BACKGROUND
the rice pathogens xanthomonas oryzae pathovar oryzae  and xanthomonas oryzae pathovar oryzicola  cause economically significant disease in many rice-growing regions of the world  <cit> .
xoo invades rice vascular tissue to cause bacterial leaf blight, whereas xoc colonizes the mesophyll parenchyma tissue to cause bacterial leaf streak.
xoo gains access to the xylem through wounds or natural openings such as hydathodes, while xoc, in contrast, enters the leaf mainly through stomata  <cit> .
xoo and xoc are closely related, infect the same host, and are often both established in the same rice fields.
the complete genome sequences of japanese xoo strain t <dig>  and korean xoo strain kacc <dig> have been published  <cit> .
the genome sequences of a third xoo strain, philippine strain pxo99a, and a strain of xoc, philippine strain bls <dig>  have recently been completed and are also publicly available, through the comprehensive microbial resource .
the genomes of xoo and xoc strains are similar with respect to size, % g+c, and gene content, but show several inversions and rearrangements and some indels relative to one another .
these bacteria constitute an excellent comparative model for understanding determinants of tissue specificity in plant-bacterial interactions.
defining differences in gene expression and gene regulation between xoo and xoc is an important step toward that goal.
dna microarray technology makes it possible to monitor the expression of thousands of genes simultaneously.
microarrays can be of two general types: 1) arrays based on in situ synthesis of oligonucleotide probes, using photochemical techniques or an ink-jet oligonucleotide synthesizer  <cit>  and 2) spotted arrays, consisting of presynthesized dna molecules or oligomers deposited onto glass slides or filter membranes  <cit> .
spotted arrays are generally less costly to produce, and because they are spotted rather than synthesized from a template, they constitute a highly flexible design platform.
currently, oligonucleotide- or amplicon-spotted microarrays representing the whole or partial genomes of the following plant pathogenic bacteria are available: pseudomonas syringae, ralstonia solanacearum, xanthomonas axonopodis, xanthomonas campestris, and xylella fastidiosa  <cit> .
these arrays, enabled by whole-genome sequence availability, have been used to study responses to environmental cues such as heat shock  <cit> , and to probe gene expression patterns related to pathogenesis  <cit> .
they have also been used to assess genome diversity of isolates of a particular organism by comparative genome hybridization  <cit> .
based on the available genome sequences, we constructed a combined xoo and xoc whole genome microarray for both pathovars of x. oryzae  that contains  <dig>  distinct 50– <dig> mer oligonucleotides, representing sequences from  <dig>  genes shared by xoo and xoc, sequences specific to  <dig>  xoo genes, sequences specific to xoc  <dig>  genes, and a control corresponding to a gene encoding hygromycin phosphotransferase not found in xoo or xoc.
we present here the details of the microarray design and optimization, and the results of a successful experiment to validate the array by comparing gene expression of strain pxo99a of xoo and strain bls <dig> of xoc  in a rich medium vs. xom <dig>  a minimal medium reported to induce the hrp  genes in xoo strain t <dig>  <cit> , and by independently assessing the expression of a subset of those genes by semi-quantitative rt-pcr.
in addition to several arbitrarily selected genes, validation by semi-quantitative rt-pcr focused on the hrp and rax genes.
the hrp genes encode a type iii secretion system , which many plant pathogenic bacteria, including xoo and xoc, require for hypersensitive reaction elicitation in resistant or non-host plants and for pathogenesis in susceptible host plants  <cit> .
expression of hrp genes is regulated by plant signals as well as in response to environmental stimuli such as carbon source, temperature, and ph  <cit> .
the rax genes are required for avrxa <dig> activity.
avrxa <dig> is a pathogen associated molecule recognized by the xa <dig> resistance protein  <cit> .
rax genes include eight genes predicted to contribute to three roles: type i secretion, sulfur metabolism and two-component regulation  <cit> .
RESULTS
quality of the xo array
we used a diagnostic method developed by rocke  <cit>  to examine the contribution of different factors to measured differences in signal intensity in  <dig> hybridizations  comparing xoo gene expression in psb vs. xom <dig> using the xo array.
this method employs analysis of variance .
anova can be used to calculate the gene expression changes in replicated array experiments and to correct systematic errors  <cit> .
factors examined were treatment, dye, sample, and error .
the treatment factor  had the largest effect whereas the dye, sample, and error effects were much smaller.
this analysis indicates that the measured significant changes in gene expression are due to the treatment and not to variability of other parameters.
the non-specific background error rate was assessed using  <dig> replicate spots of an oligonucleotide probe corresponding to the non-xo gene encoding hygromycin phosphotransferase .
across  <dig> hybridizations each using cdna prepared from xoo or xoc rna , error rates for positive artifacts were  <dig> – <dig> % and  <dig> – <dig> % respectively.
that is, less than 2% of the hph gene oligonucleotides showed over two-fold differences in signal intensity.
