BACKGROUND
plants interact with the environment in various ways and routinely face challenges from potential pathogens, but disease occurs only in limited cases as survival is a rule rather than an exception.
plants are sessile and unlike animals do not have mobile defender cells, instead they depend upon the innate immunity of each cell.
plants have preformed physical and chemical barriers and continuously produce antimicrobial compounds which are enough to deter most microbes, yet a pathogen may overcome these defenses and cause infection.
when a potential pathogen gets over these barriers and is recognized by plant cells as an invader, a rapid and coordinated induction of defense response by resistant plant prevents microbe colonization and disease development, often termed as incompatible interaction.
however if the plant is unable to recognize the pathogen or does not respond rapidly enough as in case of susceptible plants, disease spreads and is termed as compatible interaction.
recognition of the pathogen by plant triggers signal transduction cascades that leads to rapid defense mobilization  <cit> .
r-gene products have long been implicated as the receptors which directly  <cit>  or indirectly  <cit>  recognize pathogens and initiate defense response.
conversely studies have shown that there are two branches of immune system, one uses transmembrane pattern recognition receptors  that respond to pathogen associated molecular patterns  and the second, that acts largely inside the cell, uses nb-lrr type r-gene products  <cit> .
perception of an invader by host cell leads to activation of protein kinases or/and inhibition of protein phosphatases triggering the ca2+ influx which in turn leads to active oxygen species  generation, mapk activation, anion effluxes and plasma membrane depolarization  <cit> .
the prime target of such signal transduction is the cell nucleus, where modulation of numerous genes takes place to face the invasion.
the genes are coordinately activated in several waves  <cit> .
the products of immediately activated genes or primary response genes subsequently activate the secondary response genes  <cit> .
these subsequent transcriptional events reinforces and amplifies defense signals and results in production of antimicrobial metabolites, pathogenesis related proteins, enzymes of oxidative stress protection, stress related hormones, cell wall lignification and fortification and often hypersensitive response.
rice  is the major nutritional source for above  <dig> percent of the global and  <dig> percent of the asian population  <cit> .
it globally provides  <dig> percent of human per capita energy and  <dig> percent of per capita protein http://www.knowledgebank.irri.org.
but the crop yield obtained is greatly affected by various diseases of which blast, leaf blight and sheath blight are most devastating ones, resulting in a huge gap between the yield potential and the actual yield.
following blast, bacterial leaf blight of rice, caused by xanthomonas oryzae pv oryzae , is responsible for huge economic loss.
bacterial leaf blight is known to occur in all rice growing areas and is exceptionally severe in asia.
it is reported to have reduced annual production by as much as  <dig> percent in india and  <dig> percent in japan http://www.knowledgebank.irri.org.it has also been extensively studied as a model disease of rice to understand the host-pathogen interactions, bacterial pathogenesis and defense responses in monocotyledonous plants  <cit> .
the symptoms in adult plants appear as water-soaked yellowish stripes on leaf blades or starting at leaf tips which increase in length and width killing the infected leaves.
infected plants produce sterile and empty panicles and in severe cases the plant wilts and dies.
at seedling stage the disease totally eradicates the plants of wide areas leading to epidemic.
enormous effort has been put to develop resistant cultivars carrying major r-genes of which  <dig> have been identified till date  <cit> .
availability of genome sequences for both rice and xoo as well as continuing annotation projects has opened up the path for global expression studies of both contenders.
microarray technology has been excellently used to study constitutive and early defense responses in the concerned system.
previous studies have highlighted the complexity of the genetic networks involved in defense response.
ethylene and jasmonic acid as well as mapk pathways have been found to be important in case of rice-xoo interaction.
the previous studies report transcriptomic events at four hours or later after inoculation  <cit>  whereas considering the rapidity of plant defense response documented in other plant-pathogen systems  <cit>  and short generation time of xoo, it is expected that the bacterial pathogen induces host reprogramming even at prior time points.
moreover large accumulation of additional annotation data since the afore-mentioned studies is enough to justify a revisit to the problem.
in the present study, to best of our knowledge, an effort has been made for the first time to dissect the rice-bacterial interaction system at one hour after inoculation  in both resistant and susceptible hosts.
this time point was selected after deliberate consideration of the facts that xoo is known to reach early log phase in culture within one hour of growth  <cit>  and the method of inoculation used i.e.
the clipping method, deposits the pathogen directly in the infection court  <cit> .
in parsley and bean cell suspension cultures, the phenylpropanoid biosynthetic gene transcription rate was found to be maximum at around one hour when challenged with avirulent pathogen  <cit>  the present study delineates the early transcriptomic changes in response to pathogen attack in much detail and underlines the sophisticated regulatory mechanisms that are brought into play to combat the microbe invasion.
RESULTS
disease symptoms in iet <dig> and ir24
in fifty five days old plants inoculated with xanthomonas oryzae pv oryzae strain bxo <dig>  the symptoms first appear five days after inoculation  as yellowish lesions around the site of inoculation in both the cultivars.
in susceptible ir <dig> the whole leaf turned grayish yellow and dried up at  <dig> dai, lesions were visible on other leaves of the same plant as well.
in resistant iet <dig> at  <dig> dai the progression of lesion was limited to  <dig> +/-  <dig> cm from inoculation site whereas other leaves of the plant remained unaffected .
the symptoms in eighteen days old plants were also comparable with previously documented reports  <cit> .
microarray experiment and validation
agilent rice gene expression microarrays were used to examine differential transcript accumulation in resistant iet <dig> and susceptible ir <dig> cultivars at  <dig> hai with bxo <dig> or mock water treated control.
