression of ta..s in sumai during this crucial time might be a consequence of the already high transcript abundance and the subsequent induction at hai is assumed to be stimulated by further secreted fungal proteases. the early high level activity of ta..s until the timepoint hai in cv. sumai is consistent with previous data from sumai gene expression studies, demonstrating the fhb-responsive expression of several pis at already hai. in this period, an exclusive induction of the tested serine-protease inhibitor was also observed for the moderately resistant cv. dream. consequently, the early expression of wheat pi genes could be an immediate reaction to early levels of secreted fusarium tl and sl proteases which have been reported for different compatible interactions, amongst others between f. graminearum and barley as well as wheat. here, the activity of fungal proteases has been attested already at to hai, long before a corresponding expression could be observed in kernels. in fact, beside their harmful roles during the phase of a necrotrophic intracellular nutrition, fungal proteases were found to be secreted already during the earlier intercellular colonisation of spike rachis, probably to suppress certain plant defence reactions by degrading pr-proteins. in this sense, the serine-protease inhibitor ta..s1_at seems to be an interesting resistance candidate as transcript accumulations were present during the early and the later phases of fungal spike colonisation. however, this potential still needs to be confirmed in a further study. nevertheless, pis are discussed as candidates for an improved resistance strategy against grain infecting fungal pathogens and our results from qpcr and transcriptome analyses do not contradict these considerations. analysis of the detoxification mechanisms in wheat concerning fhb resistance fusarium proteases and mycotoxins act in a kind of strategic cooperation during spike and kernel colonisation by featuring complementary roles during the host defence suppression and the intracellular colonisation of spikelets. from an economic perspective, fusarium species causing fhb belong to the most important trichothecene producers and don is a predominant trichothecene toxin produced by these species. silencing the fusarium tri gene down-regulates more than genes involved in the mycotoxin production and results in a reduction of don production and pathogenicity. meanwhile, several different plant genes are known to be up-regulated at the transcriptional level in response to either don treatment or don production which are thus likely to be involved in the don-resistance. to analyze the expected impact of a specific mycotoxin defence on the general fhb resistance of cv. dream, a literature-to-transcriptome approach was used. known toxin resistance-related genes from wheat and barley were checked for homologous genes on the wheat array and their respective expression profiles in the cultivars dream and lynx. a diverse set of wheat genes could be identified as possible members of a general detoxification mechanism. those genes are listed in table including the respective literature sources. within this set, genes originate from a study of trichothecene-induced gene expression in barley. screening the expression patterns of those genes in the cv. dream vs. cv. lynx microarray data revealed for all genes similar expression patterns. they were exclusively expressed or induced in fusarium treated samples collected 72 h after infection. moreover, they were also up-regulated in both genotypes and, in addition, they were up-regulated in both genotypes and the level of up-regulation was higher in susceptible cv. lynx in all cases. however, expression differences between both genotypes never reached a level of statistical significance. finally, they were not expressed in mock control samples at all, although this observation was not reliable in the case of cv. lynx samples collected at hai due to the above mentioned restrictions. to analyse the observed congruities in more detail and to test whether or not the expression in the susceptible cv. lynx is just a temporary phenomenon, a selection of six genes representing different functional categories was forwarded to qpcr analysis using the above mentioned inoculation time-courses of the cultivar pairs dream vs. lynx and sumai vs. florence-aurore. a)class association as obtained from respective literature sources. b)gene annotations were updated by blastn analysis in the ncbi database, otherwise published annotations were considered. c)published sources: 1); 2); 3); 4); 5); 6); 7); 8); 9); 10); 11); 12). d)predicted mrp gene sequence contains est cluster: ca bj and bj303163. the analysed genes associated with don detoxification are taugt and a homologue of the barley udp-glucosyltransferase gene hvugt genes that are supposed to be involved in the resistance to don accumulation are tapdr and tamdr. as representatives of the functional categories “defence-related” and “general” a further putative serine-protease gene and a 12-oxophytodienoate reductase gene were included. the qpcr data for the winter wheat cultivars dream vs. lynx showed similar expression patterns for all tested genes as did the microarray experiments. consequently, a temporary and higher induction peak was found for lynx at hai compared to dream. on the other hand, the transcripts of all tested genes peaked at hai in the cv. dream samples, while lynx revealed suppressed or consistent inductions. in addition, a >4-fold induction was already observed before hai for most of the cv. dream alleles and the expressions were showing a general and increasing