leaf rust , caused by the 
 heteroecious basidiomycete puccinia triticina eriks , is one of the most 
 important diseases of wheat worldwide .
serious yield losses can result as a consequence of its broad 
 distribution and potential to develop rapidly under optimal environmental 
 conditions .
furthermore , the ability of p. 
 triticina to form new races that can attack previously resistant cultivars , 
 along with the capacity of fungal spores to travel long distances , can make 
 full control of leaf rust difficult .
due 
 to its importance , epidemiological , genetic 
 [ 2 , 3 ] , and molecular aspects 
 [ 4 , 5 ] of the 
 disease have been studied extensively . as a consequence , the 
 wheat - p .
triticina interaction is 
 well defined in genetic terms and stages of infection 
 [ 4 , 68 ] .
scanning electron 
 microscopy work by hu and rijkenberg   identified 
 important time points in infection structure 
 formation by p. triticinia on 
 susceptible and resistant lines of hexaploid wheat . 
 
after12  hours , the fungus has successfully 
 penetrated into the stoma , formed substomatal vesicles ( ssv ) , and primary 
 infection hyphae are visible . 

after ssv formation , the primary infection hypha grows and attaches to a mesophyll or 
 epidermal cell . at 24 hours 
 postinoculation ( hpi ) , a septum appears separating the haustorial mother cell 
 from the infection hypha after which the fungus forms haustorium and penetrates 
 the cell .
nevertheless , 
 numerous genes involved in the wheat - pathogen response have been 
 identified .
these include genes encoding 
 leaf rust resistance , antifungal hydrolases such as 
 glucanase and chitinase [ 10 , 11 ] , protein 
 kinases , and enzymes involved in the production of 
 reactive oxygen species [ 10 , 13 ] .
of 
 the leaf rust resistance genes , lr1 is described as one of those conferring 
 resistance to leaf rust low pathogenic races in wheat seedlings 
 [ 2 , 14 ] . until recently
, 
 however , molecular studies of the host response to pathogen attack were 
 restricted to the analysis of a relatively small number of genes or 
 proteins .
this has changed with the development of high - throughput technologies , such as cdna 
 and oligonucleotide microarrays that allow expression profiling of thousands of genes 
 simultaneously .
thus , microarrays 
 represent important tools for the global analysis of many plant processes , 
 including the response to pathogen attack [ 1520 ] .
there are already a number of studies that 
 have used cdna and oligonucleotide microarrays to characterize plant - pathogen 
 interactions .
arabidopsis cdna microarray analysis demonstrated a substantial 
 network of regulatory coordination among different defence signaling pathways 
 . in another study ,
microarray 
 analysis of the arabidopsis transcriptome during systemic acquired 
 resistance revealed groups of genes with common regulation patterns 
 .
for instance , oligonucleotide 
 arrays were used to analyze maize challenged with the pathogen cochliobolus 
 carbonum , barley 
 challenged by blumeria graminis f. sp .
hordei 
 , and fusarium 
  to 
 identify genes that showed expression changes during the interactions . in wheat lines carrying 
 the leaf rust resistance gene
lr1 , however , little is known 
 regarding the expression of genes during differential responses following 
 inoculation with compatible and incompatible races of the pathogenic leaf rust 
 fungus p. triticina . in this report
, we describe the development 
 and use of a wheat cdna microarray to examine changes in gene expression in the 
 wheat line rl6003 in response to challenge by compatible and incompatible races 
 of p. triticina .
wheat near isogenic line rl6003 ( thatcher * 6/centenario ) , carrying the lr1 leaf rust 
 resistance gene , was used throughout the study because thatcher is known to strongly 
 express the introgressed lr genes .
seeds were sown in 12  cm 
 diameter plastic pots filled with a 3 : 1 soil / sunshine professional growing mix 5 soil mixture 
 ( sun gro horticulture , vancouver , british columbia , canada ) , at a rate of one 
 seed per pot .
seedlings were maintained 
 in a growth room at 20c with a 16-hour photoperiod and a photon flux density 
 of approximately 145  mol ms .
plants were inoculated at the 
 2 - 3 leaf stage with urediniospores of p. triticina virulence phenotypes 
 bbb ( incompatible ) or tjb ( 7 - 2 ) ( compatible )   mixed with a 
 light mineral oil ( bayol - esso canada , oak bluff , manitoba , canada ) . 

twenty five l of a 16.6  mg 
 uredinispores / ml 
 of mineral oil inoculum were applied to each pot .
seedlings were sprayed until runoff with the 
 spore suspensions using a devilbis - type sprayer , connected to an air line and 
 operated at a pressure of 17  kpa . 

control plants were sprayed only with mineral oil , referred to as mock 
 inoculation .
the inoculated plants were 
 allowed to dry for at least 30 minutes to let the oil volatilize and then 
 incubated overnight in a 100% humid chamber ( percival model i-60d ) 
 ( percival scientific , perry , iowa , usa ) .
they were subsequently transferred to a 
 growth chamber and kept under the conditions described above . 