the number of transcripts found to be differentially expressed in resistant plants compared to susceptible ones after pathogen inoculation were  <dig>  amongst them  <dig> were found to be differentially expressed after mock treatment as well and were not taken into consideration for further analysis .
a remaining subset of  <dig> transcripts were taken to be differentially expressed due to bxo <dig> inoculation, of these  <dig> were found to be up-regulated and  <dig> down-regulated in iet <dig> as compared to ir <dig>  the microarray data has been submitted to arrayexpress and is available as e-mexp- <dig>
real-time qpcr was used to validate the microarray results.
fifteen genes were randomly selected from the differentially expressed genes.
the differential expressions of selected genes were verified by quantitative pcr.
both the up- and down-regulated genes showed same trend of expression as obtained by microarray when further analyzed by qpcr .
identification and annotation of differentially expressed transcripts
the transcripts were annotated with the help of rice annotation project database http://rapdb.dna.affrc.go.jp.
annotations could be assigned to  <dig> up- and  <dig> down-regulated transcripts.
on the basis of go functional categories transcripts were assorted into several groups.
the up-regulated transcripts were grouped into cell signaling , transcriptional factors , defense related , general stress related , transport , metabolic , structural  and miscellaneous  .
similarly down-regulated transcripts consisted of cell signaling , transcription factors , defense related , stress related , transport , metabolic , structural  and miscellaneous  .
interactions for  <dig> up-regulated  and  <dig> down-regulated  transcripts were mapped.
expression of modulated transcripts at different time points
the present  <dig> hai data set was compared to previously reported expressional studies done at  <dig> and  <dig> hai as well as in untreated plants-geo data set gse <dig>  <cit> .
amongst the  <dig> transcripts obtained from present study  <dig> showed similar trend in  <dig> or  <dig> hai data sets, further  <dig> showed greatest differential expression at  <dig> hai,  <dig> were constitutively up-regulated and  <dig> were constitutively down-regulated in all data sets .
constitutively up-regulated transcripts include wrky <dig>  dreb1b, chitinase precursor, hin <dig> and nb-arc domain containing proteins  while constitutively down-regulated transcripts include phospholipase a <dig> .
transcripts which were uniquely up-regulated at  <dig> hai include calmodulin like protein, nod  <dig> family protein and protein disulphide isomerase.
the comparison of expression data at different time points revealed that a plant maintains a continuous state of alertness as soon as it perceives a pathogen attack and coordinately modulates the expression of genes for the purpose.
while the differential expression of some genes i.e.
chitinase, wrky factors, dreb1b, phospholipase, nod family proteins may provide an advantage against pathogen, over-expression of nb-arc domain containing or calmodulin-like protein may help in rapid perception and signal transmission.
cell signaling
it was interesting to note that the largest up-regulated cluster consisted of  <dig> transcription factors and  <dig> cell signaling related proteins, transcripts which or the products of which may act as mediators to usher defense response.
in the corresponding down-regulated cluster  <dig> transcription factors and  <dig> signaling related proteins were found.
two receptor-like kinases  were found to be up-regulated , one of them bearing a lysm domain .
the lysm domains are found in a variety of peptidoglycan or chitin binding proteins and have been implicated in perception of rhizobial lipichitooligosaccaride signals  <cit>  and further elicitation of signals via its intercellular kinase domain  <cit> .
a transcript for nb-arc domain containing protein  was found to be highly up-regulated  as well.
nb-arc is an ancient highly conserved domain of a class of plant resistance proteins  <cit> .
it has a functional atpase domain and its nucleotide binding site is proposed to regulate activity of the r-protein in pathogen recognition.
serine/theonine protein kinases  have long been implicated to play a role in signaling processes concerned with self verses non-self recognition and disease resistance  <cit> .
a up-regulated stk was found to be similar to mapkkk <dig>  , which is known to be induced by pathogens  <cit> .
another mapk cascade initiating protein mapkkk <dig>   <cit>  as well as protein phosphatase 2c  or pp2c  a regulator of mapk pathway, known to be activated in stress  <cit> , was found to be up-regulated in the present case study, suggesting that there may be a remarkable fine tuning of mapk cascade at such early a time point.
calcium ion is the most important signal entity in cell, its importance is reflected in present work by differential regulation of transcripts of several associated proteins, up-regulation of a calmodulin-like protein , os12g <dig>  a calmodulin-binding protein-like protein and os06g <dig>  an ef-hand domain containing protein, similar to ccd <dig> .
studies in wheat cultured cells have revealed that ccd- <dig> mrna are strongly responsive to elicitors of snow mold and gene product ccd <dig> plays a role in elicitor provoked ca2+ mediated signal transduction  <cit> .
some ca2+ binding proteins os05g <dig>  a calcium binding protein and an ef-hand domain containing protein  were also found to be down-regulated indicating tight regulation of signaling pathways.
besides ca2+ flux transporters for several other ions across membrane were also stimulated.
these ion fluxes affect membrane potential which in turn affect uptake by other channels and activation of defense response  <cit> .
a net k+ eflux in elicitor treated cells is a common early response  <cit> .
in present data set a transcript similar to high affinity k+ uptake pump hak <dig>    <cit>  and k+ channel protein   were found to be down-regulated whereas a low to high affinity k+ pump kup <dig>   <cit>  is up-regulated implying a regulation of k+ uptake.
an additional complexity is introduced by the fact that k+ uptake is sensitive to ammonium transport and a transcript for amt2-like protein  an ammonium transporter has also been found to be up-regulated  <cit> .