trend towards the peak at hai. such a maximum induction at hai has likewise been observed for the don resistance candidate gene pdr -like in infected spikes of the chinese landrace wangshuibai which represents one of the most important genetic resources for fhb and don resistance. like the analysed genes tapdr and tamdr, pdr5-like is like a plasma membrane abc transporter which co-segregates with the don resistance qtl qfhs.ndus-6bs from cv. wangshuibai. in the cultivar pair sumai vs. florence-aurore the fusarium-induced expression levels obtained for the udp-glucosyltransferase and abc transporter genes were showing typical curve characteristics in cv. sumai samples; starting with a low level induction at hai, followed by a consistent increase up to the peak at or hai and showing a continuous downtrend thereafter. in contrast, considerable inductions for the susceptible cv. florence-aurore did not appear until to hai. interestingly, both ugt genes show induction peaks at hai in cv. sumai while both abc transporter genes peak at hai. deviating induction patterns were observed for the representatives of the functional categories “defence-related” and “general”. for all tested genes no expressions were measured from samples collected at hai. in summary, the expression profiles of genes related to detoxification in both resistant cultivars were following an early beginning steady-state model, providing transcript levels during the reported period of increased don accumulations between and hai. in contrast, both susceptible cultivars showed typically late and temporary inductions. our observations for the expressions of ugt and abc transporter genes in cv. sumai are furthermore in accordance with expression patterns previously observed for the abc transporter gene tapdr as well as the ugt gene taugt in fhb-treated spike samples of cv. wangshuibai. the tapdr gene is a member of the atp-binding cassette protein superfamily and has been identified in cv. wangshuibai due to its strong up-regulation upon don treatment as well as f. graminearum inoculation. after fungal infection, the relative amount of tapdr transcripts increased in wangshuibai at hai. the function of tapdr in fhb resistance is proposed to be don-related because gene expressions were found to peak after to 12 h of don-inoculation and declined slowly thereafter. in addition, a late expression peak was observed for the susceptible cv. alondra similar to our observations in the susceptible cv. florence-aurore. the general role of pdr transporters in the resistance to antifungal drugs was first characterized in yeast and a particular function in don resistance was confirmed based on a yeast mutant carrying a knockout variant of the pdr transporter gene resulting in a non-natural hypersensitivity to don. the second analysed transporter gene tamdr was initially isolated from wheat root apices as being induced by aluminium toxicity. however, tamdr was up-regulation together with tapdr in cv. wangshuibai and, thus, was supposed to be involved in don resistance as well. in fact, our time-course qpcr expression data were able to reveal that both genes show similar expression profiles upon fusarium infection in the resistant cultivars dream and sumai, respectively. although genotype-specific differences were present, the observed similar expression patterns indicate a possible trichothecene-responsive up-regulation for tamdr as well. using nullisomic-tetrasomic wheat lines, we have also located the tamdr allele on chromosome 5a where tapdr had already been placed before. these observations may reflect a common mechanism of transcriptional co-regulation for both genes. in general, there is accumulating evidence that gene order in eukaryotic genomes is not completely random and that pathogen-responsive as well as other genes with similar expression levels tend to be clustered within the same genomic neighbourhoods. in fact, for tapdr it was discovered that the gene expression is not induced by ja, sa and abiotic stress factors but by decreasing concentrations of al3+ and free [ca2+. this mode of regulation was also reported for the tamdr gene due to its general induction by al injury in wheat roots. both toxicities activate plant programmed cell death via an oxidative burst and both inhibit calcium channels of plasma membranes which causes a decrease of the intracellular second messenger [ca2+. in order to explain the commonalities we have found between both abc transporters in terms of induction, we suggest that don induces the expression of tapdr and tamdr indirectly via decreased levels of [ca2+. whether tamdr thus has a similar relevance for the detoxification process as can be suggested for tapdr still needs to be proven in a further study. two ugt genes supposed to be involved in the don detoxification were analysed with qpcr. quite a few of the plant ugts are related to disease resistance where they play important roles in the detoxification of exogenous compounds, for example fungal metabolites such as don. blastn analysis revealed the homology between the transcript ta..s1_at and the taugt gene which had originally been cloned from cv. wangshuibai. ta..s1_at has revealed a significant full length sequence homology to the barley ugt gene hvugt. both genes have displayed the respective characteristic qpcr expression profiles for cvs. dream and sumai as described above. however, higher induction levels were observed for the putative hvugt gene when compared to taugt at the first instance, the wheat gene taugt was the most interesting candidate since it was suggested to be an efficient candidate gene for improving don