BACKGROUND
the fungus puccinia hordei is a biotrophic pathogen causal agent of leaf rust, a serious leaf disease of barley worldwide.
this pathogen causes serious economic losses with yield reductions by up to 62%
<cit>  and adversely affects grain quality by reducing grain weight and increased levels of undesirable protein in the barley growing region of the world
<cit> .
rust fungi have a complex life cycle that involves two parasitic stages, dikaryotic and monokaryotic
<cit> .
the dikaryotic stage is the form causing rust disease by attacking mesophyll tissues until pathogen injures the epidermis to release urediospores
<cit> .
barley resistance to leaf rust pathogens is governed by major resistance  genes  that are race-specific.
most rph genes confer complete seedling resistance associated with necrosis  of the plant cells attacked by the pathogen sporelings
<cit> , while some rph resistance genes confer incomplete resistance, in which the fungus forms small uredinia surrounded by chlorotic or necrotic plant tissues.
this second resistance type is a non-hr  polygenically inherited leaf rust resistance, which was termed “partial resistance” and is not associated with plant cell necrosis
<cit> .
several seedling resistance genes were identified from cultivated and wild barley, of which  <dig> were designed rph <dig> to rph <dig> <cit> .
the resistance provided by single rph genes has often been overcome by new pathotypes, believed to have arisen after gene mutations.
as a direct consequence, the number of effective rph genes available to breeders is decreasing rapidly, suggesting the need for a new gene deployment strategy
<cit> .
the leaf rust resistance gene rph <dig>  located on chromosome 2hs
<cit> , was derived from pi  <dig>  an accession of wild barley  collected in israel.
when evaluated for its reaction toward a worldwide collection of over  <dig> p. hordei isolates, it conferred resistance to all but the isolates 90– <dig>  from israel.
rph <dig> is one of the most broadly effective resistance genes and it is therefore useful in barley breeding programs for leaf rust resistance.
barley interaction with the leaf rust pathogen represents a model to understand the molecular basis of both race-specific and partial resistance.
molecular basis of partial resistance were recently investigated using eqtl  analyses carried out in p. hordei infected doubled haploid lines and qtl-nils
<cit> .
these studies provided an overview of the responsive dynamic defence process and identified several candidate genes as being co-localized with the phenotypic qtl.
no additional published microarray studies were dedicated to barley-p. hordei interaction.
in addition to transcriptional studies, proteomic techniques can provide insight into the molecular mechanisms underpinning resistance gene-based plant defence responses.
the compatible interaction between wheat and the leaf rust pathogen p. triticina was investigated at  <dig>   <dig> and  <dig> dpi  and only at the latter time point of inoculation seven plant proteins involved in translation and stress responses were identified as pathogen-responsive
<cit> .
no published proteomic studies have been performed on barley leaves infected with leaf rust.
in this work, barley responses to leaf rust infection were investigated, in two barley near isogenic lines differing for the introgression of the broad effective leaf rust resistance gene rph <dig>  a proteomic study was performed at early  and late  infection times and, after analysis of protein pattern changes, it was observed that only at the late time points and in the resistant nil differential protein accumulation occurs in response to pathogen inoculation.
the differentially expressed proteins were involved in photosynthesis, carbon metabolism, defence responses and secondary metabolism.
RESULTS
experimental design and 2-de analysis
in this study, the defence responses to leaf rust of two near-isogenic barley lines, bowman and bowman-rph <dig> differing for the presence/absence of the broad effective leaf rust resistance gene rph <dig>  were investigated using a proteomic approach.
the utilization of nils allows to relate the pathogen-responsive changes in protein accumulation observed between the two nils to the resistance gene activity.
two time points of inoculation were selected for the analyses.
at the first time point investigated,  <dig> hours post inoculation , bibliographic data report that the leaf rust pathogen has established haustoria in the mesophyll cells and has started intercellular hyphal growth
<cit> ; brian j. steffenson, personal communication].
at  <dig> days post inoculation , the number of cells with established haustoria is higher than at  <dig> hpi and hyphae completed their intercellular growth.
since infection conditions applied in the present work are basically the same as those previously reported
<cit> , we assumed that in our experiments the differentiation of infection structures and colonization process followed the same timing as described above.
in agreement with previous observations, no disease symptoms were observed in both the nils at  <dig> hpi, while at  <dig> dpi few chlorotic areas were observed in the susceptible nil bowman only .
in order to assess the success of the inoculation process, single plants of each biological replicate were left until  <dig> dpi.
at this stage sporulating colonies were observed in bowman while bowman-rph <dig> exhibited only few chlorotic areas.
to further verify that defence responses were properly deployed in the infected leaves used for proteomic analyses, quantitative rt-pcrs were conducted on genes encoding for oxalate oxidase, an h2o <dig> generating enzyme
<cit> , and callose synthase, an enzyme involved in cell wall reinforcement
<cit> .
for both genes expression was significantly increased by inoculation at  <dig> and  <dig> dpi  and was unresponsive by  <dig> dpi, with higher transcription level in bowman compared to bowman-rph <dig> .
these results demonstrate that active defence responses were triggered in both the genotypes at the inoculation time points used to detect leaf rust infection-dependent changes in protein accumulation.
proteins obtained from the two infection time points were subjected to the proteomic analysis and three technical replicates were performed for each of two biological replicates.
in the 2-de maps, an average of  <dig> protein spots were visualized with the sypro ruby fluorescent staining.
after image and statistical analyses, no significantly differentially accumulated proteins were identified at  <dig> hpi in both genotypes .
in agreement with our results, no changes in protein accumulation were also detected at  <dig> dpi in the interaction between the leaf rust pathogen p. triticina and a susceptible wheat line
<cit> .
nevertheless, it is important to observe that at  <dig> hpi more than  <dig> genes were differentially expressed in barley leaves of genotypes carrying qtls for partial resistance to p. hordei in accordance with an active defence response at this infection time
<cit> .