error rates associated with non-spotted controls  were similarly low,  <dig> – <dig> % and  <dig> – <dig> % following hybridization with xoo- and xoc-derived cdna, respectively .
optimization of hybridization temperature and sample amounts
to optimize temperature and amount of labeled cdna sample for hybridization, array performance was assessed at  <dig>   <dig>   <dig>  and 52°c and using labeled cdna amounts of  <dig>   <dig>   <dig>   <dig>  and  <dig> pmol.
the mean signal intensity associated with the cyanine dyes and the correlation coefficients in self-self hybridizations were examined.
there were no differences in these parameters associated with using high vs. low power of the scanner photomultiplicator .
hybridization with probe .
for temperature, the best correlation coefficient  was obtained at 42°c.
a hybridization temperature of 42°c and a labeled cdna amount of  <dig> pmol resulted in the strongest signals associated with the cyanine dyes  and the highest correlation coefficients among arrays .
therefore, these parameters were used in all subsequent hybridizations.
a probes were generated from rnas extracted from xoo cultured in psb.
b pmt represents power of the scanner photomultiplicator that can influence ratio experimental estimation, dynamical range extension or saturation of highly expressed genes.
in this study, we tuned to two  pmt levels to acquire raw slide pictures.
c value of correlation coefficient in self-self hybridization.
application and validation of the array to identify xoo and xoc genes differentially expressed in a rich vs. a minimal medium
given the distinct tissue specificities of xoo and xoc, we reasoned that these two pathovars might regulate the expression of important pathogenesis-associated genes differently.
therefore, we used the microarray to assess whether xoo and xoc show distinct patterns of differential gene expression in peptone sucrose broth  vs. xom <dig>  a minimal medium reported to activate hrp gene expression in xoo, presumably by mimicking the ph and nutrient content in the apoplast  <cit> .
individually for xoo and xoc, three biological replicates  were carried out to compare gene expression in the two culture media.
average correlation coefficients across the biological replicates were  <dig>  for xoo and  <dig>  for xoc, respectively .
a correlation coefficient of xoo samples.
b correlation coefficient of xoc samples.
to identify differentially expressed genes, the lmgene package  <cit>  was used.
the resulting list of genes with significantly different expression between the two growth conditions was then refined using a false discovery rate  of 5% and a fold-change minimum of  <dig>  , resulting in  <dig> genes for xoo and  <dig> genes for xoc.
additional file  <dig> provides a complete list of the differentially expressed xoo and xoc genes, sorted according to functional category and fold-change in expression .
to validate these results, semi-quantitative rt-pcr was used to independently assess expression levels for  <dig> xoo and  <dig> xoc genes selected arbitrarily from the list .
rna samples that were used in the microarray experiment as well as rna samples extracted from three additional replicate sets of cultures were used as templates.
there was good correlation between the semi-quantitative rt-pcr and the microarray results .
although the amplitude of gene expression fold change between the two techniques is different, as might be expected since semi-quantitative rt-pcr is not a reliable measure of quantitative differences, the general trend of gene expression is consistent.
for additional verification, we performed quantitative rt-pcr on  <dig> genes from xoo and  <dig> from xoc.
in each case the results verified the expression patterns observed using semi-quantitative rt-pcr .
athe gene id is according to the primary annotation obtained from the comprehensive microbial resource.
xoo designations represent ids of genes from xanthomonas oryzae pv.
oryzae kacc  <dig> and xocorf designations represent ids of genes from xanthomonas oryzae pv.
oryzicola bls <dig>
xoo and xoc genes differentially expressed in xom <dig> relative to psb
of the differentially expressed genes,  <dig> xoo genes were up-regulated and  <dig> were down-regulated in xom <dig> as opposed to psb.
for xoc, only  <dig> and  <dig> genes were up-regulated and down-regulated, respectively, in xom <dig> .
these genes represent  <dig> functional categories, based on the tigr annotation for the genomes available through the comprehensive microbial resource  <cit>  .
the xoo genes up-regulated in xom <dig> encode primarily hypothetical proteins  and proteins involved in cellular processes ; most of the down-regulated xoo genes encode hypothetical proteins  or proteins involved in signal transduction , dna metabolism , mobile and extra-chromosomal element functions , or transport and binding .
xoc genes expressed at a higher level in xom <dig> relative to psb, as in xoo, encode hypothetical proteins  and proteins involved in cellular processes .
in contrast to xoo however, xoc genes down-regulated in xom <dig> predominantly encode proteins involved in protein synthesis .
genes for general metabolism and transport and binding proteins
many of the differentially expressed genes in xoo and xoc are involved in general metabolism, such as amino acid biosynthesis and energy metabolism.
also several genes for transport and binding proteins are differentially expressed in both strains.
differential expression of these genes likely reflects general adaptation to the different culture media related to nutrient uptake and utilization.