resistance. however, our expression data are in accordance with recent observations which have demonstrated that hvugt can protect yeast from don by converting it to don-3-glucoside while taugt was not able to convert don. in addition, with our observations in the cultivars dream and sumai hvugt has demonstrated relevant activities in a number of fhb-treated wheat cultivars as well as in barley, indicating that it be might of general relevance. hvugt and also taugt were detected as don resistance candidates in don inoculated spikes of cv. wangshuibai in a gene expression study using the affymetrix wheat gene-chip®. moreover, blastn analysis could demonstrate that hvugt has also been identified as don resistance related gene in wheat dh-lines carrying the major fhb resistance qtl fhb from cv. cm82036 as well as in two related barley transcriptome studies. finally, the gene hvugt appears to be a remarkable candidate gene for fhb resistance. it is considered relevant for a promising strategy to improve fhb resistance not only in wheat but also other cereal species. as representative for the functional category “general”, the expressions of a putative wheat gene encoding for a 12-oxophytodienoate reductase was analysed. ta..s1_at was functionally characterised by significant homology to the maize 12-oxo-phytodienoate reductase gene zmopr. the homologous barley gene was previously found to respond to pathogen-derived trichothecene accumulation. in addition to ta..s1_at, two more putative opr genes were identified as up-regulated in response to fhb: the gene zmopr and the gene zmopr. all three genes are putative wheat homologous of the opr i group members which preferentially catalyse the formation of the natural ja precursor 12-oxo-phytodienoic acid. in our qpcr analysis, the zmopr homologue ta..s1_at has shown a fhb-associated induction at hai which was common for both the resistant genotypes. this might indicate a rapid and transient up-regulation of ta..s1_at. in fact, the genes zmopr and zmopr have demonstrated a transient induction upon fusarium verticillioides infection in maize. a similar rapid and transient up-regulation caused by a variety of environmental cues including hydrogen peroxide was observed for the ta..s1_at homologous gene osopr in rice. don is known to induce the transient accumulation of h2o as the most stable compound involved in oxidative burst. indeed, yeast studies indicate detoxifying functions for opri enzymes. indications for a complex crosstalk between fungal and plant proteases and their inhibitors during fhb defence the putative wheat serine protease gene belongs to the subtilisin-like protease family and was initially detected as a gene that strictly responds to pathogen-derived trichothecene accumulation in barley. in addition, serine proteases were found to be enriched in the cv. dream transcriptome upon fhb treatment and were annotated to the go term ‘serine-type carboxypeptidase activity’. an early ta..a1_at expression was found for cv. sumai here, exclusive and equal >2-fold inductions were present at and hai. at hai, both resistant cultivars showed the highest induction level, in cv. dream even with a peak of >60-fold, while at this timepoints no expressions were found in the susceptible cultivars. an opposing effect was observed at hai, when exclusive expression was observed for both susceptible wheat cultivars, while no expression was detectable in the resistant ones. as proteolytic and protein-binding enzymes proteases feature important functions for the selective breakdown of regulatory proteins and several plant proteases have been linked to defence responses. although many questions remain unanswered concerning their mode of action, there is evidence that plant proteases, in particular subtilisin-like proteases, are involved in the crosstalk between pathogen and host. in this context, a defence-counter-defence mechanism was observed between the plant-pathogen interaction tomato/phytophthora infestans, in which both, host and pathogen are supposed to release specific sets of proteases and protease inhibitors mutually impairing each other. moreover, such counter-defence mechanism is supported by the assumption of a strong co-evolution between proteases and protease inhibitors which are mutually released during a pathogen-host interaction. it is interesting in this context, that proteases as well as protease inhibitors were enriched in the transcriptome of the resistant cultivar dream upon f. graminearum infection. regarding the role of the reported plant proteases involved in don resistance, we suggest that they do not act in response to a don accumulation but rather in response to a fusarium protease-rich environment as fusarium proteases appear together with mycotoxins during spike rachis and kernel colonisation. in addition, a specific function within a detoxification mechanism has yet not been described for plant proteases.  