BACKGROUND
pierce's disease  has been a chronic problem for california's grape industry since the 1880s.
the threat from this disease has recently become more severe with the introduction and establishment of a more effective vector, the glassy-winged sharpshooter .
the disease is caused by xylella fastidiosa, a xylem-limited, gram negative bacterium that is hosted by a wide range of plant species in and around vineyards in the southern united states and mexico  <cit> .
over the past few years, federal, state governments, and the grape industry have funded pd research.
much of this research has focused on means of controlling the vector with insecticides and natural predators as a critical first step in integrated crop management.
however, even low populations of the glassy-winged sharpshooter can have severe impact on vineyard health, thus limiting the effectiveness of predators to solve pd.
in addition, as pesticide use becomes more restricted and as pesticide resistance develops, it is likely that the ultimate solution to pd will be host resistance.
resistance to pd exists in some grape species and cultivars have been bred from these species.
for example, accessions of vitis aestivalis, v. arizonica, v. shuttleworthii, and v. smalliana are highly resistant to pd  <cit> , and breeding programs have utilized these resistant species to develop pd resistant grapes for the southeastern united states  <cit> .
efforts to breed pd resistant grapes for california are underway  <cit> .
the goals of these breeding efforts are to develop durably resistant cultivars, map and identify dna-based markers for resistance to aid in selection, and to identify resistance genes.
the introduction of pd resistance genes into wine grapes is complicated by the need for several generations of back-crossing to exclude unfavorable fruit characters associated with the resistant vitis species.
once resistance genes are identified it may be possible to directly introduce resistance into elite wine grape cultivars by transgenic technologies.
systemic infection studies under greenhouse conditions have shown differential distribution patterns of x. fastidiosa populations between resistant and susceptible genotypes and also among different organs or tissues of resistant genotypes  <cit> .
this study found that x. fastidiosa populations in the tissues of susceptible genotypes did not differ among nodes, internodes, petioles, and leaf blades.
however, the resistant genotypes had lower x. fastidiosa population levels, with highest levels in leaf blades, followed by petioles, and lowest levels in stem nodes and internodes.
differences between x. fastidiosa populations in the resistant genotypes compared to the susceptible genotypes were greatest in the stem internodes.
the inheritance of pd resistance in a v. rupestris × v. arizonica population was also evaluated by quantifying x. fastidiosa levels with elisa  <cit>  and by symptomology, including leaf scorch and a cane maturation index  <cit> .
from genotypic screening and genetic mapping studies, it was concluded that a dominant allele controls pd resistance  <cit> .
more recently, krivanek et al.
<cit>  have identified a locus that is linked to pd resistance and denoted it as 'pierce's disease resistance 1' .
these studies confirm that there is genetically based pd resistance in grapes.
they also found a range of resistance and tolerance to x. fastidiosa, which suggests that host responses to the pathogen are genotype dependent.
the results from these studies prompted investigations into molecular basis of these host-pathogen interactions, which are currently poorly understood.
functional genomic approaches provide powerful tools for identifying expressed genes.
among these techniques, expressed sequence tags ,  <cit> , serial analysis of gene expression ,  <cit>  and massively parallel signature sequencing ,  <cit> , have been successfully employed.
however due to its relative simplicity and ease, single pass est sequencing has been the most widely used method to characterize genes associated with cellular development, biotic and abiotic stress in plant research.
subtractive suppression hybridization  est cloning can be used to maximize the identification of genes involved in host responses to pathogen infection and disease development.
ssh cloning is also an effective method for cloning differentially regulated genes in cells.
this technique has been used to isolate plant genes that are expressed in response to infection  <cit> .
using molecular hybridization and subtraction techniques, the ssh cdna library approach reduces the cloning of abundantly expressed housekeeping genes or genes commonly expressed in both control and treated plants, thereby normalizing expressed cdna profiles during library construction.
as a result, it significantly enhances the chances of cloning differentially expressed genes.
this is particularly important because many pathogenesis-related genes are expressed at low levels, and can be limited to a particular tissue or cell type  <cit> .