proton influx is another important defense response and three h+ transporters were found to be up-regulated in present study , one of them was a proton-peptide symporter ptr <dig>  and other two were sugar-proton symporters stp <dig>  and plt <dig>  like proteins.
the sugar-h+ symporters probably serve dual purpose, they relocate h+ and sugars into infected cells, which behaves as a sink and draws carbon resources for energy consumption to put up defense on one hand  <cit>  while on the other hand modulate sugar signals  which are known to influence sar pathway  <cit> .
monosaccharide-h+ symportes in arabidopsis are rapidly induced by pathogenic elicitors  <cit> .
lipid signaling has emerged as an important component of stress signaling and phospatidic acid  is a key lipid second messenger.
pa is directly formed via activation of phospholipase d  and indirectly by phosphorylation of diacylglycerol  by dag kinase  or dgk   <cit> .
the dgk pathway has been known to be activated in defense response against pathogens within few hours of infection  <cit> , a transcript similar to pa generating dag kinase is upregulated in the present data set as well whereas a phospholipase a <dig> family protein  was down-regulated .
pa can be deacylated by phospholipase a <dig>  to produce lpa and free fatty acids , which are signaling compounds in plant responses to auxin  <cit> .
further a transcription factor auxin responsive factor  <dig> or arf <dig>  that binds to aux/iaa family proteins , a critical component in auxin signaling pathway, as well as an aux/iaa family protein  were found to be down-regulated  <cit> , incidentally another aux/iaa family protein  was found to be up-regulated .
this two-way modulation of aux/iaa family proteins is in agreement with previous findings.
aux/iaa genes were discovered based on their induction by auxin and some can promote auxin response, however many aux/iaa proteins actually inhibit auxin response  <cit>
the down-regulated cell signaling associated transcripts include two interesting genes a leucine-rich repeat  <dig> containing protein  and a remorin domain containing protein  .
lrr repeats occur in various proteins to provide structural framework for protein-protein interaction and when associated to kinase domain are known to function as receptors.
remorins are unique to plants and are ubiquitously expressed.
differential expression of remorins have been well documented in arabidopsis, interestingly most of the remorins were found to be down-regulated in case of bacterial-plant incompatible interactions  <cit> .
transcription factors
the host cell gears up its arsenal on receiving signals of pathogen attack through elaborate signal transduction network by modulating expression of several transcription factors.
these act as hubs that further modulate defense, hormone and growth related genes.
the up-regulated cluster consists of several transcription factors some of them are r2r <dig> type myb transcription factor  related to stress response  <cit> , myc7e  related to aba response, osnac protein , dreb1b, ethylene responsive factor  family protein , wrky <dig>  .
wrky <dig> is a member of the well documented wrky family related to regulation of many cellular processes including defense and is known in turn to be modulated by mapk cascade  <cit> .
osnac is induced by abiotic and biotic stress as well as aba, jasmonic acid and hydrogen peroxide  <cit>  and plays a role in activation of defensin  <cit> .
dreb1b responds to variety of stress thus inducing expression of pr genes  <cit> .
erf <dig> is induced by both ethylene and ja  <cit> .
it integrates ja and ethylene signaling  <cit>  and both signaling pathways are required simultaneously  <cit>  for erf <dig> induction.
the tight co-regulation of erf <dig> by both stress hormones is quite understandable considering the fact that of all the total number of defense related genes induced by ethylene and ja about 80% are erf <dig> mediated.
there are also reports of erf <dig> being under mapk cascade control mediated by ein <dig> and ein <dig>  <cit> .
the most important genes up-regulated by erf <dig> are basic chitinase, defensins and glutathione synthases  which have been found to be up-regulated in present study as well .
ethylene and ja play a pivotal role in plant defense, plants with impaired ethylene signal transmission exhibit susceptibility to necrotrophic bacterium  <cit> .
ethylene induces cell-wall strengthening, xylem occlusion response, phenylpropanoid derived phytoalexin production, pr-genes including beta-glucanase and chitinase  <cit>  both of which have been found to be up-regulated in the present study.
while ethylene and ja have synergistic functions aba is antagonistic to both, it interferes at different levels with ethylene and ja signaling  <cit> .
the transcription factors found to be down-regulated were auxin response factor  <dig>  aux/iaa and b <dig> transcription factor .
all three are growth related and are expressed in all organs, auxin response factor  <dig> is required for normal growth  <cit>  and b <dig> belongs to a family of plant transcription factors with various roles in plant development.
metabolism
secondary metabolites play an important role in plant defense  <cit> .
the present study also documents similar findings; there was up-regulation of seven genes related to flavanoid biosynthesis , three cytochrome p <dig> monoxygenases , a flavanoid 3-monoxygenase , a flavanone 3-hydroxylase-like protein   <cit>  and a glutathione s-transferase-gst  <dig>  .
gst proteins are known to act as escort proteins in flavanoid transport.
wd- <dig> repeat like domain containing protein  was down regulated .
wd- <dig> repeat containing protein have been found to be necessary for anthocyanin biosynthesis at the dfr  step in arabidopsis leaves but does not seem to affect upstream genes involved in flavanoid bio-synthesis.
it appears that flavanoid biosynthesis pathway is modulated to produce excess flavanoides rather than anthocyanins.
the flavanoides may produce lignins to strengthen cell wall or phytoalexins the classical anti-microbial plant compound  <cit> .
transcripts for several well studied pathogen induced genes were found to be up-regulated these include: a chitinase , a chitinase precursor oschib , an aspartic protease os11g <dig>  as well as two harpin-induced  <dig>  domain containing proteins  .
chitinases are induced by environmental stress and considered to play a role in active or passive defense  <cit> .