four cdna libraries were constructed from 
 either thatcher lr1 leaf tissue 
 ( 4-leaf stage ) sampled 24 hours after inoculation with p. triticina 
 race 
 bbb or developing seeds of  glenlea  sampled at 5 , 15 , or 25 days 
 postanthesis .
plasmid dna , pbk - cmv 
 ( stratagene , la jolla , calif , usa ) , and psport 6.0 ( invitrogen , burlington , 
 ontario , canada ) , respectively , for the leaf and seed libraries , were isolated 
 using the perfect prep direct bind kit ( eppendorf , hamburg , germany ) , adapted 
 for the qiagen 3000 liquid handling robot ( qiagen , mississauga , ontario , canada ) . 

est sequencing was performed with m13f and 
 m13r universal primers using the big dye v2.0 ( abi , foster city , calif , usa ) 
 and resolved on an abi 377 genetic analyzer . 

high - quality sequence was obtained using the softwares phred and 
 cross_match ( default settings except for minscore = 20 and minmatch = 12 ) 
 .
a total of 24  959 ests were 
 obtained from the libraries and were submitted to genbank ( accessions 
 be417910-be418911 , bg903972-bg910140 , bq619578-bq620868 , bq235898-bq252370 , es316459 ) .
the 
 sequences were assembled using cap3 software ( default parameters except overlap 
 length cutoff = 40 and overlap identity cutoff = 95 ) 
 , and in 
 combination with some manual annotations , a unigene set of approximately 
 12  500 sequences was identified .
plasmids of the unigene set 
 members were used as template for pcr amplification of the inserts using 
 universal m13 primers .
amplicons were 
 visualized on agarose gels and selected for use on the microarray based on 
 their quality and uniqueness .
a subset 
 of 7728 selected amplicons was consolidated to 384-well plates and purified 
 using multiscreen 384 pcr filtration plates ( millipore , nepean , ontario , canada ) .
aliquots of the purified products were run on 
 1% ( w / v ) agarose gels , sized and quantified using known amounts of 
 lambda hind iii markers .
they were then diluted in 
 1  microspotting 
 plus solution ( telechem , sunnyvale , calif , usa ) 
 to a final concentration of 100  ng/l . 
 a total of 384 controls provided with the spotreport-3 ( stratagene ) kit , 
 consisting of arabidopsis cab , rbcl 
 and rca genes , human -cell actin gene , cot-1 , ssdna and polya , as well as 
 wheat cab , rbcl , rca , and 
 human -cell receptor genes , were also included as were 96 empty 
 wells .
all 8208 samples were printed by 
 telechem onto superamine substrates according to the company standard 
 procedures ( www.arrayit.com ) in 48 blocks of a 19  18 array using side - by - side double 
 spotting .
approximately 1  g of leaf tissue was frozen in liquid n2 and ground to a fine powder with a mortar and 
 pestle .
total rna was extracted using the rnawiz isolation reagent ( ambion , austin , tex , usa ) and fursther 
 purified using rneasy columns ( qiagen ) , according to manufacturer 's 
 instructions .
fluorescent probes were synthesized by reverse transcription of the rna in the presence of 
 cy3-dctp or cy5-dctp ( amersham biosciences , baie - d'urf , quebec , canada ) , using a modification of the protocol for the preparation of unamplified cdna 
 .
each labeling reaction was performed in a 
 40  l volume containing 10  g total rna , 0.75  g oligo(dt)1218 
 ( invitrogen ) , 500  m each of datp , dgtp , and dttp , 50  m dctp , 25  m of either 
 cy3-dctp or cy5-dctp , 8  l of 5  superscript ii 
 first strand buffer ( invitrogen ) , 10  mm dithiothreitol , and 40 u of rnasin 
 rnase inhibitor ( promega , madison , wis , usa ) .
the reaction mixture was heated 
 at 65c for 5 minutes and then cooled 
 to 42c .
four hundred units of 
 superscript ii reverse transcriptase ( invitrogen ) were added , and the labeling 
 reaction was allowed to proceed for 2 hours at 42c .
the rna was then degraded by treatment with 5  l of 50 mm edta ( ph 8) 
 and 2  l of 10 n naoh at 65c for 20 minutes .
after the addition of 4  l of 5 m acetic 
 acid , the mock and tjb ( mt ) or mock and bbb ( mb ) samples were pooled , and the 
 cdna was precipitated by the addition of one volume of isopropanol .
the pellet was washed with 70% ethanol , air 
 dried , resuspended in 5  l of nuclease - free water , and added to 60  l of 
 digeasy hyb solution ( roche , indianapolis , ind , usa ) containing 0.45  g of 
 yeast trna and 0.45  g of denatured salmon sperm dna .
slides were prehybridized by 
 incubating for 45 minutes at 42c in a preheated solution of 5  ssc , 0.1% ( w / v ) sodium dodecyl 
 sulfate , and 1% 
 ( w / v ) bovine serum albumin .
denatured dual cy3/cy5-labeled target cdnas 
 were added directly to each slide and sealed under a 22  mm  60  mm 
 hydrophobic coverslip ( sigma - aldrich , oakville , ontario , canada ) .
microarray 
 slides were placed on top of 
 support slides in a slide staining box ( diamed , west chester , pa , usa ) containing 
 approximately 20  ml digeasy hyb at the bottom , and incubated at 
 42c for 18 to 20 hours .
after hybridization , the 
 slides were quickly rinsed with 1  ssc ( until 
 coverslips fell off ) , washed three times with 1  
 ssc and 0.1% sds ( w / v ) for 10 minutes at 50c , then washed three more times 
 with 0.1 ssc for 1 minute at room temperature .
the slides were dried by 
 centrifugation and scanned for fluorescence emission using a genepix 4000b 
 scanner ( axon instruments , union city , calif , usa ) . 