chemotaxis and bacterial motility
a number of genes in xoo and xoc that are involved in motility and chemotaxis were up-regulated in xom <dig> , for example, in xoo, chemotaxis genes cher , encoding a methyl transferase, chew , encoding a coupling protein, and chey , encoding a two-component response regulator, and in xoc, flagellar basal body and motor switch genes, flgf  and flin .
the expression of chemoreceptor genes tsr and mcp was up-regulated in response to xom <dig> in both xoo and xoc.
the chemoreceptors encoded by these genes perceive environmental chemicals and activate genes such as pil and fli that are involved in movement  <cit> .
consistent with this finding, pil, fli, and another gene involved in bacterial movement, flg, were also up-regulated in xom <dig> both in xoo and xoc.
these data are consistent with the fact that nutrient conditions, especially the type of carbon source, are involved in the regulation of bacterial motility  <cit> .
moreover, the abundance of genes involved in bacterial movement that are up-regulated in xom <dig> suggests that xoo and xoc may activate genes for motility in the nutrient-limited environments of the rice xylem and mesophyll tissue and that these genes may be important for virulence.
this notion is supported by the recent report that mutations in genes for twitching motility resulted in reduced virulence in xoc  <cit>  and by the fact that bacterial chemotaxis has essential roles in virulence in ralstonia solanacearum  <cit> .
signal transduction genes
two-component systems  are widespread signal transducers in prokaryotes that enable these organisms to respond to environmental stimuli through changes in gene expression  <cit> .
environmental cues are typically perceived through a sensor histidine kinase associated with the cell membrane.
the second component is a response regulator, which upon activation by the sensor kinase activates downstream components of the response pathway.
in many cases, signalling through a single two-component system results in a coordinated change in expression of multiple genes  <cit> .
in xoo, expression of the paired sensor kinase and response regulator genes phop  and phoq  is down-regulated in xom <dig>  phop-phoq is a two-component system that governs virulence, mediates the adaptation to mg2+-limiting environment and regulates numerous cellular activities in salmonella and other species  <cit> .
we showed recently that phop and phoq in xoo are required for activation of hrp genes and avrxa <dig> activity, and full virulence  <cit> .
differential expression of phop-phoq was not detected in xoc, raising the possibility that these genes are regulated differently by xoo and xoc in planta as well, and therefore may play a role in their distinctive pathogenicity.
another difference was a ggdef domain protein  down-regulated in xom <dig> in xoo but not in xoc.
recently, several ggdef, eal, and hd-gyp domain proteins of x. campestris were shown to play a role in virulence.
they are hypothesized to compose a network of signal transduction systems for response to different environmental cues to modulate the level of the second messenger cyclic di-gmp  <cit> .
rax genes
we specifically examined expression levels of the rax  genes in xoo and xoc, and assessed expression of several of these genes independently by rt-pcr  because avrxa <dig> produced by xoo is an important determinant for race-specific interactions and is postulated to be involved in bacterial cell-cell communication  <cit> .
the rax genes are expressed constitutively in rich medium in xoo  <cit> .
they are highly conserved in xoo and xoc, but it is not known whether they are expressed in xoc and whether xoc produces avrxa <dig>  no significant differences in expression of any of the rax genes in xoo and xoc greater than  <dig>  fold were observed in the microarray data, and the rt-pcr results confirmed this observation, except for xoo raxc which showed a slight down regulation in xom <dig>  these results suggest that the expression of rax genes is largely unaffected by nutrient status.
their expression in xoc raises the possibility that like xoo, xoc produces avrxa <dig> or a similar molecule.
aresults of microarray analysis, as described in text.
a log <dig> ratio of  <dig>  is equivalent to a  <dig>  fold relative increase in expression.
aresults of rt-pcr, as described in text.
+, up-regulated in xom2; -, down-regulated in xom2;  <dig>  no change in transcript abundance detected, blank, not tested.
hrp genes
we also examined expression of hrp genes, which as described above, are essential for pathogenicity in both xoo and xoc.
expression of xoo hpa <dig> , hrpg , hrcc , and hrpe  were up-regulated in xom <dig>  consistent with the report by tsuge et al.
<cit>  which showed that several hrp loci in xoo strain t <dig> are induced in xom <dig>  no other xoo hrp genes represented on the array showed induction greater than  <dig>  fold in xom <dig>  however, when several were examined by semi-quantitative rt-pcr each was detectably up-regulated .
the tsuge et al.
study reported > <dig> fold induction for some hrp genes in xom <dig>  it should be noted, however, that the authors used a gus reporter, and therefore measured accumulated enzyme activity, which does not necessarily correlate quantitatively with microarray results, which measure accumulation of mrna.
also, there may be differences in the response of the two strains, t <dig> and pxo99a, used here, to xom <dig>  clearly though, semi-quantitative rt-pcr appears to have been a more sensitive assay for some of the hrp genes represented on the array.