these genes are less likely to be represented in a library if standard est cloning methods are used.
recently completed est projects have greatly contributed to the total number of developmentally regulated vitis ests available in the public domain  <cit> .
further, there is information on microarray gene expression associated with viral infection  <cit>  and on individual ests involved in host defense such as nonspecific lipid-transfer proteins   <cit>  and phytoalexin  <cit> .
however, to date information on ests expressed in response to the x. fastidiosa challenge is lacking.
the goal of this study was to characterize the molecular events in the grape/x.
fastidiosa interaction using the ssh technique to compare populations of mrna from highly resistant and susceptible grape genotypes from a grape mapping population being used to characterize pd resistance derived from a v. arizonica × v. candicans hybrid  <cit> .
for instance, the identified putative genes that are associated with host defense and/or resistance responses in this study can be used to develop molecular markers for pd resistance genetic mapping project.
they are also useful for molecular-assistant-selection if they are found to be tightly linked to the pd resistance genes.
to maximize cloning expression profiles associated with the host-pathogen interaction, a time course sampling scheme was designed tissue specific cdna libraries were constructed from stem, leaf and shoot tissues.
this report provides the transcriptome analysis of contrasting genotypes in response to x. fastidiosa infection among different tissues and provides ests associated with this host-pathogen interaction.
RESULTS
sequencing and assembly
a total of  <dig>  ests with an average of  <dig> ests per library were sequenced from the  <dig> ssh libraries.
the average size of the est was  <dig> bp with  <dig>  sequences of  <dig> bp or more.
the number of ests sequenced from each library varied from  <dig> to  <dig> sequences .
transcript redundancy in the est collection was reduced by first comparing clusters within each library and then among all  <dig> libraries.
these comparisons resulted in the assembling of  <dig>  unique sequences including  <dig> clusters  and  <dig> singleton ests .
the percentage of unique sequences in each library varied from  <dig>  to  <dig> % .
in the resistant genotype 9621- <dig>  transcript diversity from leaf and shoot tissues was reduced from  <dig>  to  <dig> % and from  <dig>  to  <dig> %, respectively, after infection by x. fastidiosa.
however the opposite results were observed in the stem tissue where transcript diversity increased from  <dig>  to  <dig> % .
in the susceptible genotype, on the other hand, transcript diversity was reduced in infected leaf and stem tissues and also in the non-infected shoot tissue .
in order to assess the number of unique and overlapping transcripts among the  <dig> libraries, four comparisons were made: those derived from resistant infected -libraries ; those derived from resistant control -libraries ; those derived from susceptible infected -libraries ; and those from susceptible control -libraries .
there were a total of  <dig> contigs  <dig>   <dig>   <dig> and  <dig> that were further assembled into the four respective classes  <dig> ,  <dig> ,  <dig>  and  <dig> .
these sequences were later used to construct the  <dig> non-redundant contigs for all  <dig> libraries .
singletons were not included in this analysis.
contigs were grouped as present in one, two, three or all the four classes .
the number of non-overlapping sequences in the above four classes was  <dig> ,  <dig> ,  <dig>  and  <dig> , respectively.
only  <dig> sequences were common among all four classes;  <dig> contigs had ests that were expressed in the two control classes  and  <dig> had ests common between the two infected classes  .
the distribution also included  <dig> contigs that were made from si and rc classes and  <dig> contigs that were made from ri and sc classes.
after this analysis, 72% of the  <dig> unique contigs belonged to one of the above four class, while the remaining 28% were overlaps.
functional annotation of the ests and comparative expression analysis
comparison of the  <dig>  non-redundant sequences from the ssh libraries against the non-redundant protein database  of the ncbi revealed that  <dig> sequences have significant similarity  to existing sequences and  <dig>  were unique.
only two ests showed significant similarity to x. fastidiosa .
complete details of the blast results are available through our website  <cit> .
when these  <dig>  sequences were passed through the ht-go-fat toolkit and blast searched against the supplied database,  <dig> sequences generated a hit, out of which  <dig> had at least one go term .