aspartic protease is a major family of protease enzymes, expression of an aspartic protease was found to be up-regulated in case of incompatible interaction between potato and the fungus phytophthora infestans  <cit> .
hin <dig> is induced by bacterial effector, harpin through mapk activity  <cit> .
genes related to lipid metabolism were also found to differentially regulated, a lipase   was up-regulated whereas a gene related to lipid biosynthesis, an acyl carrier protein  was down-regulated .
the cell lipid metabolism perhaps is so diverted to provide ingredients and energy for mounting defense respose.
a lipoxygenases , chloroplast precursor of lipoxygenase lox <dig> , was also found to be significantly up-regulated.
lipoxygenases are key enzymes of lipid metabolism and ja biosynthesis  <cit> .
lox <dig> is required for wound induced ja accumulation and is involved in early defense response to pathogens  <cit> .
the expression of lox <dig> in turn is enhanced by ja through a positive feedback loop.
the up-regulation of ja producing enzyme indicates the important role played by ja signaling in the xanthomonas-rice incompatible interaction.
along with chloroplastid lipoxygenase other plastidial proteins including tic <dig>  and a protein similar to 1-deoxy-d-xylulose 5-phosphate synthase  <dig> precursor or dxps  were up-regulated  as well.
tic <dig> is a nadph-dependent dehydrogenase and its dehydrogenase activity is affected by calmodulin.
it is associated with tic translocon on the stomatal side of the plastidial inner envelope.
it may serve as a switch to differentially integrate redox signals from inside of chloroplast with calcium signals outside and influence the activity and/or specificity of tic translocon  <cit> .
defense involves induction as well as repression of several proteins.
in order to meet the demand the cell modulates several components of transcriptional, translational and post-translational modification machinery.
present work documents up-regulation of nuclease <dig>  a ribosomal t <dig> family protein , ribosomal protein s8e domain containing protein , ribosomal protein s <dig> domain containing protein  and a protein related to arp <dig>  .
arp <dig> is a component of chromatin modifying complex implicated in maintaining state of gene activation  <cit> .
while several others were down regulated , these included tfiih domain containing protein , methyltransferase type  <dig> domain containing protein , rna-binding protein 8a-like protein , pre-mrna slicing factor srp <dig> , ribosomal protein s <dig>  and an adenine salvage related protein aprt <dig> .
transcripts for f-box/lrr-repeat max <dig> homolog  and ring-h <dig> finger protein atl <dig>  were found to be up-regulated.
f-box/lrr-repeat proteins function as substrate recruiting subunit of scf-type ubiquitin e <dig> ligases  <cit> .
atl is a multigenic family of putative ring-type e <dig> ubiquitin ligases  <cit> , the specificity determinants that mediate the transfer of ubiquitin to the ε-amino group of target proteins resulting in mono-ubiquitination, additional ubiquitin moieties are transferred to the target protein by e <dig>  a multiubiquitin chain assembly factor  <cit> .
while multi-ubiquitination generally tag proteins for degradation, mono-ubiquitination of a target results in non-proteolytic events such as changes in protein activity, histone modification, localization or protein-protein interactions  <cit> .
incidentally beta  <dig> subunit of 20s proteosome , the core complex of the 26s proteasome, was also found to be up-regulated implying modulation of ubiquitin mediated protein degradation.
protein transport apparently has also been affected, the relocation of proteins to new sites for defense was evident by the down-regulation of b <dig> protein   involved in peptide transport from er to golgi and up-regulation of a dnaj like protein , two peptide disulphide isomerases  like proteins  and a peptide-proton symporter ptr <dig> .
dnaj and pdi are molecular chaperones and rapid induction of pdi in wheat after fungal inoculation during early response has been previously documented  <cit> .
CONCLUSIONS
although massive efforts have been put in past to annotate and characterize gene functions, it was difficult to assign role to many differentially expressed transcripts due to lack of information.
moreover numerous transcripts have been annotated as proteins containing domains having diverse functional roles but their specific roles remain elusive.
nevertheless through analysis of present dataset and annotations of transcripts differentially expressed at  <dig> hai it was found that as the plant faces the pathogenic challenge it suspends its growth till it can spare the resources, thus there was up-regulation of defense related genes and loss of growth related ones.
in resistant plant host cell recognizes the pathogen through plasma membrane or/and cytoplasm located receptors  and initiates diverse signaling pathways including mapk cascade, ca2+ signaling, ionic fluxes, lipid and sugar signaling.
these signals are ultimately transduced to nucleus resulting in up-regulation of several defense and stress related transcription factors and down-regulation of growth and development related ones.
the transcription factors in turn modulate the expression of down-stream genes resulting in metabolic modulations.
thus ethylene and jasmonic acid responses are activated while auxin signaling is repressed .
protein turn-out and trafficking are adjusted to churn out a proteome suited to serve the defensive needs.
primary metabolites are channeled to provide energy and raw materials for defense and secondary metabolism is diverted to produce pathogen deterrents .
amongst the modulated transcripts the receptor like proteins i.e.
lysm and nb-arc domain containing proteins are attractive candidates as r-genes and hence also for transgenic modifications for resistance development.
a nod  <dig> family protein was another interesting transcript found to be up-regulated.
this family of proteins has been implicated in nodule development but their role in pathogenesis remains elusive, in depth studies may reveal the specific advantage that this protein may provide to host cells in host-pathogen interaction.
the gene modulations undertaken by plant cells at such early a stage highlight the ability of plant cells to rapidly mount a complex defense response.
the resistant plant unleashes a diverse arsenal in a highly coordinated manner, no wonder that the susceptible plant which lags behind falls to disease.