in stark contrast to the results with xoo, in xoc, expression increase of greater than  <dig>  fold in xom <dig> in the microarray experiment was only observed for hpa <dig> .
upregulation was not detected for any of several xoc hrp genes tested by semi-quantitative rt-pcr with the exception of hrpe, for which increased expression in xom <dig> was detectable by this method.
xiao et al  <cit> , using rt-pcr, observed hpa <dig> expression in strain rs <dig> of xoc in a synthetic medium they named xom <dig>  but not in nutrient broth .
xom <dig> is identical to xom <dig> except that it substitutes fe-edta for fe-edta.
the authors also reported expression of the gfp gene cloned downstream of the hrpx promoter in cultures grown in xom <dig> but not nb.
the reporter construct itself was positioned downstream of the lac promoter, so this finding is difficult to interpret, but may indicate a difference in hrp regulation between rs <dig> and bls <dig>  the strain used here.
unfortunately, no other hrp genes were tested in rs <dig>
our observation that, in xoc cultured in xom <dig>  hrp genes other than hpa <dig> and hrpe, were not induced, including genes encoding the key hrp regulators hrpg, a member of the ompr family of response regulators of two-component systems  <cit> , and hrpx, an arac-type transcriptional activator that is the target of hrpg  <cit>  underscores the fact that hrp genes are regulated differently in xoo vs. xoc, and reveals that the differences reside at or upstream of hrpg, perhaps at the level of environmental sensing.
this in vitro finding in turn suggests that hrp gene expression may differ for these pathogens in in planta environments , an exciting possibility that remains to be tested, and that may provide clues to tissue specificity.
the fact that xoc hpa <dig> and hrpe are induced in xom <dig>  despite lack of induction of hrpg, hrpx, and other hrp genes, indicates that hpa <dig> and hrpe are under different or additional regulatory controls from the other hrp genes.
curiously, both genes encode extracellular proteins.
hrpe is the main structural component of the hrp pilus  <cit> , and hpa <dig> is a hrp-secreted protein with similarity to harpins  <cit> , glycine-rich proteins that may assist in type iii delivery of effectors into plant cells  <cit> .
the differential regulation we have detected here for hpa <dig> and hrpe may reflect differential regulation in planta.
this possibility fosters the intriguing speculation that the corresponding proteins accumulate early in the plant-bacterial interaction for rapid deployment once the rest of the type iii secretion apparatus is assembled.
tsuge et al   <cit>  demonstrated that induction of hrpg in xom <dig> was partially dependent on the trh  gene, and that trh was also required for wildtype levels of hpa <dig> expression in planta, but that trh mutation did not result in a measurable difference in virulence.
in light of our uncoupling of hpa <dig> and hrp expression in xoc, it may be informative to assay the effect of the trh mutation in xoo on the expression of other hrp genes in planta.
clearly, however, the possibility of multiple pathways for activation of hrp gene expression under different conditions exists.
CONCLUSIONS
in contrast to other large scale approaches to the study of gene expression in plant pathogenic bacteria, including cdna-aflp  <cit>  and in vivo expression technology , the whole genome microarray allows for genome-wide profiling of transcript levels under different conditions and over time.
cost, flexibility, sensitivity, and specificity are important factors that affect the utility of an array.
in this study, we designed and constructed a microarray for xoo and xoc based on spotted 50–70-mer oligonucleotides.
this platform is a relatively low cost and flexible, with good sensitivity  <cit> .
using the picky software, we were able to maximize specificity of probes on the array.
our initial experiments with the xo array and validation of select gene expression values by semi-quantitative rt-pcr demonstrate that the array generates robust and reliable data, though it may not be as sensitive as rt-pcr for some genes.
by comparing gene expression in xoo and xoc cultured in psb vs. xom <dig>  these experiments also provide insight and prompt new hypotheses regarding differential regulation of genes between xoo and xoc that may contribute to their distinct pathogenic characteristics.
it is important to note that the artificial minimal medium xom <dig> cannot be presumed to be an accurate proxy for in planta conditions.
for example, although we have demonstrated that the component or properties of xom <dig> that induce in vitro hrp gene expression in xoo are not effective for xoc, it is not clear whether these component or properties are the same as those that induce xoo hrp gene expression in planta, or whether in fact xoo and xoc respond to different, or identical, cues in the host.
nonetheless, the results presented provide several candidate genes whose expression it will be important to compare in planta, and whose regulation it will be important to elucidate, toward gaining a detailed understanding of xoo and xoc pathogenicity that can then be used to develop more effective and environmentally-sound disease management practices.