photomultiplier ( pmt ) voltages were adjusted manually to balance the 
 amount of red and green signals in the images obtained .
feature intensities were quantified 
 using genepix pro 5.1 array analysis software and acuity 4.0 ( axon 
 instruments ) .
microarray spots flagged 
 as  bad ,   not found ,  or with specific unwanted feature 
 parameters were removed automatically by imposing stringent filtering criteria 
 to the entire microarray dataset ( acuity criteria dia 60 and circularity 
 80 ) .
the log2 median ( logm ) 
 ratio of expression values for genes , incompatible versus mock ( mb ) and 
 compatible versus mock ( mt ) , that met the filtration criteria and were found in 
 both treatment sets , was subjected to further statistical 
 analysis .
an analysis of variance using the sas procedure mixed ( sas , cary , nc , usa ) 
 was carried out on the ( logm ) ratio data for incompatible ( mb ) and compatible 
 ( mt ) interactions , and contrasts were used to identify genes with significant 
 differences in expression at one or more time points as defined by their p 
 values .
the model included compatibility 
 type ( mb or mt ) , time , and the interaction between compatibility type and 
 time .
main plots were replicate by 
 interaction type ( representing mb or mt pairs of batches of inoculum ) in a 
 randomized complete block arrangement , and subplots were individual 
 slides .
genes with a significant f value 
 ( f  .055 ) for a contrast between interaction type by time were selected 
 and verified by resequencing the dna used to create the spot on the 
 microarray .
in addition to the anova , a t 
  test was used to determine at which time point(s ) the treatments yielded 
 significantly different expression values ( p  .05 ) .
putative gene function was assigned 
 based on blastx analysis against the genbank nonredundant protein database 
 .
the best hit with an e value less than or equal to 1  10 was used to assign the est annotation . in some cases ,
the second best hit had a more informative description of 
 the gene function and was used to assign an annotation . if a significant blastx hit could not be 
 found , then the annotation from the dana - farber cancer institute ( dfci ) wheat 
 gene index cluster that contained the est was used http://compbio.dfci.harvard.edu/tgi/ , 
 if available .
gene ontology ( go ) 
 annotation was performed by using blastx to compare wheat genes to the 
 predicted protein sequences for arabidopsis 
 thaliana from the arabidopsis information resource ( tair ) genome release version 6 .
the best hit with an e value less than or equal to 1 10 was used to 
 assign the ests an arabidopsis homolog and the tair annotation tool was used to place the genes into go 
 categories .
to unravel global changes in the 
 host transcriptome during leaf rust interactions , wheat rl6003 , which carries 
 the lr1 resistance gene , was challenged with two different 
 races of the leaf rust fungus , puccinia 
 triticina . virulence phenotype bbb 
 carries the avirulence gene avr lr1 and provokes an incompatible reaction on rl6003 , while race tjb lacks the 
 avirulence gene and therefore produces a compatible interaction .
the tjb 
 interaction displayed a compatible  3 + 4-  infection type 
 characterized by large pustules and 
 abundant sporulation while interaction with bbb yielded an incompatible  ; 1-  infection type 
 ( figure 1 ) .
the presence of small 
 pustules or no sporulation was typical of a hypersensitive response ( hr ) .
no symptoms were observed on mock - inoculated 
 seedlings ( figure 1 ) . to identify genes
whose expression profile can distinguish compatible from incompatible 
 interactions , we designed an experiment using the pathosystem outlined in the 
 previous section : one host genotype , rl6003 , inoculated with the two rust 
 races , bbb and tjb , each compared to a mock control sample .
infection ensued and samples were 
 taken at four time points : 3 , 6 , 12 , and 24 hpi .
sequences on the 
 microarray were taken from an assembly of approximately 12  500 unigenes 
 generated from an est set of 24  959 sequences .
parameters to the assembly program , cap3 , were the same as those used by tigr for its unigene assemblies 
 ( www.tigr.org ) .
these parameters favoured assembly of homoeologous genes into single 
 sequences without the inclusion of paralogous genes .
spots  met the 
 filtration criteria outlined in the materials and methods , 4439 of which were 
 shared between the mb and mt datasets and were used for statistical 
 analysis .
statistical analysis of the 
 microarray data using sas and confirmation of the identity of ests by 
 sequencing revealed 192 genes that were significantly differentially regulated 
 across at least one of the four times points between the compatible and 
 incompatible interactions ( p .055 for the pathogen type by time 
 interaction anova effect)supplementary table 1   
 available online at 
 doi:10.1155/2007/17542 ) .
seventy one 
 percent of the genes had different expression values between the two treatments , 
 as determined using tests ( p  .05 ) , at a single time point , 18% 
 of genes showed differential expression at 2 time points , and 1% at 3 time 
 points , while no genes in this set were differentially expressed at all four of 
 the time points investigated . at 6 hpi ,
46% of the genes were differentially 
 expressed , followed by 40% at 24 hpi .
the 3- and 12-hour time points showed much less differential gene 
 expression at 11% and 10% of the genes , respectively . using blast analysis
, we were able 
 to assign , with various degrees of confidence , a potential function to 150 of 
 the differentially regulated genes . 