 plants interact with the environment in various ways and routinely face challenges from potential pathogens, but disease occurs only in limited cases as survival is a rule rather than an exception. plants are sessile and unlike animals do not have mobile defender cells, instead they depend upon the innate immunity of each cell. plants have preformed physical and chemical barriers and continuously produce antimicrobial compounds which are enough to deter most microbes, yet a pathogen may overcome these defenses and cause infection. when a potential pathogen gets over these barriers and is recognized by plant cells as an invader, a rapid and coordinated induction of defense response by resistant plant prevents microbe colonization and disease development, often termed as incompatible interaction. however if the plant is unable to recognize the pathogen or does not respond rapidly enough as in case of susceptible plants, disease spreads and is termed as compatible interaction. recognition of the pathogen by plant triggers signal transduction cascades that leads to rapid defense mobilization. r-gene products have long been implicated as the receptors which directly or indirectly recognize pathogens and initiate defense response. conversely studies have shown that there are two branches of immune system, one uses transmembrane pattern recognition receptors that respond to pathogen associated molecular patterns and the second, that acts largely inside the cell, uses nb-lrr type r-gene products. perception of an invader by host cell leads to activation of protein kinases or/and inhibition of protein phosphatases triggering the ca2+ influx which in turn leads to active oxygen species generation, mapk activation, anion effluxes and plasma membrane depolarization. the prime target of such signal transduction is the cell nucleus, where modulation of numerous genes takes place to face the invasion. the genes are coordinately activated in several waves. the products of immediately activated genes or primary response genes subsequently activate the secondary response genes. these subsequent transcriptional events reinforces and amplifies defense signals and results in production of antimicrobial metabolites, pathogenesis related proteins, enzymes of oxidative stress protection, stress related hormones, cell wall lignification and fortification and often hypersensitive response. rice is the major nutritional source for above percent of the global and percent of the asian population. it globally provides percent of human per capita energy and percent of per capita protein http://www.knowledgebank.irri.org. but the crop yield obtained is greatly affected by various diseases of which blast, leaf blight and sheath blight are most devastating ones, resulting in a huge gap between the yield potential and the actual yield. following blast, bacterial leaf blight of rice, caused by xanthomonas oryzae pv oryzae, is responsible for huge economic loss. bacterial leaf blight is known to occur in all rice growing areas and is exceptionally severe in asia. it is reported to have reduced annual production by as much as percent in india and percent in japan http://www.knowledgebank.irri.org.it has also been extensively studied as a model disease of rice to understand the host-pathogen interactions, bacterial pathogenesis and defense responses in monocotyledonous plants. the symptoms in adult plants appear as water-soaked yellowish stripes on leaf blades or starting at leaf tips which increase in length and width killing the infected leaves. infected plants produce sterile and empty panicles and in severe cases the plant wilts and dies. at seedling stage the disease totally eradicates the plants of wide areas leading to epidemic. enormous effort has been put to develop resistant cultivars carrying major r-genes of which have been identified till date. availability of genome sequences for both rice and xoo as well as continuing annotation projects has opened up the path for global expression studies of both contenders. microarray technology has been excellently used to study constitutive and early defense responses in the concerned system. previous studies have highlighted the complexity of the genetic networks involved in defense response. ethylene and jasmonic acid as well as mapk pathways have been found to be important in case of rice-xoo interaction. the previous studies report transcriptomic events at four hours or later after inoculation whereas considering the rapidity of plant defense response documented in other plant-pathogen systems and short generation time of xoo, it is expected that the bacterial pathogen induces host reprogramming even at prior time points. moreover large accumulation of additional annotation data since the afore-mentioned studies is enough to justify a revisit to the problem. in the present study, to best of our knowledge, an effort has been made for the first time to dissect the rice-bacterial interaction system at one hour after inoculation in both resistant and susceptible hosts. this time point was selected after deliberate consideration of the facts that xoo is known to reach early log phase in culture within one hour of growth and the method of inoculation used i.e. the clipping method, deposits the pathogen directly in the infection court. in parsley and bean cell suspension cultures, the phenylpropanoid biosynthetic gene transcription rate was found to be maximum at around one hour when challenged with avirulent pathogen the present study delineates the early transcriptomic changes in response to pathogen attack in much detail and underlines the sophisticated regulatory mechanisms that are brought into play to combat the microbe invasion.  disease symptoms in iet and ir24 in fifty five days old plants inoculated with xanthomonas oryzae pv oryzae strain bxo the symptoms first appear five days after inoculation as yellowish lesions around the site of inoculation in both the cultivars. in susceptible ir the whole leaf turned grayish yellow and dried up at dai, lesions were visible on other leaves of the same plant as well. in resistant iet at dai the progression of lesion was limited to +/- cm from inoculation site whereas other leaves of the plant remained unaffected. the symptoms in eighteen days old plants were also comparable with previously documented reports. microarray experiment and validation agilent rice gene expression microarrays were used to examine differential transcript accumulation in resistant iet and susceptible ir cultivars at hai with bxo or mock water treated control. the number of transcripts found to be differentially expressed in resistant plants compared to susceptible ones after pathogen inoculation were amongst them were found to be differentially expressed after mock treatment as well and were not taken into consideration for further analysis. a remaining subset of transcripts were taken to be differentially expressed due to bxo inoculation, of these were found to be up-regulated and down-regulated in iet as compared to ir the microarray data has been submitted to arrayexpress and is available as e-mexp- real-time qpcr was used to validate