 the rice pathogens xanthomonas oryzae pathovar oryzae and xanthomonas oryzae pathovar oryzicola cause economically significant disease in many rice-growing regions of the world. xoo invades rice vascular tissue to cause bacterial leaf blight, whereas xoc colonizes the mesophyll parenchyma tissue to cause bacterial leaf streak. xoo gains access to the xylem through wounds or natural openings such as hydathodes, while xoc, in contrast, enters the leaf mainly through stomata. xoo and xoc are closely related, infect the same host, and are often both established in the same rice fields. the complete genome sequences of japanese xoo strain t and korean xoo strain kacc have been published. the genome sequences of a third xoo strain, philippine strain pxo99a, and a strain of xoc, philippine strain bls have recently been completed and are also publicly available, through the comprehensive microbial resource. the genomes of xoo and xoc strains are similar with respect to size, % g+c, and gene content, but show several inversions and rearrangements and some indels relative to one another. these bacteria constitute an excellent comparative model for understanding determinants of tissue specificity in plant-bacterial interactions. defining differences in gene expression and gene regulation between xoo and xoc is an important step toward that goal. dna microarray technology makes it possible to monitor the expression of thousands of genes simultaneously. microarrays can be of two general types: 1) arrays based on in situ synthesis of oligonucleotide probes, using photochemical techniques or an ink-jet oligonucleotide synthesizer and 2) spotted arrays, consisting of presynthesized dna molecules or oligomers deposited onto glass slides or filter membranes. spotted arrays are generally less costly to produce, and because they are spotted rather than synthesized from a template, they constitute a highly flexible design platform. currently, oligonucleotide- or amplicon-spotted microarrays representing the whole or partial genomes of the following plant pathogenic bacteria are available: pseudomonas syringae, ralstonia solanacearum, xanthomonas axonopodis, xanthomonas campestris, and xylella fastidiosa. these arrays, enabled by whole-genome sequence availability, have been used to study responses to environmental cues such as heat shock, and to probe gene expression patterns related to pathogenesis. they have also been used to assess genome diversity of isolates of a particular organism by comparative genome hybridization. based on the available genome sequences, we constructed a combined xoo and xoc whole genome microarray for both pathovars of x. oryzae that contains distinct 50– mer oligonucleotides, representing sequences from genes shared by xoo and xoc, sequences specific to xoo genes, sequences specific to xoc genes, and a control corresponding to a gene encoding hygromycin phosphotransferase not found in xoo or xoc. we present here the details of the microarray design and optimization, and the results of a successful experiment to validate the array by comparing gene expression of strain pxo99a of xoo and strain bls of xoc in a rich medium vs. xom a minimal medium reported to induce the hrp genes in xoo strain t, and by independently assessing the expression of a subset of those genes by semi-quantitative rt-pcr. in addition to several arbitrarily selected genes, validation by semi-quantitative rt-pcr focused on the hrp and rax genes. the hrp genes encode a type iii secretion system, which many plant pathogenic bacteria, including xoo and xoc, require for hypersensitive reaction elicitation in resistant or non-host plants and for pathogenesis in susceptible host plants. expression of hrp genes is regulated by plant signals as well as in response to environmental stimuli such as carbon source, temperature, and ph. the rax genes are required for avrxa activity. avrxa is a pathogen associated molecule recognized by the xa resistance protein. rax genes include eight genes predicted to contribute to three roles: type i secretion, sulfur metabolism and two-component regulation.  quality of the xo array we used a diagnostic method developed by rocke to examine the contribution of different factors to measured differences in signal intensity in hybridizations comparing xoo gene expression in psb vs. xom using the xo array. this method employs analysis of variance. anova can be used to calculate the gene expression changes in replicated array experiments and to correct systematic errors. factors examined were treatment, dye, sample, and error. the treatment factor had the largest effect whereas the dye, sample, and error effects were much smaller. this analysis indicates that the measured significant changes in gene expression are due to the treatment and not to variability of other parameters. the non-specific background error rate was assessed using replicate spots of an oligonucleotide probe corresponding to the non-xo gene encoding hygromycin phosphotransferase. across hybridizations each using cdna prepared from xoo or xoc rna, error rates for positive artifacts were – % and – % respectively. that is, less than 2% of the hph gene oligonucleotides showed over two-fold differences in signal intensity. error rates associated with non-spotted controls were similarly low, – % and – % following hybridization with xoo- and xoc-derived cdna, respectively. optimization of hybridization temperature and sample amounts to optimize temperature and amount of labeled cdna sample for hybridization, array performance was assessed at and 52°c and using labeled cdna amounts of and pmol. the mean signal intensity associated with the cyanine dyes and the correlation coefficients in self-self hybridizations were examined. there were no differences in these parameters associated with using high vs. low power of the scanner photomultiplicator. hybridization with probe. for temperature, the best correlation coefficient was obtained at 42°c. a hybridization temperature of 42°c and a labeled cdna amount of pmol resulted in the strongest signals associated with the cyanine dyes and the highest correlation coefficients among