forty two gene sequences did not share sequence homology to any 
 sequences in the genbank nonredundant protein database at our threshold e value ( 1  10  5 ) , and therefore , no annotation could be assigned .
gene ontology ( go ) annotation was performed using the tair annotation 
 tool and the genes were placed into goslim categories ( figure 2 ) .
molecular function assignment revealed 
 members in a broad range of categories ( figure 2(a ) ) including those genes with putative hydrolase activity , nucleotide binding activity , and protein binding 
 activity .
the differentially regulated 
 genes were also members of a variety of cellular components with a large 
 representation from the chloroplast and mitochondria ( figure 2(b ) ) .
the biological process go assignment 
 illustrated that genes involved in protein metabolism , electron transport or 
 energy pathways , and response to abiotic / biotic stimuli were well represented 
 in our dataset ( figure 2(c ) ) . of the 7728 cdnas 
 spotted on the chip
, 2142 came from the p. triticina challenged est library raising the 
 possibility that some of the cdnas spotted to the array were fungal in 
 origin .
of the 2142 ests , only 2 
 sequences found a significant match ( e value < 1  10 ) against the 
 phytopathogenic fungi and oomycete est database available from cogeme ( http://cogeme.ex.ac.uk ) 
 and had no match against ests from non - p .
comparison of the 2142 ests to an in - house p. triticinia library 
 of over 40  000 ests ( bakkeren , unpublished ) found that 22 had significant 
 matches to fungal sequence using blast ( e value < 1  10 ) . of these ,
only one was found to be 
 differentially expressed in this study , and closer examination revealed that 
 the sequence ( talr1013e06r ) was a gene from a conserved pathway and had 
 stronger homology to a rice gene than to the fungal sequence .
a number of genes involved in the 
 energy status of the cell were differentially regulated .
for example , the interaction between 
 rl6003 
 and an incompatible or compatible rust race produced a differential expression 
 pattern for ribulose 1,5-bisphosphate carboxylase / oxygenase ( rubisco ) small 
 subunit ( talr1140f12a , talr1150f09f ) .
rubisco is the main source of energy production 
 for the plant cell , generating atp and reductive potential ( nadph ) through 
 photosynthesis . in the incompatible 
 interaction , the expression of the gene
was reduced at 6 hpi but returned to 
 levels identical to the mock 
 inoculation at 12 and 24 hpi .
the compatible interaction , however , was 
 characterized by a strong increase in the transcription of the rubisco small 
 subunit at 24 hpi ( figure 3 ) .
along with differential expression of rubisco , there was a change in the transcription of 
 its major regulatory protein , rubisco activase ( talr1173g10f , talr1175g08f ) 
 . at 3 hpi
, there was a small 
 reduction in the transcription of rubisco activase relative to basal levels for 
 both the compatible and incompatible interactions . at 6 hpi
the plants involved in an 
 incompatible interaction continued to repress the expression of rubisco 
 activase , while in the compatible interaction , expression of the transcript 
 returned to near basal levels .
chlorophyll a / b - binding protein 
 genes ( talr1131b12a , talr1142e01a , and talr1167e07a ) , an ftsh like 
 aaa - metalloprotease ( talr1134f10f ) , and a photosystem ii phosphoprotein 
 ( talr1130a12a ) were down regulated at 6 hpi in the incompatible interaction 
 relative to basal and compatible interaction levels . in both treatments ,
the expression of these 
 genes was at basal levels for the other time points .
an important enzyme involved in 
 photorespiration , glycolate oxidase ( talr1162d03a ) , was also found to be 
 differentially regulated .
its expression 
 was repressed at 3 and 6 hpi in plants involved in an incompatible interaction 
 but not in a compatible one .
the 
 production of reactive oxygen species ( ros ) is central to the defence mounted 
 by plants in reaction to challenge with an incompatible pathogen [ 31 , 32 ] . shortly after challenge
, the plant produces 
 an  oxidative burst  which is believed to have three roles : ( i ) induces 
 damage on the invading organism , ( ii ) bolsters structural defences , and ( iii ) 
 acts as a biochemical signal to induce other defence mechanisms in the 
 plant .
the production of ros presents a 
 challenge to a plant because of the damage these compounds can cause to its own 
 proteins , dna , and other cellular components .
the plant must therefore invoke a balanced system that produces ros for 
 defence at the same time as a number of antioxidants to protect against 
 oxidative damage .
ascorbate peroxidase 
 ( apx : tae25014d09f ) , a central component of the ros scavenging system , was 
 differentially expressed in the compatible and incompatible interactions . at 6 hpi ,
transcription of apx in plants 
 challenged by the incompatible race was significantly upregulated relative to 
 transcription in those challenged by the compatible race 
 ( figure 4 ) .