these findings underline, as expected, that a longer time is required to detect the response at protein level.
moreover, because of the proteomic approach adopted, only a part of the proteome was investigated in this study and this do not permit a satisfactory comparison between transcriptomic and proteomic data.
figure
<dig> reports 2-de representative maps of proteins isolated from both the bowman and the bowman-rph <dig> in mock-inoculated and leaf rust inoculated conditions at  <dig> dpi.
eighteen protein spots showing significant differential accumulation in response to pathogen infection were identified at  <dig> dpi; differential accumulation was claimed only for spots in which normalized volumes of six replicates for each condition was showing an average fold change in their relative volumes of at least two folds .
in the susceptible nil  at  <dig> dpi only a few differences were detectable .
in particular, two spots  were more abundant and one spot  was less abundant in the rust inoculated sample with respect to the mock inoculated one.
in the resistant nil  a total of  <dig> spots were significantly down accumulated or absent  in the inoculated samples with respect to its control, while the levels of three protein spots rose in the inoculated sample with respect to the control condition .
<dig> and figure
<dig>  indicate the spots, identified by lc-esi-ms/ms, showing significant changes of at least two-fold in their relative volumes  after  <dig> dpi.
proteins that increased or decreased after this treatment are reported in red or in green, respectively.
spot  <dig> is highlighted in blue, indicating its absence in the bowman-rph <dig> inoculated sample.
statistical information about lc-esi-ms/ms analysis are reported in additional files
<dig> and
<dig>
abb.
: abbreviation.
a: experimental molecular weight and pi.
b: theoretical molecular weight and pi.
c: amino acid coverage .
d: annotation obtained by blast-p alignment analysis against the viridiplantae subset of the nr-database at ncbi.
e: value referred to the mature form of the protein.
<dig> and figure
2; proteins were ordered into five functional classes, as indicated in table
<dig>
identification of rph15-resistance gene-related proteins
the  <dig> spots  of interest were characterized by lc-esi-ms/ms.
the differentially modulated proteins could be grouped into the following five different functional classes: photosynthesis and energy metabolism, carbohydrate metabolism, protein biosynthesis and degradation, defence responses and secondary metabolism.
photosynthesis and energy metabolism
twelve of the identified proteins corresponded to photosynthetic or chloroplast-related proteins, including rubisco large chain  and rubisco activase, while one spot represented an atp synthase protein .
rubisco is the most expressed protein in leaves, with about 30% of total leaf proteins
<cit>  and an increase of rubisco degraded forms was generally observed in wheat leaves after fusarium graminearum infection
<cit> .
many spots were identified in the present work as degradation products of rulc; the experimental molecular weights were significantly lower than their theoretical values , thus indicating a degradation of rulc in the samples analyzed
<cit> .
degradation of photosynthetic apparatus may contribute to restrict pathogen growth into barley cells by promoting the activation of defences in non-infected cells because small peptide and amino acids derived from degradation can be directed to other metabolic pathways involved in defence
<cit> .
we observed higher levels of rulc fragments in the control samples than in the inoculated tissues of bowman-rph <dig>  supporting that turnover of rulc was markedly reduced in this genotype after infection.
interestingly, no statistically significant differences in rulc fragments accumulation were observed between control and inoculated conditions in bowman.
to confirm the proteomics data, a western blot analysis using an anti rulc antibody was performed on the protein extracts of the two nils under control and inoculated conditions .
results from this experiment support a reduction of rulc degraded forms  occurring on infected bowman-rph <dig> with respect to control tissues.
in bowman-rph <dig> inoculated sample was in fact evaluated a reduction of  <dig> %,  <dig> %,  <dig> % and  <dig> % of the rulc degraded forms with respect to the control sample respectively for the major bands of the degradation forms numbered from  <dig> to  <dig> .
a similar rulc degradation trend was observed in rice as induced by bacterial blight infection, in lesion mimic mutants
<cit>  and in wheat after fusarium graminearum infection
<cit> .
taken together, our results support the conclusion that in the resistant genotype bowman-rph <dig> the rubisco integrity was more preserved after infection with respect to control sample.
rubisco activase  regulates rubisco activity by hydrolysing atp to promote the dissociation of inhibitory sugar phosphates
<cit> .
ruacs accumulation is reduced in the bowman-rph <dig> nil infected sample  .
in the susceptible nil bowman, no substantial alterations of rulc and ruacs were observed.
it can be hypothesized that this effect on ruacs is linked to a different strategy to modulate the phothosynthesis in bowman-rph <dig>  the genotype in which the pathogen negatively affect rubisco degradation.
spot  <dig> represents an atp-synthase β chain.
this protein is involved in photosynthesis and oxidative respiration to drive synthesis of atp in chloroplasts and mitochondria
<cit> .
accumulation of this protein was down-regulated after infection in the resistant nil only .
a decrease of proteins related to carbon metabolism and photosynthesis that include rulc, ruacs and atp synthase has been also observed in orange  leaves after infection with the biotrophic bacterial pathogen xanthomonas citri pv.
citri <cit> .
similarly, a down regulation of atp-synthase subunits was observed in grape  response to flavescence dorée infection
<cit> .
similar observations were obtained at the transcriptomic level for barley leaves infected with the biotrophic powdery mildew fungus
<cit>  and after an extensive analysis of the effects on photosynthetic genes of several biotic stresses in eight plant species
<cit> .
these investigations suggest that a slow turnover of many photosynthetic proteins represent an adaptive consequence resulting in reduced energy supply when plants are facing a biotic attack and require to re-direct resources in immediate defence needs
<cit> .
the reduced accumulation of these three proteins in the resistant nil is therefore possibly consistent with a re-direction of the metabolism to fuel defence responses.
nevertheless, down accumulation of rulc, ruacs and atp synthase proteins was also observed in the leaves of rice spotted leaf  <dig> mutants that undergo to spontaneous programmed cell death , caused by oxidative burst and membrane damage, in the absence of pathogen infection
<cit> .