arrays. therefore, these parameters were used in all subsequent hybridizations. a probes were generated from rnas extracted from xoo cultured in psb. b pmt represents power of the scanner photomultiplicator that can influence ratio experimental estimation, dynamical range extension or saturation of highly expressed genes. in this study, we tuned to two pmt levels to acquire raw slide pictures. c value of correlation coefficient in self-self hybridization. application and validation of the array to identify xoo and xoc genes differentially expressed in a rich vs. a minimal medium given the distinct tissue specificities of xoo and xoc, we reasoned that these two pathovars might regulate the expression of important pathogenesis-associated genes differently. therefore, we used the microarray to assess whether xoo and xoc show distinct patterns of differential gene expression in peptone sucrose broth vs. xom a minimal medium reported to activate hrp gene expression in xoo, presumably by mimicking the ph and nutrient content in the apoplast. individually for xoo and xoc, three biological replicates were carried out to compare gene expression in the two culture media. average correlation coefficients across the biological replicates were for xoo and for xoc, respectively. a correlation coefficient of xoo samples. b correlation coefficient of xoc samples. to identify differentially expressed genes, the lmgene package was used. the resulting list of genes with significantly different expression between the two growth conditions was then refined using a false discovery rate of 5% and a fold-change minimum of, resulting in genes for xoo and genes for xoc. additional file provides a complete list of the differentially expressed xoo and xoc genes, sorted according to functional category and fold-change in expression. to validate these results, semi-quantitative rt-pcr was used to independently assess expression levels for xoo and xoc genes selected arbitrarily from the list. rna samples that were used in the microarray experiment as well as rna samples extracted from three additional replicate sets of cultures were used as templates. there was good correlation between the semi-quantitative rt-pcr and the microarray results. although the amplitude of gene expression fold change between the two techniques is different, as might be expected since semi-quantitative rt-pcr is not a reliable measure of quantitative differences, the general trend of gene expression is consistent. for additional verification, we performed quantitative rt-pcr on genes from xoo and from xoc. in each case the results verified the expression patterns observed using semi-quantitative rt-pcr. athe gene id is according to the primary annotation obtained from the comprehensive microbial resource. xoo designations represent ids of genes from xanthomonas oryzae pv. oryzae kacc and xocorf designations represent ids of genes from xanthomonas oryzae pv. oryzicola bls xoo and xoc genes differentially expressed in xom relative to psb of the differentially expressed genes, xoo genes were up-regulated and were down-regulated in xom as opposed to psb. for xoc, only and genes were up-regulated and down-regulated, respectively, in xom. these genes represent functional categories, based on the tigr annotation for the genomes available through the comprehensive microbial resource. the xoo genes up-regulated in xom encode primarily hypothetical proteins and proteins involved in cellular processes; most of the down-regulated xoo genes encode hypothetical proteins or proteins involved in signal transduction, dna metabolism, mobile and extra-chromosomal element functions, or transport and binding. xoc genes expressed at a higher level in xom relative to psb, as in xoo, encode hypothetical proteins and proteins involved in cellular processes. in contrast to xoo however, xoc genes down-regulated in xom predominantly encode proteins involved in protein synthesis. genes for general metabolism and transport and binding proteins many of the differentially expressed genes in xoo and xoc are involved in general metabolism, such as amino acid biosynthesis and energy metabolism. also several genes for transport and binding proteins are differentially expressed in both strains. differential expression of these genes likely reflects general adaptation to the different culture media related to nutrient uptake and utilization. chemotaxis and bacterial motility a number of genes in xoo and xoc that are involved in motility and chemotaxis were up-regulated in xom, for example, in xoo, chemotaxis genes cher, encoding a methyl transferase, chew, encoding a coupling protein, and chey, encoding a two-component response regulator, and in xoc, flagellar basal body and motor switch genes, flgf and flin. the expression of chemoreceptor genes tsr and mcp was up-regulated in response to xom in both xoo and xoc. the chemoreceptors encoded by these genes perceive environmental chemicals and activate genes such as pil and fli that are involved in movement. consistent with this finding, pil, fli, and another gene involved in bacterial movement, flg, were also up-regulated in xom both in xoo and xoc. these data are consistent with the fact that nutrient conditions, especially the type of carbon source, are involved in the regulation of bacterial motility. moreover, the abundance of genes involved in bacterial movement that are up-regulated in xom suggests that xoo and xoc may activate genes for motility in the nutrient-limited environments of the rice xylem and mesophyll tissue and that these genes may be important for virulence. this notion is supported by the recent report that mutations in genes for twitching motility resulted in reduced virulence in xoc and by the fact that bacterial chemotaxis has essential roles in virulence in ralstonia solanacearum. signal transduction genes two-component systems are widespread signal transducers in prokaryotes that enable these organisms to respond to environmental stimuli through changes in gene expression. environmental cues are typically perceived through a sensor histidine kinase associated with the cell membrane. the second component is a response