another critical protein for maintaining redox balance in the face of oxidative stress is 
 glutathione , which acts as ubiquitous supplier of reduction power for cellular 
 processes .
glutathione is a conjugate of 
 cysteine and glutamate and therefore relies on both of these amino acids for 
 synthesis .
cysteine synthase 
 ( tae25012d06r ) is the final catabolic step in the production of cysteine and 
 was found to be differentially regulated between the two treatments ( figure 4 ) .
plants in an incompatible 
 interaction increased the transcription of this gene at 6 hpi , while those in a 
 compatible interaction reduced it . at 
 the 12-hour time point ,
the incompatible plants had reduced transcription of 
 the gene relative to basal levels while the compatible plants had upregulated 
 transcription .
recognition of p. triticina infection by rl6003 occurs through a gene - for - gene 
 system where resistance only occurs if the plant possesses an r - gene encoding a 
 product able to detect the presence or action of a specific avirulence factor 
 ( avr - gene ) produced by the pathogen [ 3335 ] .
if either of these components is missing , the plant is unable to mount 
 an effective defence against the invader .
r - genes identified to date fall into five different categories and 
 commonly contain a leucine - rich repeat region ( lrr ) , a domain known to be 
 involved in protein - protein interactions .
a significant portion of the microarray cdna content used in this study was 
 derived from a library of rl6003 ( thatcher lr1 ) 
 leaf tissue collected 24 hpi with an incompatible p. triticina race . although 
 we have recently confirmed ( by cloning of lr1 in a separate study ) that lr1 was not 
 present on the array ( unpublished data ) , three of the differentially regulated 
 genes identified in our experiment nevertheless contained lrr domains , two of 
 which are of the nbs - lrr class of r - genes ( tae05012c12f , talr1134c01r ) .
there did not appear to be a coordinated 
 pattern of expression among these three genes at the time points tested 
 ( supplementary table 1 ) .
recently , a cyclophilin was found to be necessary for host - pathogen recognition in a. thaliana .
a cyclophilin - like protein ( tae25043b07r ) 
 was differentially regulated in our experiment and it shared an expression 
 profile very much like that of one of the identified nbs - lrr genes 
 ( tae05012c12f , figure 5 ) .
two other disease - associated genes were also identified , an mlo4-like gene ( talr1140f02a ) and a multidrug resistant - associated 
 protein 1-(mrp1- ) like gene 
 ( talr1143b08f ) .
they were generally repressed in this 
 treatment , with the strongest repression at 6 hpi . in the compatible interaction ,
initial 
 repression of these genes occurred at 3 and 12 hpi , but returned to near - basal 
 levels at the other time points ( figure 6 ) .
these include ubiquitin associated proteins ( tae05010c02f , tae15029b06r , 
 and tae012c02a ) , a ubiquitin conjugating enzyme ( tae25042d03a ) , and components 
 of ubiquitin ligase complexes ( tae05014d09f , tae15001b10r ) .
there did not appear to be a coordinated 
 expression pattern among the differentially expressed ubiquitination 
 components .
in addition to genes already mentioned , a large collection of genes with an 
 identified role in disease or stress response were significantly differentially 
 regulated between plants challenged with incompatible and compatible races of p. triticina .
components of the shikimate - phenylpropanoid 
 pathway , caffeoyl - coa - o - methyltransferase ( tae05029c04f ) , 3-deoxy - d - arabino - heptulosonate 
 7-phosphate synthase ( talr1013e06r ) , farnesyl - pyrophosphate synthetase 
 ( talr1161d04r ) , and udp - glucose glucosyltransferase 1 ( talr1148b05f ) , revealed 
 differential transcription patterns as did genes involved in signal 
 transduction ( myb - like transcription factor tae05012h07f , wrky11 transcription 
 factor talr1159c08f , calmodulin talr1021e02r ,   mitogen activated protein 
 kinase kinase talr1106b01r , and phosphatidylinositol-4-phosphate 5-kinase - like gene tae05025c08f ) , and 
 stress - associated genes such as heat shock proteins ( hps70tae05013a12r , hsp80tae05005c08a ) , 
 osmotic control genes ( betaine aldehyde dehydrogenase 
tae25007b01r , 
 s - adenosylhomocysteine hydrolase 2talr1174g05r , 
 clc - f chloride channel 
to gain 
 insight into the transcriptional changes that occur in incompatible and 
 compatible reactions of the wheat isogenic line , rl6003 , challenged with 
 avirulent and virulent races of the leaf rust pathogen p. triticina , we conducted a cdna microarray gene - profiling 
 experiment . in this study
, we used an 
 anova to identify 192 wheat genes that showed differential expression profiles 
 resulting from two different pathogen challenges .
our results revealed a 
 clear differentiation between incompatible and compatible interactions and led 
 to the identification of differentially regulated genes putatively involved in 
 defence reactions .
we examined four 
 time points early in the infection stages of virulent and avirulent races of p. triticinia on the wheat line 
 rl6003 . at 6 and 24
hpi , 46% and 40% of 
 the differentially expressed genes , respectively , were found to have different 
 degrees of change between the two pathogen treatments .