since the rph <dig> resistance most likely involves an hr-dependent pcd response, similarly to that of most rph genes conferring complete resistance
<cit> , it cannot be excluded that the down-regulation of photosynthetic proteins observed in the resistant nil could also depend from cellular damage caused by the hr response.
an higher accumulation of rulc degraded forms was observed for bowman-rph <dig> with respect to bowman in the control samples .
also for ruacs and agppase, higher accumulation was present in bowman-rph <dig> than in bowman control samples.
conversely, this behaviour was not observed for several other protein spots , indicating a process specific for proteins involved in the photosynthetic apparatus.
since data are deriving from six replicates for each experiment  and sd detected for these proteins was low, it can be postulated that the observed trend represents a genuine differential accumulation among bowman and bowman-rph <dig> control samples ascribable to an unknown alteration of the cellular homeostasis orchestrated by the rph <dig> resistance gene or by another tightly associated sequence.
carbohydrate metabolism
two infection-modulated proteins identified in this study are related to sugar metabolism: sucrose synthase  and adenosine diphosphate glucose pyrophosphatase .
susy accumulation was significantly increased in infected leaves of the resistant nil .
susy catalyzes the reversible reaction from sucrose to fructose and udp-glucose and an increase in this activity usually results in decreased starch biosynthesis.
in addition, susy is the key enzyme of symplastic sucrose unloading and in concert with invertases, it can modulate the sink capacity of plant tissues
<cit> .
susy has a dual role in producing both cytosolic adpg, directed to starch biosynthesis, and udpg, necessary for cell wall material and glycoprotein biosynthesis
<cit> .
defense-related susy activity may serve to allocate sucrose into callose deposition and other carbohydrate-consuming defense reactions, as supported by the observation of an increased susy activity in the resistant response of tobacco plants to phytophthora nicotianae <cit>  and in several other plant–pathogen interactions .
a leaf rust infection-dependent induction of a callose synthase gene was observed in the present work , suggesting re-directioning of sugars to structural components of the cell wall to constitute physical barriers to the infection.
a consistent decrease in adp-glucose pyrophosphatase  abundance  was observed in infected tissues of the resistant genotype only.
agppase catalyzes adp-glucose breakdown to produce amp and g1p, and its reduced accumulation/activity drastically lead to a reduction in starch biosynthesis
<cit> .
there are increasing evidences that the availability of soluble carbohydrates is a major factor in determining plant resistance to infections.
the results of plant-pathogen interactions also depend on the rapid mobilization of carbohydrates and on the reprogramming of the carbon flow from sucrose to hexoses
<cit> .
the data about susy and agppase suggest that in the resistant genotype under infection there could be a redirection of primary metabolism that leads to a reduction of starch biosynthesis in order to provide a strong supply to the hexoses pool.
to further verify the role of sugar metabolism in rph15-mediated response to leaf rust, an evaluation of reducing sugars and sucrose was conducted on control and inoculated conditions in both nils  .
while sucrose content was not affected by infection, reducing sugars were slightly but significantly increased  by inoculation in the resistant nil and significantly decreased in the susceptible one.
the reducing sugars increase supports a re-direction of the hexoses pool to defence pathways activated in the resistant nil in response to the leaf rust pathogen infection.
the data are the means ± se of three experiments run in triplicate .
different letters on the values indicate significant difference p-values  of the t-tests performed comparing control and inoculated samples.
protein degradation
protein spot number  <dig>  was characterized as a leucine aminopeptidase  protein.
lap <dig> accumulation was observed in the inoculated sample of the resistant nil only.
aminopeptidases represent plant responses to wound and pathogen stresses
<cit>  and their activity  is involved in the turnover of unfolded or damaged proteins that accumulate as a result of the oxidative burst.
furthermore, the role of aminopeptidases in the genesis of bioactive peptides in animals
<cit>  and, more recently, in plants
<cit>  highlighted their possible contribution in defence responses signalling.
the modulation of the levels of lap <dig> observed was rph <dig> resistance gene-dependent, consequently this protein could have a role both at the level of protein turnover, since the rph <dig> resistance response would most likely involve an hr-associated oxidative burst, and/or in resistance gene signalling.
the fact that lap <dig> is more abundant in bowman-rph <dig> line under infection reinforces the idea that protein degradation could be one of the main mechanisms contributing to resistance deployment.
defence responses
two protein spots  were respectively identified as chitinase and chitinase iii-like.
protein abundance significantly decreased in the inoculated samples of the resistant nil only at  <dig> dpi, while in the susceptible nil the levels of chitinases were not affected.
for both chitinases, the level of accumulated proteins was however constitutively higher in mock-inoculated tissues of the resistant nil with respect to the susceptible one.
this behaviour supports the possibility that a higher constitutive level of these defence proteins is associated with resistance and it operates as an early defence barriers against the pathogen infection.
similar results were found in the interaction between wheat and septoria tritici where a higher chitinase activity, measured on whole leaf extracts, was observed in control with respect to inoculated plants during the first  <dig> dpi but the enzymatic activity did not correlate well with resistance in the host or the infection course of the pathogen
<cit> .
plant chitinases, however, are classified into seven classes , on the basis of their structure, substrate specificity, mechanisms of catalysis, and sensitivity to inhibitors, with several members for each class
<cit> .
therefore it cannot be excluded that in the present work only two specific members of this class of defence proteins with peculiar roles in defence were detected.
secondary metabolism
spot  <dig> was identified as a flavonoid 7-o-methyltransferase and the production of this protein was enhanced in infected leaves of the resistant nil while in the susceptible nil infection resulted in a significant down accumulation of the protein .
this enzyme, that accumulated faster in incompatible interactions between barley and powdery mildew with respect to compatible interactions, was demonstrated to preferentially catalyze the methylation of the flavone apigenin, most likely leading to the production of phytoalexins
<cit> .