the microarray results. fifteen genes were randomly selected from the differentially expressed genes. the differential expressions of selected genes were verified by quantitative pcr. both the up- and down-regulated genes showed same trend of expression as obtained by microarray when further analyzed by qpcr. identification and annotation of differentially expressed transcripts the transcripts were annotated with the help of rice annotation project database http://rapdb.dna.affrc.go.jp. annotations could be assigned to up- and down-regulated transcripts. on the basis of go functional categories transcripts were assorted into several groups. the up-regulated transcripts were grouped into cell signaling, transcriptional factors, defense related, general stress related, transport, metabolic, structural and miscellaneous. similarly down-regulated transcripts consisted of cell signaling, transcription factors, defense related, stress related, transport, metabolic, structural and miscellaneous. interactions for up-regulated and down-regulated transcripts were mapped. expression of modulated transcripts at different time points the present hai data set was compared to previously reported expressional studies done at and hai as well as in untreated plants-geo data set gse. amongst the transcripts obtained from present study showed similar trend in or hai data sets, further showed greatest differential expression at hai, were constitutively up-regulated and were constitutively down-regulated in all data sets. constitutively up-regulated transcripts include wrky dreb1b, chitinase precursor, hin and nb-arc domain containing proteins while constitutively down-regulated transcripts include phospholipase a. transcripts which were uniquely up-regulated at hai include calmodulin like protein, nod family protein and protein disulphide isomerase. the comparison of expression data at different time points revealed that a plant maintains a continuous state of alertness as soon as it perceives a pathogen attack and coordinately modulates the expression of genes for the purpose. while the differential expression of some genes i.e. chitinase, wrky factors, dreb1b, phospholipase, nod family proteins may provide an advantage against pathogen, over-expression of nb-arc domain containing or calmodulin-like protein may help in rapid perception and signal transmission. cell signaling it was interesting to note that the largest up-regulated cluster consisted of transcription factors and cell signaling related proteins, transcripts which or the products of which may act as mediators to usher defense response. in the corresponding down-regulated cluster transcription factors and signaling related proteins were found. two receptor-like kinases were found to be up-regulated, one of them bearing a lysm domain. the lysm domains are found in a variety of peptidoglycan or chitin binding proteins and have been implicated in perception of rhizobial lipichitooligosaccaride signals and further elicitation of signals via its intercellular kinase domain. a transcript for nb-arc domain containing protein was found to be highly up-regulated as well. nb-arc is an ancient highly conserved domain of a class of plant resistance proteins. it has a functional atpase domain and its nucleotide binding site is proposed to regulate activity of the r-protein in pathogen recognition. serine/theonine protein kinases have long been implicated to play a role in signaling processes concerned with self verses non-self recognition and disease resistance. a up-regulated stk was found to be similar to mapkkk, which is known to be induced by pathogens. another mapk cascade initiating protein mapkkk as well as protein phosphatase 2c or pp2c a regulator of mapk pathway, known to be activated in stress, was found to be up-regulated in the present case study, suggesting that there may be a remarkable fine tuning of mapk cascade at such early a time point. calcium ion is the most important signal entity in cell, its importance is reflected in present work by differential regulation of transcripts of several associated proteins, up-regulation of a calmodulin-like protein, os12g a calmodulin-binding protein-like protein and os06g an ef-hand domain containing protein, similar to ccd. studies in wheat cultured cells have revealed that ccd- mrna are strongly responsive to elicitors of snow mold and gene product ccd plays a role in elicitor provoked ca2+ mediated signal transduction. some ca2+ binding proteins os05g a calcium binding protein and an ef-hand domain containing protein were also found to be down-regulated indicating tight regulation of signaling pathways. besides ca2+ flux transporters for several other ions across membrane were also stimulated. these ion fluxes affect membrane potential which in turn affect uptake by other channels and activation of defense response. a net k+ eflux in elicitor treated cells is a common early response. in present data set a transcript similar to high affinity k+ uptake pump hak and k+ channel protein were found to be down-regulated whereas a low to high affinity k+ pump kup is up-regulated implying a regulation of k+ uptake. an additional complexity is introduced by the fact that k+ uptake is sensitive to ammonium transport and a transcript for amt2-like protein an ammonium transporter has also been found to be up-regulated. proton influx is another important defense response and three h+ transporters were found to be up-regulated in present study, one of them was a proton-peptide symporter ptr and other two were sugar-proton symporters stp and plt like proteins. the sugar-h+ symporters probably serve dual purpose, they relocate h+ and sugars into infected cells, which behaves as a sink and draws carbon resources for energy consumption to put up defense on one hand while on the other hand modulate sugar signals which are known to influence sar pathway. monosaccharide-h+ symportes in arabidopsis are rapidly induced by pathogenic elicitors. lipid signaling has emerged as an important component of stress signaling and phospatidic acid is a key lipid second messenger. pa is directly formed via activation of phospholipase d and indirectly by phosphorylation of diacylglycerol by dag kinase or dgk. the dgk pathway has been known to be activated in defense response against pathogens within few hours of infection, a transcript similar to pa generating dag kinase is upregulated in the present data set as well whereas a phospholipase a family protein was down-regulated. pa can be deacylated by phospholipase a to produce lpa and free fatty acids, which are signaling compounds in plant responses to auxin. further a transcription factor auxin responsive factor or arf that binds to aux/iaa family proteins, a critical component in auxin signaling pathway, as well as an aux/iaa family protein were found to be down-regulated, incidentally another aux/iaa family protein was found to be up-regulated. this two-way modulation of aux/iaa family proteins is in agreement with previous findings. aux/iaa genes were discovered based on their induction by auxin and some can promote auxin response, however many aux/iaa proteins