in contrast , only 11% and 10% of genes were 
 differentially expressed at 3 and 12 hpi . upon inoculation ,
p. triticinia urediospores germinate within hours on the wheat leaf surface , form a germ 
 tube , and grow until a stomatal guard cell is reached . at this point
, the fungus forms an appressorium 
 over the stoma , enters by force into the substomatal space , and creates a 
 substomatal vesicle .
the fungal 
 growth into the intercellular space occurs at 6 hpi   and coincides with the 
 highest differential gene expression in our study . at 24  hours ,
when 40% of the identified genes 
 reveal changes in expression , the fungus has already produced a septum which 
 separates the primary infection hypha from the newly formed haustorial mother 
 cell which initiates contact with adjacent mesophyll or epidermal cells as 
 shown by hu and rijkenberg .
those 
 authors have studied the infection process of p. triticinia on susceptible ( thatcher ) and resistant wheat lines 
 ( rl6040 which contains the lr19 leaf 
 rust resistance gene and rl6043 which contains the lr21 leaf rust resistance gene ) of wheat and no significant 
 differences were observed between the three lines during these early infection 
 events .
moreover , very similar results 
 were obtained during p. triticinia infection of nonhost species such as maize , oat , sorghum , and barley .
based on these studies , it is likely that the 
 growth of the two races of p. triticinia used on rl6003 in this study invoke very similar responses during the early 
 infection stages as that of the p. 
 triticinia races used by hu and rijkenberg on other resistant wheat lines 
 .
if this is the case , rl6003 was 
 responding to the leaf rust pathogen before any cellular penetration had 
 occurred .
this suggests that the plant 
 is able to perceive fungal movements on the leaf surface and past the guard 
 cells , possibly through cell membrane rearrangements or cell wall degradation 
 products caused by compounds excreted by the fungus .
our early stage data for differential 
 expression of host genes contrast the findings of caldo et al .   where no 
 differences in expression among barley genes were observed up to 16 hpi after 
 treatment with blumeria graminis f. 
 sp.hordei .
those authors found differential gene 
 expression only during the stages involving haustoria - plant epidermal membrane 
 contact .
however , differential plant 
 gene expression has been detected in other plant systems during the initial 
 hours of pathogen infection [ 19 , 
 40 , 41 ] .
this study is therefore the first to show such early differential gene 
 expressions in wheat using the pathogen p. 
 triticinia .
resistance of wheat genotypes to leaf rust pathogens can often be attributed to a 
 gene - for - gene interaction . in 
 general , its visible defence response , the hypersensitive response ( hr ) , 
 reflects a multitude of metabolic changes in affected cells [ 42 , 43 ] and 
 results in localized plant cell death . rl6003
challenged with an incompatible race of p. triticinia exhibits an hr ( figure 1 ) and it is believed to be 
 mediated through pathogen elicitor detection by lr1 .
one of the hallmarks of 
 the hr disease response is the generation of reactive oxygen species ( ros ) in 
 an oxidative burst across the plasmalemma of plants cells , perturbing the redox 
 state of cells and allowing controlled oxidation , which may have an immediate 
 antimicrobial effect [ 4345 ] . in 
 asparagus sprengeri mesophyll ,
hr 
 induction by a g - protein activator , mastoparan ( mp ) , resulted in significant 
 changes in photosynthesis .
the 
 authors demonstrated that during the elicitor - induced hr and oxidative burst , 
 light was stimulating the hr , that o2 evolution ceased due to a 
 disruption of photosystem ii ( psii ) and electron transport and that 
 photosynthesis was eventually inhibited by mp .
recent work to identify ests involved in a 
 rice - rice blast fungus interaction found that transcription of photosynthetic 
 genes , such as ribulose 1,5-bisphosphatase carboxylase ( rubisco ) , photosystem 
 i-(psi- ) associated genes , psii - associated genes , and chlorophyll a / b - binding 
 protein ( cab ) genes , were suppressed in both resistant and susceptible 
 interactions . in soybean , it has 
 been shown that in addition to known defence regulated genes , challenge with pseudomonas syringae induced a rapid 
 downregulation of photosynthesis and that this effect was hr specific 
 . in their study , zou et al
. 
 found that chloroplast - related genes had reduced expression 8 hours after 
 challenge and found a decrease in psii activity and an interruption in the 
 photosynthetic electron transport chain .
it is speculated that the over - reduction of psii components and an 
 interruption of electron transport result in electron leakage and the formation 
 of ros [ 19 , 41 , 46 ] .
our data also 
 identified photosynthesis - related genes that were downregulated during pathogen 
 challenge ( figure 3 ) .
six hours after inoculation , 
 we observed a coordinated decrease in transcription of these genes in the 
 resistant but not susceptible interactions .
if this decrease in photosynthetic gene expression is linked or due to 
 the hr as observed in other studies [ 40 , 41 , 46 , 47 ] , one may reasonably ask 
  what would be the purpose  ?
six hours 
 into the infection , the fungus has just entered the substomatal space and has 
 yet to directly invade a mesophyll or epidermal cell .
if the decrease in photosynthesis is used to 
 generate ros for an oxidative burst , then this burst does not likely have any 
 antimicrobial activity since p. 
 triticinia growth is identical in the first 24 hours of infection in 
 susceptible and resistance reactions with wheat .