regulator, which upon activation by the sensor kinase activates downstream components of the response pathway. in many cases, signalling through a single two-component system results in a coordinated change in expression of multiple genes. in xoo, expression of the paired sensor kinase and response regulator genes phop and phoq is down-regulated in xom phop-phoq is a two-component system that governs virulence, mediates the adaptation to mg2+-limiting environment and regulates numerous cellular activities in salmonella and other species. we showed recently that phop and phoq in xoo are required for activation of hrp genes and avrxa activity, and full virulence. differential expression of phop-phoq was not detected in xoc, raising the possibility that these genes are regulated differently by xoo and xoc in planta as well, and therefore may play a role in their distinctive pathogenicity. another difference was a ggdef domain protein down-regulated in xom in xoo but not in xoc. recently, several ggdef, eal, and hd-gyp domain proteins of x. campestris were shown to play a role in virulence. they are hypothesized to compose a network of signal transduction systems for response to different environmental cues to modulate the level of the second messenger cyclic di-gmp. rax genes we specifically examined expression levels of the rax genes in xoo and xoc, and assessed expression of several of these genes independently by rt-pcr because avrxa produced by xoo is an important determinant for race-specific interactions and is postulated to be involved in bacterial cell-cell communication. the rax genes are expressed constitutively in rich medium in xoo. they are highly conserved in xoo and xoc, but it is not known whether they are expressed in xoc and whether xoc produces avrxa no significant differences in expression of any of the rax genes in xoo and xoc greater than fold were observed in the microarray data, and the rt-pcr results confirmed this observation, except for xoo raxc which showed a slight down regulation in xom these results suggest that the expression of rax genes is largely unaffected by nutrient status. their expression in xoc raises the possibility that like xoo, xoc produces avrxa or a similar molecule. aresults of microarray analysis, as described in text. a log ratio of is equivalent to a fold relative increase in expression. aresults of rt-pcr, as described in text. +, up-regulated in xom2; -, down-regulated in xom2; no change in transcript abundance detected, blank, not tested. hrp genes we also examined expression of hrp genes, which as described above, are essential for pathogenicity in both xoo and xoc. expression of xoo hpa, hrpg, hrcc, and hrpe were up-regulated in xom consistent with the report by tsuge et al. which showed that several hrp loci in xoo strain t are induced in xom no other xoo hrp genes represented on the array showed induction greater than fold in xom however, when several were examined by semi-quantitative rt-pcr each was detectably up-regulated. the tsuge et al. study reported > fold induction for some hrp genes in xom it should be noted, however, that the authors used a gus reporter, and therefore measured accumulated enzyme activity, which does not necessarily correlate quantitatively with microarray results, which measure accumulation of mrna. also, there may be differences in the response of the two strains, t and pxo99a, used here, to xom clearly though, semi-quantitative rt-pcr appears to have been a more sensitive assay for some of the hrp genes represented on the array. in stark contrast to the results with xoo, in xoc, expression increase of greater than fold in xom in the microarray experiment was only observed for hpa. upregulation was not detected for any of several xoc hrp genes tested by semi-quantitative rt-pcr with the exception of hrpe, for which increased expression in xom was detectable by this method. xiao et al, using rt-pcr, observed hpa expression in strain rs of xoc in a synthetic medium they named xom but not in nutrient broth. xom is identical to xom except that it substitutes fe-edta for fe-edta. the authors also reported expression of the gfp gene cloned downstream of the hrpx promoter in cultures grown in xom but not nb. the reporter construct itself was positioned downstream of the lac promoter, so this finding is difficult to interpret, but may indicate a difference in hrp regulation between rs and bls the strain used here. unfortunately, no other hrp genes were tested in rs our observation that, in xoc cultured in xom hrp genes other than hpa and hrpe, were not induced, including genes encoding the key hrp regulators hrpg, a member of the ompr family of response regulators of two-component systems, and hrpx, an arac-type transcriptional activator that is the target of hrpg underscores the fact that hrp genes are regulated differently in xoo vs. xoc, and reveals that the differences reside at or upstream of hrpg, perhaps at the level of environmental sensing. this in vitro finding in turn suggests that hrp gene expression may differ for these pathogens in in planta environments, an exciting possibility that remains to be tested, and that may provide clues to tissue specificity. the fact that xoc hpa and hrpe are induced in xom despite lack of induction of hrpg, hrpx, and other hrp genes, indicates that hpa and hrpe are under different or additional regulatory controls from the other hrp genes. curiously, both genes encode extracellular proteins. hrpe is the main structural component of the hrp pilus, and hpa is a hrp-secreted protein with similarity to harpins, glycine-rich proteins that may assist in type iii delivery of effectors into plant cells. the differential regulation we have detected here for hpa and hrpe may reflect differential regulation in planta. this possibility fosters the intriguing speculation that the corresponding proteins accumulate early in the plant-bacterial interaction for rapid deployment once the rest of the type iii secretion apparatus is assembled. tsuge et al demonstrated that induction of hrpg in xom was partially dependent on the trh gene, and that trh was also required for wildtype levels of hpa expression in planta, but that trh mutation did not result in a measurable difference in virulence. in light of our uncoupling of hpa and hrp expression in xoc, it may be informative to assay the effect of the trh mutation in xoo on the expression of other hrp genes in planta. clearly, however, the possibility of multiple pathways for activation of hrp gene expression under different conditions exists.  