our transcriptome study provides evidence for the existence of a biphasic defence reaction against fhb in wheat. jasmonate and ethylene regulated non-specific antifungal protections are supplemented by host gene networks associated to the accumulation of f. graminearum-derived trichothecenes and subtilisin-like proteases. using a literature-to-transcriptome approach, genes described as related to don resistance were identified due to analogies in their microarray expression profiles which hence, may belong to a detoxification pathway that is active in different resistant wheat cultivars as well as in barley. our qpcr expression analyses of seven wheat genes associated with the suppression of fungal virulence factors have demonstrated similar fhb-responsive inductions in the cultivars dream and sumai moreover, an earlier first induction and a steady-state level of expression were found to be associated with fhb resistance, while fhb-responsive gene expression in susceptible cultivars was typically late and temporary. these results will help not only to understand changes in overall gene expression in wheat during fusarium infection, but will also help to identify potential targets for development of disease control strategies. in fact, genes interesting for further investigations in this direction were identified in both wheat defence mechanisms. these are, nsltp, defensin and mjrp genes as well as the pdr-transporter gene tamdr, the ugt gene hvugt and the putative serine-protease inhibitor gene ta..s1_at. the last three genes have shown regulations in response to fhb in the cultivars dream and sumai in general, the identification of resistance candidate genes that are commonly active in different resistant wheat and barley cultivars is an important result with regard to the development of novel strategies against fhb severity and grain toxin contamination. 
the gene expressions at and 72 h after inoculation with fusarium were used to trace possible defence mechanisms and associated genes. among differentially expressed genes, two putative main defence mechanisms were found in the fhb-resistant dream cultivar. especially abc transporter, udp-glucosyltransferase, protease and protease inhibitor genes associated with the defence mechanism against fungal virulence factors are apparently active in different resistant genetic backgrounds, according to reports on other wheat cultivars and barley. another mechanism based on the targeted suppression of essential fusarium virulence factors comprising proteases and mycotoxins was found to be an essential, induced defence of general relevance in wheat. fusarium head blight caused by fusarium species like f. graminearum is a devastating disease of wheat worldwide. resistant varieties are the only effective way to control this disease, but the molecular events leading to fhb resistance are still poorly understood. a non-specific mechanism was based on several defence genes probably induced by jasmonate and ethylene signalling, including lipid-transfer protein, thionin, defensin and gdsl-like lipase genes. transcriptional profiling was conducted for the winter wheat cultivars dream and lynx. moreover, similar inductions upon fungal infection were frequently observed among fhb-responsive genes of both mechanisms in the cultivars dream and sumai mycotoxins such as deoxynivalenol produced by the fungus affect plant and animal health, and cause significant reductions of grain yield and quality. a comparative qpcr was carried out for selected genes to analyse the respective expression patterns in the resistant cultivars dream and sumai. this was further supported in our qpcr experiments on seven genes originating from this mechanism which revealed similar activities in the resistant cultivars dream and sumai finally, the combination of early-stage and steady-state induction was associated with resistance, while transcript induction generally occurred later and temporarily in the susceptible cultivars.
 fusarium head blight caused by fusarium species like f. graminearum is a devastating disease of wheat worldwide. mycotoxins such as deoxynivalenol produced by the fungus affect plant and animal health, and cause significant reductions of grain yield and quality. resistant varieties are the only effective way to control this disease, but the molecular events leading to fhb resistance are still poorly understood. transcriptional profiling was conducted for the winter wheat cultivars dream and lynx. the gene expressions at and 72 h after inoculation with fusarium were used to trace possible defence mechanisms and associated genes. a comparative qpcr was carried out for selected genes to analyse the respective expression patterns in the resistant cultivars dream and sumai.  among differentially expressed genes, two putative main defence mechanisms were found in the fhb-resistant dream cultivar. both are defined base on their specific mode of resistance. a non-specific mechanism was based on several defence genes probably induced by jasmonate and ethylene signalling, including lipid-transfer protein, thionin, defensin and gdsl-like lipase genes. additionally, defence-related genes encoding jasmonate-regulated proteins were up-regulated in response to fhb. another mechanism based on the targeted suppression of essential fusarium virulence factors comprising proteases and mycotoxins was found to be an essential, induced defence of general relevance in wheat. moreover, similar inductions upon fungal infection were frequently observed among fhb-responsive genes of both mechanisms in the cultivars dream and sumai  especially abc transporter, udp-glucosyltransferase, protease and protease inhibitor genes associated with the defence mechanism against fungal virulence factors are apparently active in different resistant genetic backgrounds, according to reports on other wheat cultivars and barley. this was further supported in our qpcr experiments on seven genes originating from this mechanism which revealed similar activities in the resistant cultivars dream and sumai finally, the combination of early-stage and steady-state induction was associated with resistance, while transcript induction generally occurred later and temporarily in the susceptible cultivars. the respective mechanisms are attractive for advanced studies aiming at new resistance and toxin management strategies. 