one of the key enzymes involved in removing h2o2 , 
 apx , was identified as being differentially expressed in our study .
although apx has been shown to be 
 translationally inhibited during programmed cell death , based on its 
 expression profile in this study , we speculate that the cellular h2o2 concentration increased 6 hours after challenge with an incompatible race of p. triticinia and that apx levels were 
 likely increased in an attempt by the cell to curtail oxidative damage . if ros are generated at this stage , it is 
 more likely that they act on nonhost defence responses and have a role in 
 cellular fortification , intercellular signaling , or act to change the redox 
 state of the cell for regulatory purposes . additional experiments to measure ros generation during the wheat - rust 
 interaction would be needed to determine if an oxidative burst is occurring at 
 6 hpi .
the interruption 
 in the electron transport chain in the chloroplast during the hr is believed to 
 be mediated by a zn - dependent aaa - metalloprotease called ftsh . during stress , a component of psii , d1 ,
can 
 become damaged and has to be replaced by a newly synthesized d1 protein in 
 order to restore psii function .
ftsh 
 degrades damaged d1 , allowing for a functional version of the protein to take 
 its place [ 49 , 50 ] .
we identified a wheat 
 aaa - metalloprotease ftsh - like gene ( talr113410f ) with reduced expression in the 
 incompatible reactions at 3 and 6 hpi .
where the 
 expression of a chloroplast ftsh protein was reduced and electron transport 
 interrupted 6 hours following the induction of an hr by tobacco mosaic 
 virus .
those authors believed that the 
 disruption of photosynthesis perturbed cellular homeostasis ( possibly by 
 consuming reductive power ) which accelerated the hr .
they suggested that it was also possible that 
 reducing photosynthesis reduced sugar production , limiting the food source for 
 the invading pathogen .
it seems likely 
 that the downregulation of photosynthesis and the interruption of the electron 
 transport chain may be conserved features of the defence response to pathogen 
 challenges as it is shared by rice , tobacco , and soybean   and we 
 observed evidence of this mechanism in wheat .
plant responses to 
 biotic stress involve multiple interlinked regulatory pathways that transduce 
 suitable signals for efficient defence reactions 
 [ 33 , 34 , 
 42 , 51 , 
 52 ] .
in addition to the ros pathway described 
 earlier , our microarray profiling detected a number of genes in the shikimate 
 and phenylpropanoid pathways that were differentially regulated .
the shikimate pathway leads to the 
 biosynthesis of phenylpropanoid , phytoalexins ( terpenoids , flavonoids ) , lignin , 
 and salicylate which are main secondary metabolites of the disease resistance 
 machinery [ 18 , 53 
 56 ] .
our analysis identified that 
 3-deoxy - d - arabino heptulosonate-7-phosphate synthase , the entry point into the 
 shikimate pathway , was differentially regulated between the two pathogen treatments . at 6 hpi
, transcription was found to be 
 increased in the incompatible interaction but the greatest difference occurred 
 at 24 hpi when the expression was increased in the compatible interaction and 
 downregulated in the incompatible interaction .
other components downstream in the shikimate - phenylpropanoid pathway 
 were also differentially regulated , such as 3-beta hydroxysteroid 
 dehydrogenase - isomerase , which is involved in metabolism of alkaloids , 
 caffeoyl - coa o - methyltransferase , which is involved in lignin biosynthesis and 
 cell wall fortification , and farnesyl - pyrophosphate synthetase , which is 
 involved in the biosynthesis of terpenoid compounds .
all of the genes identified in the 
 shikimate - phenylpropanoid pathway had their strongest expression in plants 
 challenged with a compatible pathogen .
this may illustrate an important distinction in the lr1 pathosystem in which the plants involved in an incompatible 
 interaction dedicate cellular resources to redox systems , as evidenced by early 
 expression of ros enzymes and downregulating photosynthetic genes , while plants 
 in a compatible interaction trigger nonspecific defence responses , such as 
 cellular fortification and alterations in their secondary metabolism . in our data
, we 
 identified three genes that contain lrr domains , regions involved in 
 protein - protein interactions , and two of the three were of the nbs - lrr class , 
 with homology to putative r - genes , although neither of them encodes the lr1 gene ( unpublished data ) .
one of the nbs - lrr genes ( tae05012c12f ) 
 shares homology with the rice protein xa1 , which confers bacterial 
 blight resistance .
interestingly , 
 yoshimura et al .   showed that xa1 expression was induced by wounding and pathogen attack , which prompted the authors to speculate that   xa1 
 may be involved in enhancing the disease response to bacterial blight .
although it is not responsible for avr lr1 recognition , this xa1-like nbs - lrr gene may be activated to serve a similar purpose , which is the enhancement of the defence response ; it is possible that other , yet undescribed , fungal factors present in race bbb interact with this resistance - like gene .
plants in the incompatible interaction showed 
 a spike of induction of this gene at 6 hpi , which coincided with the expression of other defence systems genes .
it was 
 also recently found that a cyclophilin protein plays a role in an r - gene signaling pathway .
the bacterial effector , avrrpt2 from pseudomonas syringae ,   relies on an arabidopsis cyclophilin 
 to initiate its protease activity .