since methylated flavonoids have been found to have more antifungal activity than their unmethylated precursors
<cit> , it is plausible that the protein accumulation in bowman-rph <dig> represents a resistance gene-mediated defence response coupled to pathogen recognition, while in the susceptible nil a successful leaf rust colonization is associated to the suppression of defence responses leading to reduction of 7-o-methyltransferase protein.
several fungal pathogens demonstrate to secrete protein effectors capable in suppressing resistance gene-based and basal defence responses
<cit> ; it is therefore possible that p. hordei secreted effectors contribute to pathogen proliferation and disease development during the interaction with the susceptible nil.
transcript analysis and correlation with proteomic data
eight genes involved in protein accumulation changes were evaluated at the transcription level by quantitative rt-pcr  using rnas obtained from inoculation time course experiments that included also the inoculations time points used for proteomics analyses .
<dig>  error bars represent sd across all rt-pcr replicates .
statistical significance of differential expression for the tested genes was evaluated with a wilcoxon two group test .
no or weak correlations between mrna and protein levels were observed for the lap <dig>  atp synthase and susy.
for the last gene, only a weak correlation with the proteomic data was observed in bowman-rph <dig> being susy transcription up regulated until  <dig> dpi but then down regulated.
this transcriptional response is coherent with both the observed increase of susy protein  and reducing sugars  at  <dig> dpi.
in bowman, susy gene expression increased from  <dig> to  <dig> dpi suggesting a delayed plant response to the fungus.
nevertheless, bibliographic data indicate that pathogen responsiveness of susy transcription is highly variable ranging from down regulation during a barley-b.
graminis f. sp.
tritici non-host interaction
<cit>  to enhanced transcription observed in arabidopsis plants infected with plasmodiophora brassicae <cit>  and in phytoplasma-infected grape plants
<cit> .
in agreement with the proteomic data, expression of ruacs and agppase genes decreased at  <dig> to  <dig> dpi, while transcript levels of the two chitinase genes tested decreased in the resistant nil starting from  <dig> dpi.
proteomics and transcription analyses both suggested an up-regulation of flavonoid 7-o-methyltransferase in bowman-rph <dig> after leaf rust infection while the gene transcription was repressed by pathogen infection in the susceptible nil.
an up-regulation for the flavonoid 7-o-methyltransferase gene was observed also during a transcript profiling study of the broad spectrum race-nonspecific leaf rust resistance gene lr <dig> after interaction with p. triticina, but not in the compatible interaction or in the race-specific resistance gene lr <dig> <cit> , thus supporting a possible important role of this enzyme in defence responses involving lignin biosynthesis and production of phytoalexins antimicrobial compounds
<cit> .
in our experiments we observed that, for the time points subjected to parallel proteomics and transcriptomic analyses,  <dig> % of the protein changes correlate with the transcriptomic data, since for five genes out of  <dig> analyzed, an agreement was observed between protein accumulation and transcriptional change at the corresponding time points.
higher level on incongruent expression between mrnas and proteins was however frequently observed by other groups, in other species and experimental conditions
<cit>  and is most likely a result of the biology of gene expression which includes various levels of regulation during protein synthesis: post-transcriptional, translational, and post-translational.
thus, integrated analysis of both mrnas and proteins is crucial to gain further insights into complex biological systems.
CONCLUSIONS
in this work, proteomics and a complementary transcriptomic approaches were applied to two nils differing for the presence of the highly effective broad spectrum rph <dig> leaf rust resistance gene.
the genetic materials analysed allows to relate the observed responses to the presence/absence of the resistance gene.
we observed that the susceptible nil was basically unresponsive to pathogen infection at the time points analyzed, indicating that basal defences are suppressed and/or delayed by pathogen effectors or by the lacking of effective signalling pathways.
in a study on the susceptible interaction between the leaf rust pathogen p. triticina and wheat, changes in proteome were identified only after  <dig> dpi
<cit> , indicating that a similar delay in defence towards leaf rusts could represent a common feature of susceptible genotypes.
in bowman, a substantial transcripts down regulation in infected tissues, with respect to control samples, was observed for susy, lap <dig> and chitinases, while down regulation of both, protein and transcripts, was observed for flavonoid 7-o-methyltransferase, supporting a suppression of defence responses by pathogen effectors.
in the resistant genotype we observed a reduction of rulc degradation products together with a down regulation of ruacs and atp synthase.
these responses could be associated to the re-direction of the metabolism to sustain defence responses and, possibly, to cellular damage caused by hr responses.
protein changes for bowman-rph <dig> also supports a i) carbohydrate metabolism variation leading to a reduction in starch biosynthesis possibly connected to allocation of sugars into carbohydrate-consuming defence reactions , ii) an increase of proteins related to proteolitic activities and iii) accumulation of enzymatic proteins involved in phytoalexin and lignin production.
these responses were observed in the resistant nil only and are therefore dependent from the resistance pathways activated, upon pathogen recognition, by the rph15-mediated resistance signalling.
since none of these changes were highlighted at  <dig> hpi, it can be assumed that accumulation of defence-related proteins starts in the interval between  <dig> hpi and  <dig> dpi.
in conclusion, the proteomic approach adopted, complemented with transcriptomic analysis, provided a picture about the timing, entity and the nature of differential defence responses in basal resistance and rph15-gene mediated resistance.