actually inhibit auxin response the down-regulated cell signaling associated transcripts include two interesting genes a leucine-rich repeat containing protein and a remorin domain containing protein. lrr repeats occur in various proteins to provide structural framework for protein-protein interaction and when associated to kinase domain are known to function as receptors. remorins are unique to plants and are ubiquitously expressed. differential expression of remorins have been well documented in arabidopsis, interestingly most of the remorins were found to be down-regulated in case of bacterial-plant incompatible interactions. transcription factors the host cell gears up its arsenal on receiving signals of pathogen attack through elaborate signal transduction network by modulating expression of several transcription factors. these act as hubs that further modulate defense, hormone and growth related genes. the up-regulated cluster consists of several transcription factors some of them are r2r type myb transcription factor related to stress response, myc7e related to aba response, osnac protein, dreb1b, ethylene responsive factor family protein, wrky. wrky is a member of the well documented wrky family related to regulation of many cellular processes including defense and is known in turn to be modulated by mapk cascade. osnac is induced by abiotic and biotic stress as well as aba, jasmonic acid and hydrogen peroxide and plays a role in activation of defensin. dreb1b responds to variety of stress thus inducing expression of pr genes. erf is induced by both ethylene and ja. it integrates ja and ethylene signaling and both signaling pathways are required simultaneously for erf induction. the tight co-regulation of erf by both stress hormones is quite understandable considering the fact that of all the total number of defense related genes induced by ethylene and ja about 80% are erf mediated. there are also reports of erf being under mapk cascade control mediated by ein and ein. the most important genes up-regulated by erf are basic chitinase, defensins and glutathione synthases which have been found to be up-regulated in present study as well. ethylene and ja play a pivotal role in plant defense, plants with impaired ethylene signal transmission exhibit susceptibility to necrotrophic bacterium. ethylene induces cell-wall strengthening, xylem occlusion response, phenylpropanoid derived phytoalexin production, pr-genes including beta-glucanase and chitinase both of which have been found to be up-regulated in the present study. while ethylene and ja have synergistic functions aba is antagonistic to both, it interferes at different levels with ethylene and ja signaling. the transcription factors found to be down-regulated were auxin response factor aux/iaa and b transcription factor. all three are growth related and are expressed in all organs, auxin response factor is required for normal growth and b belongs to a family of plant transcription factors with various roles in plant development. metabolism secondary metabolites play an important role in plant defense. the present study also documents similar findings; there was up-regulation of seven genes related to flavanoid biosynthesis, three cytochrome p monoxygenases, a flavanoid 3-monoxygenase, a flavanone 3-hydroxylase-like protein and a glutathione s-transferase-gst. gst proteins are known to act as escort proteins in flavanoid transport. wd- repeat like domain containing protein was down regulated. wd- repeat containing protein have been found to be necessary for anthocyanin biosynthesis at the dfr step in arabidopsis leaves but does not seem to affect upstream genes involved in flavanoid bio-synthesis. it appears that flavanoid biosynthesis pathway is modulated to produce excess flavanoides rather than anthocyanins. the flavanoides may produce lignins to strengthen cell wall or phytoalexins the classical anti-microbial plant compound. transcripts for several well studied pathogen induced genes were found to be up-regulated these include: a chitinase, a chitinase precursor oschib, an aspartic protease os11g as well as two harpin-induced domain containing proteins. chitinases are induced by environmental stress and considered to play a role in active or passive defense. aspartic protease is a major family of protease enzymes, expression of an aspartic protease was found to be up-regulated in case of incompatible interaction between potato and the fungus phytophthora infestans. hin is induced by bacterial effector, harpin through mapk activity. genes related to lipid metabolism were also found to differentially regulated, a lipase was up-regulated whereas a gene related to lipid biosynthesis, an acyl carrier protein was down-regulated. the cell lipid metabolism perhaps is so diverted to provide ingredients and energy for mounting defense respose. a lipoxygenases, chloroplast precursor of lipoxygenase lox, was also found to be significantly up-regulated. lipoxygenases are key enzymes of lipid metabolism and ja biosynthesis. lox is required for wound induced ja accumulation and is involved in early defense response to pathogens. the expression of lox in turn is enhanced by ja through a positive feedback loop. the up-regulation of ja producing enzyme indicates the important role played by ja signaling in the xanthomonas-rice incompatible interaction. along with chloroplastid lipoxygenase other plastidial proteins including tic and a protein similar to 1-deoxy-d-xylulose 5-phosphate synthase precursor or dxps were up-regulated as well. tic is a nadph-dependent dehydrogenase and its dehydrogenase activity is affected by calmodulin. it is associated with tic translocon on the stomatal side of the plastidial inner envelope. it may serve as a switch to differentially integrate redox signals from inside of chloroplast with calcium signals outside and influence the activity and/or specificity of tic translocon. defense involves induction as well as repression of several proteins. in order to meet the demand the cell modulates several components of transcriptional, translational and post-translational modification machinery. present work documents up-regulation of nuclease a ribosomal t family protein, ribosomal protein s8e domain containing protein, ribosomal protein s domain containing protein and a protein related to arp. arp is a component of chromatin modifying complex implicated in maintaining state of gene activation. while several others were down regulated, these included tfiih domain containing protein, methyltransferase type domain containing protein, rna-binding protein 8a-like protein, pre-mrna slicing factor srp, ribosomal protein s and an adenine salvage related protein aprt. transcripts for f-box/lrr-repeat max homolog and ring-h finger protein atl were found to be up-regulated. f-box/lrr-repeat proteins function as substrate recruiting subunit of scf-type ubiquitin e ligases. atl is a multigenic family of putative ring-type e ubiquitin ligases, the specificity determinants that mediate the transfer of ubiquitin to the ε-amino group of target proteins resulting in mono-ubiquitination, additional ubiquitin moieties are transferred to the target protein by e a multiubiquitin chain assembly factor. while multi-ubiquitination generally tag proteins for degradation, mono-ubiquitination of a target results in non-proteolytic events such as changes in protein activity, histone modification, localization or protein-protein interactions. incidentally beta subunit of 20s proteosome, the core complex of the 26s proteasome, was also found to be up-regulated implying modulation of ubiquitin mediated protein degradation. protein transport apparently has also been affected, the relocation of proteins to new sites for defense was evident by the down-regulation of b protein involved in peptide transport from er to golgi and up-regulation of a dnaj like protein, two peptide disulphide isomerases like proteins and a peptide-proton symporter ptr. dnaj and pdi are molecular chaperones and rapid induction of pdi in wheat after fungal inoculation during early response has been previously documented.  