in contrast to other large scale approaches to the study of gene expression in plant pathogenic bacteria, including cdna-aflp and in vivo expression technology, the whole genome microarray allows for genome-wide profiling of transcript levels under different conditions and over time. cost, flexibility, sensitivity, and specificity are important factors that affect the utility of an array. in this study, we designed and constructed a microarray for xoo and xoc based on spotted 50–70-mer oligonucleotides. this platform is a relatively low cost and flexible, with good sensitivity. using the picky software, we were able to maximize specificity of probes on the array. our initial experiments with the xo array and validation of select gene expression values by semi-quantitative rt-pcr demonstrate that the array generates robust and reliable data, though it may not be as sensitive as rt-pcr for some genes. by comparing gene expression in xoo and xoc cultured in psb vs. xom these experiments also provide insight and prompt new hypotheses regarding differential regulation of genes between xoo and xoc that may contribute to their distinct pathogenic characteristics. it is important to note that the artificial minimal medium xom cannot be presumed to be an accurate proxy for in planta conditions. for example, although we have demonstrated that the component or properties of xom that induce in vitro hrp gene expression in xoo are not effective for xoc, it is not clear whether these component or properties are the same as those that induce xoo hrp gene expression in planta, or whether in fact xoo and xoc respond to different, or identical, cues in the host. nonetheless, the results presented provide several candidate genes whose expression it will be important to compare in planta, and whose regulation it will be important to elucidate, toward gaining a detailed understanding of xoo and xoc pathogenicity that can then be used to develop more effective and environmentally-sound disease management practices. 
we constructed a 50– mer oligonucleotide microarray based on available genome data for xoo and xoc and compared gene expression in xoo strains pxo99a and xoc strain bls grown in the rich medium psb vs. xom a minimal medium previously reported to induce hrp genes in xoo strain t we describe here the construction and validation of a two-genome microarray for the two pathovars of x. oryzae. three biological replicates of the microarray experiment to compare global gene expression in representative strains of xoo and xoc grown in psb vs. xom were carried out. the non-specific error rate and the correlation coefficients across biological replicates and among duplicate spots revealed that the microarray data were robust. microarray analysis revealed that using representative strains, a greater number of xoo genes than xoc genes are differentially expressed in xom relative to psb, and that these include hrp genes and other genes important in interactions with rice. both pathogens depend on hrp genes for type iii secretion to infect their host. xoo and xoc are closely related but xoo invades rice vascular tissue to cause bacterial leaf blight, a serious disease of rice in many parts of the world, and xoc colonizes the mesophyll parenchyma to cause bacterial leaf streak, a disease of emerging importance. the differentially expressed genes represent functional categories. an exception was the rax genes, which are required for production of the host resistance elicitor avrxa and which were expressed constitutively in both pathovars. genes of xoo and genes of xoc were differentially expressed in the two media with a false discovery rate of 5% and with a minimum fold-change of. semi-quantitative-rt-pcr assays confirmed differential expression of each of genes each for xoo and xoc selected for validation.
 xanthomonas oryzae pv. oryzae and x. oryzae pv. oryzicola are bacterial pathogens of the worldwide staple and grass model, rice. xoo and xoc are closely related but xoo invades rice vascular tissue to cause bacterial leaf blight, a serious disease of rice in many parts of the world, and xoc colonizes the mesophyll parenchyma to cause bacterial leaf streak, a disease of emerging importance. both pathogens depend on hrp genes for type iii secretion to infect their host. we constructed a 50– mer oligonucleotide microarray based on available genome data for xoo and xoc and compared gene expression in xoo strains pxo99a and xoc strain bls grown in the rich medium psb vs. xom a minimal medium previously reported to induce hrp genes in xoo strain t  three biological replicates of the microarray experiment to compare global gene expression in representative strains of xoo and xoc grown in psb vs. xom were carried out. the non-specific error rate and the correlation coefficients across biological replicates and among duplicate spots revealed that the microarray data were robust. genes of xoo and genes of xoc were differentially expressed in the two media with a false discovery rate of 5% and with a minimum fold-change of. semi-quantitative-rt-pcr assays confirmed differential expression of each of genes each for xoo and xoc selected for validation. the differentially expressed genes represent functional categories.  we describe here the construction and validation of a two-genome microarray for the two pathovars of x. oryzae. microarray analysis revealed that using representative strains, a greater number of xoo genes than xoc genes are differentially expressed in xom relative to psb, and that these include hrp genes and other genes important in interactions with rice. an exception was the rax genes, which are required for production of the host resistance elicitor avrxa and which were expressed constitutively in both pathovars. 