 the fungus puccinia hordei is a biotrophic pathogen causal agent of leaf rust, a serious leaf disease of barley worldwide. this pathogen causes serious economic losses with yield reductions by up to 62% and adversely affects grain quality by reducing grain weight and increased levels of undesirable protein in the barley growing region of the world. rust fungi have a complex life cycle that involves two parasitic stages, dikaryotic and monokaryotic. the dikaryotic stage is the form causing rust disease by attacking mesophyll tissues until pathogen injures the epidermis to release urediospores. barley resistance to leaf rust pathogens is governed by major resistance genes that are race-specific. most rph genes confer complete seedling resistance associated with necrosis of the plant cells attacked by the pathogen sporelings, while some rph resistance genes confer incomplete resistance, in which the fungus forms small uredinia surrounded by chlorotic or necrotic plant tissues. this second resistance type is a non-hr polygenically inherited leaf rust resistance, which was termed “partial resistance” and is not associated with plant cell necrosis. several seedling resistance genes were identified from cultivated and wild barley, of which were designed rph to rph. the resistance provided by single rph genes has often been overcome by new pathotypes, believed to have arisen after gene mutations. as a direct consequence, the number of effective rph genes available to breeders is decreasing rapidly, suggesting the need for a new gene deployment strategy. the leaf rust resistance gene rph located on chromosome 2hs, was derived from pi an accession of wild barley collected in israel. when evaluated for its reaction toward a worldwide collection of over p. hordei isolates, it conferred resistance to all but the isolates 90– from israel. rph is one of the most broadly effective resistance genes and it is therefore useful in barley breeding programs for leaf rust resistance. barley interaction with the leaf rust pathogen represents a model to understand the molecular basis of both race-specific and partial resistance. molecular basis of partial resistance were recently investigated using eqtl analyses carried out in p. hordei infected doubled haploid lines and qtl-nils. these studies provided an overview of the responsive dynamic defence process and identified several candidate genes as being co-localized with the phenotypic qtl. no additional published microarray studies were dedicated to barley-p. hordei interaction. in addition to transcriptional studies, proteomic techniques can provide insight into the molecular mechanisms underpinning resistance gene-based plant defence responses. the compatible interaction between wheat and the leaf rust pathogen p. triticina was investigated at and dpi and only at the latter time point of inoculation seven plant proteins involved in translation and stress responses were identified as pathogen-responsive. no published proteomic studies have been performed on barley leaves infected with leaf rust. in this work, barley responses to leaf rust infection were investigated, in two barley near isogenic lines differing for the introgression of the broad effective leaf rust resistance gene rph a proteomic study was performed at early and late infection times and, after analysis of protein pattern changes, it was observed that only at the late time points and in the resistant nil differential protein accumulation occurs in response to pathogen inoculation. the differentially expressed proteins were involved in photosynthesis, carbon metabolism, defence responses and secondary metabolism.  experimental design and 2-de analysis in this study, the defence responses to leaf rust of two near-isogenic barley lines, bowman and bowman-rph differing for the presence/absence of the broad effective leaf rust resistance gene rph were investigated using a proteomic approach. the utilization of nils allows to relate the pathogen-responsive changes in protein accumulation observed between the two nils to the resistance gene activity. two time points of inoculation were selected for the analyses. at the first time point investigated, hours post inoculation, bibliographic data report that the leaf rust pathogen has established haustoria in the mesophyll cells and has started intercellular hyphal growth; brian j. steffenson, personal communication]. at days post inoculation, the number of cells with established haustoria is higher than at hpi and hyphae completed their intercellular growth. since infection conditions applied in the present work are basically the same as those previously reported, we assumed that in our experiments the differentiation of infection structures and colonization process followed the same timing as described above. in agreement with previous observations, no disease symptoms were observed in both the nils at hpi, while at dpi few chlorotic areas were observed in the susceptible nil bowman only. in order to assess the success of the inoculation process, single plants of each biological replicate were left until dpi. at this stage sporulating colonies were observed in bowman while bowman-rph exhibited only few chlorotic areas. to further verify that defence responses were properly deployed in the infected leaves used for proteomic analyses, quantitative rt-pcrs were conducted on genes encoding for oxalate oxidase, an h2o generating enzyme, and callose synthase, an enzyme involved in cell wall reinforcement. for both genes expression was significantly increased by inoculation at and dpi and was unresponsive by dpi, with higher transcription level in bowman compared to bowman-rph. these results demonstrate that active defence responses were triggered in both the genotypes at the inoculation time points used to detect leaf rust infection-dependent changes in protein accumulation. proteins obtained from the two infection time points were subjected to the proteomic analysis and three technical replicates were performed for each of two biological replicates. in the 2-de maps, an average of protein spots were visualized with the sypro ruby fluorescent staining. after image and statistical analyses, no significantly differentially accumulated proteins were identified at hpi in both genotypes. in agreement with our results, no changes in protein accumulation were also detected at dpi in the interaction between the leaf rust pathogen p. triticina and a susceptible wheat line. nevertheless, it is important to observe that at hpi more than genes were differentially expressed in barley leaves of genotypes carrying qtls for partial resistance to p. hordei in accordance with an active defence response at this infection time. these findings underline, as expected, that a longer time is required to detect the response at protein level. moreover, because of the proteomic approach adopted, only a part of the proteome was investigated in this study and this do not permit a satisfactory comparison between transcriptomic and proteomic data. figure reports 2-de representative maps of proteins isolated from both the bowman and the bowman-rph in mock-inoculated and leaf rust inoculated conditions at dpi. eighteen protein spots showing significant differential accumulation in response to pathogen infection were identified at dpi; differential accumulation was claimed only for spots in which normalized volumes of six replicates for each condition was showing an average fold change in their relative volumes of at least two folds. in the susceptible nil at dpi only a few differences were detectable. in particular, two spots were more abundant and one spot was less abundant in the rust inoculated