although massive efforts have been put in past to annotate and characterize gene functions, it was difficult to assign role to many differentially expressed transcripts due to lack of information. moreover numerous transcripts have been annotated as proteins containing domains having diverse functional roles but their specific roles remain elusive. nevertheless through analysis of present dataset and annotations of transcripts differentially expressed at hai it was found that as the plant faces the pathogenic challenge it suspends its growth till it can spare the resources, thus there was up-regulation of defense related genes and loss of growth related ones. in resistant plant host cell recognizes the pathogen through plasma membrane or/and cytoplasm located receptors and initiates diverse signaling pathways including mapk cascade, ca2+ signaling, ionic fluxes, lipid and sugar signaling. these signals are ultimately transduced to nucleus resulting in up-regulation of several defense and stress related transcription factors and down-regulation of growth and development related ones. the transcription factors in turn modulate the expression of down-stream genes resulting in metabolic modulations. thus ethylene and jasmonic acid responses are activated while auxin signaling is repressed. protein turn-out and trafficking are adjusted to churn out a proteome suited to serve the defensive needs. primary metabolites are channeled to provide energy and raw materials for defense and secondary metabolism is diverted to produce pathogen deterrents. amongst the modulated transcripts the receptor like proteins i.e. lysm and nb-arc domain containing proteins are attractive candidates as r-genes and hence also for transgenic modifications for resistance development. a nod family protein was another interesting transcript found to be up-regulated. this family of proteins has been implicated in nodule development but their role in pathogenesis remains elusive, in depth studies may reveal the specific advantage that this protein may provide to host cells in host-pathogen interaction. the gene modulations undertaken by plant cells at such early a stage highlight the ability of plant cells to rapidly mount a complex defense response. the resistant plant unleashes a diverse arsenal in a highly coordinated manner, no wonder that the susceptible plant which lags behind falls to disease. 
xanthomonas oryzae pv oryzae is a devastating pathogen of rice and has been extensively studied as a model pathogen of monocotyledons. moreover the past endeavors have addressed the middle or late defense responses of the rice plant whereas in the present study an attempt has been made to investigate the early defense responses taking place immediately after inoculation. expressional data and interaction maps were used to develop a hypothetical scheme of the molecular events taking place during early defense response. moreover adjustments in expression levels of components of primary as well as secondary metabolism, protein trafficking and turnout were apparent, highlighting the complexity of defense response. in resistant plants as compared to susceptible ones genes were found to be differentially expressed. components of the calcium and lipid signaling as well as mapk cascade were modulated, by signals from surface receptors and cytosolic r-proteins, to arouse jasmonic acid and ethylene signaling and suppress auxin signaling through various transcription factors. expressional studies in both the contenders have been undertaken in past to understand the molecular mechanism underlying the compatible and incompatible interactions in the pathosystem. network analysis with the differential transcripts showed up-regulation of major clusters of cell signaling proteins and transcription factors while growth and basal metabolic components were largely found to be down-regulated. abscisic acid modulation was also evident through the expression regulation of transcription factors involved with its functions.
 xanthomonas oryzae pv oryzae is a devastating pathogen of rice and has been extensively studied as a model pathogen of monocotyledons. expressional studies in both the contenders have been undertaken in past to understand the molecular mechanism underlying the compatible and incompatible interactions in the pathosystem. continuous update on database and gene annotations necessitates constant updating on the roles of the new entities as well as reinterpretation of regulations of the previous ones. moreover the past endeavors have addressed the middle or late defense responses of the rice plant whereas in the present study an attempt has been made to investigate the early defense responses taking place immediately after inoculation.  microarray was used to study the transcriptional modulations in eighteen days old rice seedling leaves of both susceptible and resistant genotypes one hour after inoculation. in resistant plants as compared to susceptible ones genes were found to be differentially expressed. annotations could be assigned to up- and down-regulated transcripts and gene interaction maps were generated for transcripts. expressional data and interaction maps were used to develop a hypothetical scheme of the molecular events taking place during early defense response. network analysis with the differential transcripts showed up-regulation of major clusters of cell signaling proteins and transcription factors while growth and basal metabolic components were largely found to be down-regulated.  this study provides an understanding of the early defense signaling in rice cells. components of the calcium and lipid signaling as well as mapk cascade were modulated, by signals from surface receptors and cytosolic r-proteins, to arouse jasmonic acid and ethylene signaling and suppress auxin signaling through various transcription factors. abscisic acid modulation was also evident through the expression regulation of transcription factors involved with its functions. moreover adjustments in expression levels of components of primary as well as secondary metabolism, protein trafficking and turnout were apparent, highlighting the complexity of defense response. 