once activated , avrrpt2 cleaves rin4 , which is then recognized by the nbs - lrr 
 protein rps2 and triggers an incompatible response . in our study ,
a wheat cyclophilin had a 
 nearly identical expression pattern to that of the xa1-like gene ( figure 5 ) and may indicate that they operate in the same pathway and that this method of avr - gene activation described in a. thaliana exists in wheat .
the recognition of 
 invading pathogens activates a signal cascade that leads to a change in gene 
 expression .
a number of important components 
 in the transduction of stress and disease signals have been identified .
these include several mitogen - activated 
 protein ( map ) kinases , map kinase kinases , and transcription factors [ 58 , 59 ] .
our data revealed coordinated expression 
 among components involved in map pathway signaling , transcription factors , and 
 genes that have been found to be expressed during a disease response .
their coordinated expression may indicate 
 that these genes operate in the same signal transduction pathway .
plant defence 
 response to pathogen attack can also include the modification of proteins by 
 ubiquitin [ 60 , 61 ] .
many members of the 
 ubiquitination cascade were found differentially regulated in our experiment , 
 including ubiquitin activating enzymes , ubiquitin conjugating enzymes , and 
 components of scf ubiquitin ligase complexes . despite the lack of a unified expression profile among the members of 
 the pathway ,
it is clear that the process of protein modification by ubiquitin 
 plays a role in the disease response of wheat to races of p. triticinia .
there were a 
 number of genes identified in this study that have protein products regulated 
 by changes in the redox state of the cell : apx , glycolate oxidase , 
 rubisco activase , rubisco , plastid glucose-6-phosphate dehydrogenase , and 3-deoxy - d arabino 
 heptosonate-7 phosphate synthase .
it is likely that , in addition to regulating protein activity , the 
 reductive power of the cell plays an important role in regulating the 
 transcription of these genes , possibly through a feedback mechanism involving 
 an interruption in the photosynthetic apparatus .
we also observed that a number of genes were 
 differentially expressed from basal levels in both treatments but at different 
 time points . in the incompatible response , 
 these genes were differentially regulated relative to basal level at 6 hpi 
 while in the compatible response the transcriptional differences occurred at 12 
 hpi .
the reason for this difference is 
 unknown but it is tempting to speculate that the coordinated expression of 
 pathways at 6 hpi rather than simply the induction or suppression of defence 
 pathways at early stages of infection might determine the outcome of the 
 pathogenic interaction .
a microarray 
 experiment examining gene expression differences between compatible and 
 incompatible interactions of p. syringae and a. thaliana found that the 
 expression of genes in the early hours of the incompatible infection resembled 
 the expression of the same genes at much later stages in the compatible interaction 
 .
they stated that this may indicate 
 that incompatible and compatible interactions share signaling pathways but are 
 induced at different times .
to determine 
 if this is the case between wheat and leaf rust as well , additional expression 
 profiling experiments over a broader time span would need to be performed .
it is not known 
 that the gene expression changes that were measured at the early stages of 
 infection lead to the induction of hr .
it could be that the induction did not occur until later in the 
 infection process .
the possibility exists that this study did not measure 
 thatcher lr1 undergoing compatible 
 and incompatible reactions due to the presence or absence of the avr lr1 gene , but instead measured 
 differential expression due to other genetic difference that exists between 
 fungal races bbb and tjb .
however , given 
 that many of the genes identified in this study are differentially expressed 
 during the hr and gene - for - gene recognition responses in other systems , it is 
 likely that the start of the incompatible and compatible reactions due 
  to avr lr1 lr1 was being observed . taken together , this study allowed us to show that phenotypic differences between the 
 incompatible and compatible interactions in the wheat near - isogenic line 
 rl6003/p .
triticinia pathosystem are 
 also reflected by differential expression of mrna transcripts that occur very 
 early after infection .
enzymes involved 
 in photosynthesis and in the scavenging of reactive oxygen species , signal 
 transduction and ubiquitination , as well as those of the 
 shikimate - phenylpropanoid pathway , are strongly implicated as key determinants 
 in rl6003 metabolism against incompatible pathogenic fungi .
these findings indicate that synergistic and 
 conserved strategies are utilized by the incompatible wheat host to fight 
 against p. triticina .
 in this study , we detail the construction of a custom cdna spotted microarray containing 7728 wheat ests and the use of the array to identify host genes that are differentially expressed upon challenges with leaf rust fungal pathogens . 
 wheat cultivar rl6003 ( thatcher lr1 ) was inoculated with puccinia triticina virulence phenotypes bbb ( incompatible ) or tjb ( 7 - 2 ) ( compatible ) and sampled at four different time points ( 3 , 6 , 12 , and 24 hours ) after inoculation . 
 transcript expression levels relative to a mock treatment were measured . 
 one hundred ninety two genes were found to have significantly altered expression between the compatible and incompatible reactions . among those were genes involved in photosynthesis , the production of reactive oxygen species , ubiquitination , signal transduction , as well as in the shikimate / phenylpropanoid pathway . 
 these data indicate that various metabolic pathways are affected , some of which might be used by rl6003 to mount a coordinated defense against an incompatible fungal pathogen . 
1. INTRODUCTION
2. MATERIALS AND METHODS
3. RESULTS
4. DISCUSSIONS