sample with respect to the mock inoculated one. in the resistant nil a total of spots were significantly down accumulated or absent in the inoculated samples with respect to its control, while the levels of three protein spots rose in the inoculated sample with respect to the control condition. and figure indicate the spots, identified by lc-esi-ms/ms, showing significant changes of at least two-fold in their relative volumes after dpi. proteins that increased or decreased after this treatment are reported in red or in green, respectively. spot is highlighted in blue, indicating its absence in the bowman-rph inoculated sample. statistical information about lc-esi-ms/ms analysis are reported in additional files and abb.: abbreviation. a: experimental molecular weight and pi. b: theoretical molecular weight and pi. c: amino acid coverage. d: annotation obtained by blast-p alignment analysis against the viridiplantae subset of the nr-database at ncbi. e: value referred to the mature form of the protein. and figure 2; proteins were ordered into five functional classes, as indicated in table identification of rph15-resistance gene-related proteins the spots of interest were characterized by lc-esi-ms/ms. the differentially modulated proteins could be grouped into the following five different functional classes: photosynthesis and energy metabolism, carbohydrate metabolism, protein biosynthesis and degradation, defence responses and secondary metabolism. photosynthesis and energy metabolism twelve of the identified proteins corresponded to photosynthetic or chloroplast-related proteins, including rubisco large chain and rubisco activase, while one spot represented an atp synthase protein. rubisco is the most expressed protein in leaves, with about 30% of total leaf proteins and an increase of rubisco degraded forms was generally observed in wheat leaves after fusarium graminearum infection. many spots were identified in the present work as degradation products of rulc; the experimental molecular weights were significantly lower than their theoretical values, thus indicating a degradation of rulc in the samples analyzed. degradation of photosynthetic apparatus may contribute to restrict pathogen growth into barley cells by promoting the activation of defences in non-infected cells because small peptide and amino acids derived from degradation can be directed to other metabolic pathways involved in defence. we observed higher levels of rulc fragments in the control samples than in the inoculated tissues of bowman-rph supporting that turnover of rulc was markedly reduced in this genotype after infection. interestingly, no statistically significant differences in rulc fragments accumulation were observed between control and inoculated conditions in bowman. to confirm the proteomics data, a western blot analysis using an anti rulc antibody was performed on the protein extracts of the two nils under control and inoculated conditions. results from this experiment support a reduction of rulc degraded forms occurring on infected bowman-rph with respect to control tissues. in bowman-rph inoculated sample was in fact evaluated a reduction of %, %, % and % of the rulc degraded forms with respect to the control sample respectively for the major bands of the degradation forms numbered from to. a similar rulc degradation trend was observed in rice as induced by bacterial blight infection, in lesion mimic mutants and in wheat after fusarium graminearum infection. taken together, our results support the conclusion that in the resistant genotype bowman-rph the rubisco integrity was more preserved after infection with respect to control sample. rubisco activase regulates rubisco activity by hydrolysing atp to promote the dissociation of inhibitory sugar phosphates. ruacs accumulation is reduced in the bowman-rph nil infected sample. in the susceptible nil bowman, no substantial alterations of rulc and ruacs were observed. it can be hypothesized that this effect on ruacs is linked to a different strategy to modulate the phothosynthesis in bowman-rph the genotype in which the pathogen negatively affect rubisco degradation. spot represents an atp-synthase β chain. this protein is involved in photosynthesis and oxidative respiration to drive synthesis of atp in chloroplasts and mitochondria. accumulation of this protein was down-regulated after infection in the resistant nil only. a decrease of proteins related to carbon metabolism and photosynthesis that include rulc, ruacs and atp synthase has been also observed in orange leaves after infection with the biotrophic bacterial pathogen xanthomonas citri pv. citri. similarly, a down regulation of atp-synthase subunits was observed in grape response to flavescence dorée infection. similar observations were obtained at the transcriptomic level for barley leaves infected with the biotrophic powdery mildew fungus and after an extensive analysis of the effects on photosynthetic genes of several biotic stresses in eight plant species. these investigations suggest that a slow turnover of many photosynthetic proteins represent an adaptive consequence resulting in reduced energy supply when plants are facing a biotic attack and require to re-direct resources in immediate defence needs. the reduced accumulation of these three proteins in the resistant nil is therefore possibly consistent with a re-direction of the metabolism to fuel defence responses. nevertheless, down accumulation of rulc, ruacs and atp synthase proteins was also observed in the leaves of rice spotted leaf mutants that undergo to spontaneous programmed cell death, caused by oxidative burst and membrane damage, in the absence of pathogen infection. since the rph resistance most likely involves an hr-dependent pcd response, similarly to that of most rph genes conferring complete resistance, it cannot be excluded that the down-regulation of photosynthetic proteins observed in the resistant nil could also depend from cellular damage caused by the hr response. an higher accumulation of rulc degraded forms was observed for bowman-rph with respect to bowman in the control samples. also for ruacs and agppase, higher accumulation was present in bowman-rph than in bowman control samples. conversely, this behaviour was not observed for several other protein spots, indicating a process specific for proteins involved in the photosynthetic apparatus. since data are deriving from six replicates for each experiment and sd detected for these proteins was low, it can be postulated that the observed trend represents a genuine differential accumulation among bowman and bowman-rph control samples ascribable to an unknown alteration of the cellular homeostasis orchestrated by the rph resistance gene or by another tightly associated sequence. carbohydrate metabolism two infection-modulated proteins identified in this study are related to sugar metabolism: sucrose synthase and adenosine diphosphate glucose pyrophosphatase. susy accumulation was significantly increased in infected leaves of the resistant nil. susy catalyzes the reversible reaction from sucrose to fructose and udp-glucose and an increase in this activity usually results in decreased starch biosynthesis. in addition, susy is the key enzyme of symplastic sucrose unloading and in concert with invertases, it can modulate the sink capacity of plant tissues. susy has a dual role in producing both cytosolic adpg, directed to starch biosynthesis, and udpg, necessary for cell wall material and glycoprotein biosynthesis. defense-related susy activity may serve to allocate sucrose into callose deposition and other carbohydrate-consuming defense reactions, as supported by the observation of an increased susy activity in the resistant response of tobacco plants to phytophthora nicotianae and in several other plant–pathogen interactions. a leaf rust infection-dependent induction of a callose synthase gene was observed in the present work, suggesting re-directioning of sugars to structural components of the cell wall to constitute physical barriers to the infection. a consistent decrease in adp-glucose pyrophosphatase abundance was observed in infected tissues of the resistant genotype only. agppase catalyzes adp-glucose breakdown to produce amp and g1p, and its reduced accumulation/activity drastically lead to a reduction in starch biosynthesis. there are increasing evidences that the availability of soluble carbohydrates is a major factor in determining plant resistance to infections. the results of plant-pathogen interactions also depend on the rapid mobilization of carbohydrates and on the reprogramming of the carbon flow from sucrose to hexoses. the data about susy and agppase suggest that in the resistant genotype under infection there could be a redirection of primary metabolism that leads to a reduction of starch biosynthesis in order to provide a strong supply to the hexoses pool. to further verify the role of sugar metabolism in rph15-mediated response to leaf rust, an evaluation of reducing sugars and sucrose was conducted on control and inoculated conditions in both nils. while sucrose content was not affected by infection, reducing sugars were slightly but significantly increased by inoculation in the resistant nil and significantly decreased in the susceptible one. the reducing sugars increase supports a re-direction of the hexoses pool to defence pathways activated in the resistant nil in response to the leaf rust pathogen infection. the data are the means ± se of three experiments run in triplicate. different letters on the values indicate significant difference p-values of the t-tests performed comparing control and inoculated samples. protein degradation protein spot number was characterized as a leucine aminopeptidase protein. lap accumulation was observed in the inoculated sample of the resistant nil only. aminopeptidases represent plant responses to wound and pathogen stresses and their activity is involved in the turnover of unfolded or damaged proteins that accumulate as a result of the oxidative burst. furthermore, the role of aminopeptidases in the genesis of bioactive peptides in animals and, more recently, in plants highlighted their possible contribution in defence responses signalling. the modulation of the levels of lap observed was rph resistance gene-dependent, consequently this protein could have a role both at the level of protein turnover, since the rph resistance response would most likely involve an hr-associated oxidative burst, and/or in resistance gene signalling. the fact that lap is more abundant in bowman-rph line under infection reinforces the idea that protein degradation could be one of the main mechanisms contributing to resistance deployment. defence responses two protein spots were respectively identified as chitinase and chitinase iii-like. protein abundance significantly decreased in the inoculated samples of the resistant nil only at dpi, while in the susceptible nil the levels of chitinases were not affected. for both chitinases, the level of accumulated proteins was however constitutively higher in mock-inoculated tissues of the resistant nil with respect to the susceptible one. this behaviour supports the possibility that a higher constitutive level of these defence proteins is associated with resistance and it operates as an early defence barriers against the pathogen infection. similar results were found in the interaction between wheat and septoria tritici where a higher chitinase activity, measured on whole leaf extracts, was observed in control with respect to inoculated plants during the first dpi but the enzymatic activity did not correlate well with resistance in the host or the infection course of the pathogen. plant chitinases, however, are classified into seven classes, on the basis of their structure, substrate specificity, mechanisms of catalysis, and sensitivity to inhibitors, with several members for each class. therefore it cannot be excluded that in the present work only two specific members of this class of defence proteins with peculiar roles in defence were detected. secondary metabolism spot was identified as a flavonoid 7-o-methyltransferase and the production of this protein was enhanced in infected leaves of the resistant nil while in the susceptible nil infection resulted in a significant down accumulation of the protein. this enzyme, that accumulated faster in incompatible interactions between barley and powdery mildew with respect to compatible interactions, was demonstrated to preferentially catalyze the methylation of the flavone apigenin, most likely leading to the production of phytoalexins. since methylated flavonoids have been found to have more antifungal activity than their unmethylated precursors, it is plausible that the protein accumulation in bowman-rph represents a resistance gene-mediated defence response coupled to pathogen recognition, while in the susceptible nil a successful leaf rust colonization is associated to the suppression of defence responses leading to reduction of 7-o-methyltransferase protein. several fungal pathogens demonstrate to secrete protein effectors capable in suppressing resistance gene-based and basal defence responses; it is therefore possible that p. hordei secreted effectors contribute to pathogen proliferation and disease development during the interaction with the susceptible nil. transcript analysis and correlation with proteomic data eight genes involved in protein accumulation changes were evaluated at the transcription level by quantitative rt-pcr using rnas obtained from inoculation time course experiments that included also the inoculations time points used for proteomics analyses. error bars represent sd across all rt-pcr replicates. statistical significance of differential expression for the tested genes was evaluated with a wilcoxon two group test. no or weak correlations between mrna and protein levels were observed for the lap atp synthase and susy. for the last gene, only a weak correlation with the proteomic data was observed in bowman-rph being susy transcription up regulated until dpi but then down regulated. this transcriptional response is coherent with both the observed increase of susy protein and reducing sugars at dpi. in bowman, susy gene expression increased from to dpi suggesting a delayed plant response to the fungus. nevertheless, bibliographic data indicate that pathogen responsiveness of susy transcription is highly variable ranging from down regulation during a barley-b. graminis f. sp. tritici non-host interaction to enhanced transcription observed in arabidopsis plants infected with plasmodiophora brassicae and in phytoplasma-infected grape plants. in agreement with the proteomic data, expression of ruacs and agppase genes decreased at to dpi, while transcript levels of the two chitinase genes tested decreased in the resistant nil starting from dpi. proteomics and transcription analyses both suggested an up-regulation of flavonoid 7-o-methyltransferase in bowman-rph after leaf rust infection while the gene transcription was repressed by pathogen infection in the susceptible nil. an up-regulation for the flavonoid 7-o-methyltransferase gene was observed also during a transcript profiling study of the broad spectrum race-nonspecific leaf rust resistance gene lr after interaction with p. triticina, but not in the compatible interaction or in the race-specific resistance gene lr, thus supporting a possible important role of this enzyme in defence responses involving lignin biosynthesis and production of phytoalexins antimicrobial compounds. in our experiments we observed that, for the time points subjected to parallel proteomics and transcriptomic analyses, % of the protein changes correlate with the transcriptomic data, since for five genes out of analyzed, an agreement was observed between protein accumulation and transcriptional change at the corresponding time points. higher level on incongruent expression between mrnas and proteins was however frequently observed by other groups, in other species and experimental conditions and is most likely a result of the biology of gene expression which includes various levels of regulation during protein synthesis: post-transcriptional, translational, and post-translational. thus, integrated analysis of both mrnas and proteins is crucial to gain further insights into complex biological systems.  in this work, proteomics and a complementary transcriptomic approaches were applied to two nils differing for the presence of the highly effective broad spectrum rph leaf rust resistance gene. the genetic materials analysed allows to relate the observed responses to the presence/absence of the resistance gene. we observed that the susceptible nil was basically unresponsive to pathogen infection at the time points analyzed, indicating that basal defences are suppressed and/or delayed by pathogen effectors or by the lacking of effective signalling pathways. in a study on the susceptible interaction between the leaf rust pathogen p. triticina and wheat, changes in proteome were identified only after dpi, indicating that a similar delay in defence towards leaf rusts could represent a common feature of susceptible genotypes. in bowman, a substantial transcripts down regulation in infected tissues, with respect to control samples, was observed for susy, lap and chitinases, while down regulation of both, protein and transcripts, was observed for flavonoid 7-o-methyltransferase, supporting a suppression of defence responses by pathogen effectors. in the resistant genotype we observed a reduction of rulc degradation products together with a down regulation of ruacs and atp synthase. these responses could be associated to the re-direction of the metabolism to sustain defence responses and, possibly, to cellular damage caused by hr responses. protein changes for bowman-rph also supports a i) carbohydrate metabolism variation leading to a reduction in starch biosynthesis possibly connected to allocation of sugars into carbohydrate-consuming defence reactions, ii) an increase of proteins related to proteolitic activities and iii) accumulation of enzymatic proteins involved in phytoalexin and lignin production. these responses were observed in the resistant nil only and are therefore dependent from the resistance pathways activated, upon pathogen recognition, by the rph15-mediated resistance signalling. since none of these changes were highlighted at hpi, it can be assumed that accumulation of defence-related proteins starts in the interval between hpi and dpi. in conclusion, the proteomic approach adopted, complemented with transcriptomic analysis, provided a picture about the timing, entity and the nature of differential defence responses in basal resistance and rph15-gene mediated resistance. 
the aim of the present work was to study the rph15-based defence responses using a proteomic approach. the identified proteins can be related to modulation of the photosynthetic apparatus components, re-direction of the metabolism to sustain defence responses and deployment of defence proteins. the identification of leaf rust infection-modulated defence responses restricted to the resistant nil support the hypothesis that basal defence responses of bowman, but not the rph resistance gene-based ones, are suppressed or delayed by pathogen effectors to levels below the detection power of the adopted proteomic approach. protein pattern changes in response to the leaf rust pathogen infection were investigated in two barley near isogenic lines, bowman and bowman-rph, differing for the introgression of the leaf rust resistance gene rph two infection time points, hours and four days post inoculation, were analysed. protein spots were characterized by lc-ms/ms analysis and found to be involved in photosynthesis and energy metabolism, carbohydrate metabolism, protein degradation and defence. no statistically significant differences were identified at the early time point, while at dpi eighteen protein spots were significantly up or down regulated with a fold-change equal or higher than two in response to pathogen infection. leaf rust, caused by the biotrophic fungal pathogen puccinia hordei, is one of the most important foliar disease of barley and represents a serious threat in many production regions of the world. the leaf rust resistance gene rph is of outstanding interest for resistance breeding because it confers resistance to over puccinia hordei isolates collected from around the world. almost all the pathogen-responsive proteins were identified in the bowman-rph resistant nil. proteomic data were complemented by transcriptional analysis of the respective genes. additionally, rph15-mediated resistance processes identified mainly resides on a modulation of primary metabolism, affecting photosyntesis and carbohydrate pool.
 leaf rust, caused by the biotrophic fungal pathogen puccinia hordei, is one of the most important foliar disease of barley and represents a serious threat in many production regions of the world. the leaf rust resistance gene rph is of outstanding interest for resistance breeding because it confers resistance to over puccinia hordei isolates collected from around the world. molecular and biochemical mechanisms responsible for the rph effectiveness are currently not investigated. the aim of the present work was to study the rph15-based defence responses using a proteomic approach.  protein pattern changes in response to the leaf rust pathogen infection were investigated in two barley near isogenic lines, bowman and bowman-rph, differing for the introgression of the leaf rust resistance gene rph two infection time points, hours and four days post inoculation, were analysed. no statistically significant differences were identified at the early time point, while at dpi eighteen protein spots were significantly up or down regulated with a fold-change equal or higher than two in response to pathogen infection. almost all the pathogen-responsive proteins were identified in the bowman-rph resistant nil. protein spots were characterized by lc-ms/ms analysis and found to be involved in photosynthesis and energy metabolism, carbohydrate metabolism, protein degradation and defence. proteomic data were complemented by transcriptional analysis of the respective genes. the identified proteins can be related to modulation of the photosynthetic apparatus components, re-direction of the metabolism to sustain defence responses and deployment of defence proteins.  the identification of leaf rust infection-modulated defence responses restricted to the resistant nil support the hypothesis that basal defence responses of bowman, but not the rph resistance gene-based ones, are suppressed or delayed by pathogen effectors to levels below the detection power of the adopted proteomic approach. additionally, rph15-mediated resistance processes identified mainly resides on a modulation of primary metabolism, affecting photosyntesis and carbohydrate pool. barleyleaf rustresistance generph15proteomicsnear isogenic lines 