based on the generated go information, these  <dig> sequences were divided in to the three principal go categories: molecular function , cellular component  and biological process  .
under the molecular function category, ligand binding and carrier protein contributed for 27% of the total contigs followed by the ribosomal coding transcripts 15% .
transport sequences 24% followed by signal transduction and defense response sequences 19% accounted for the majority of those in the biological process category, while many of the sequences in the cellular component category were in the chloroplast 30%, membrane and nucleus subsections 26% .
more than half of the sequences  did not match sequences in the existing databases  and other sequences were divided among the three principle categories.
the full list of gene annotation along with the corresponding go terms can be queried through our website  <cit> .
non-redundant sequences  from each individual library were analyzed using the go classification.
in order to address the issue of uneven est numbers from each library, we compared relative abundance of the gene function categories based on their relative proportions from different library types .
among the leaf tissue libraries, the non-infected leaf rc library was significantly different from the other leaf libraries because of the higher percentage of ests representing signal transduction and defense response , xenobiotic metabolism , nutrient reservoir activity , hydrolase activity and hydrolyzing o-glycosyl compounds .
infected leaf  libraries showed multi-fold over expression of the monoxygenase/oxydoreductase activity  related ests compared to the non-infected leaf libraries .
comparison of the four stem libraries showed that the si library differed significantly for ests related to xenobiotic metabolism , nutrient reservoir activity  and monooxygenase/oxidoreductase activity .
in contrast, ligand binding/carrier est category was markedly lower than for that of the other three libraries.
control libraries from both the genotypes had a higher percentage of the transport related ests  compared to the infected libraries .
among the four shoot libraries, the ri was significantly different for ests of protein modification and targeting  and monooxygenase/oxidoreductase activity  in comparison to the other three libraries while the si shoot library differed for protein kinase and phosphatase activity ests compared to the other three libraries.
interestingly, stem libraries showed a higher percentage of the signal transduction and defense-related response ests than the leaf and shoot libraries .
with the exception of the rc leaf library, chloroplast related ests were abundant in the leaf libraries.
in order to evaluate the diversity and specificity of the transcripts that were specific to a physiological condition, individual library specific ests were studied.
there were  <dig> singleton ests and  <dig> contigs that fell into this category.
the stem libraries showed reduced transcript diversity following x. fastidiosa infection in both the resistant  and susceptible selections .
while the control libraries had a wide range of functional ests including pathogen related  proteins in both the selections, infected libraries were enriched with pr proteins.
the resistant infected libraries also were more diverse than the susceptible infected libraries.
the resistant stem infected library had transcripts encoding pr protein such as β 1– <dig> glucanase and  <dig> kda proline-rich protein, primary cell wall modifying proteins such as, xyloglucan endotransglycosylase , endoxyloglucan transferase , and metabolic enzymes such as cinnamoyl-coa reductase, isopropylmalate dehydrogenase, glutamate decarboxylase, 3-hydroxybutyryl-coa dehydrogenase, pep carboxylase, quinine reductase, and auxin responsive factor that appeared following x. fastidiosa infection.
on the other hand, the susceptible infected stem library was over represented by transcripts encoding pr proteins such as pr-23s np <dig> protein precursor and osmotin-like protein tpm- <dig>  glucan  <dig> -beta-glucosidase, seed storage legumin like protein and proteolytic pathway proteins such as aspartic protease, beta <dig> proteasome subunit and 20s proteasome beta subunit  that were absent in the control library.
the infected leaf libraries were free of any known transcripts of pr proteins, with control libraries having a greater percentage of transcripts encoding unknown proteins compared to the infected libraries.
only the si shoot library had pathogen responsive ests  and an f-box/lrr-repeat protein-20).
the ri shoot library did not have any of the above transcripts in this given transcriptome set.
real-time quantitative rt-pcr analysis of the differential expression
rt-pcr analysis of  <dig> out of the  <dig> selected ests confirmed differential expression under the conditions studied.
four out of the eight ests had greater expression in the susceptible variety, with gradual accumulation of the transcript as the disease progressed .
expression of these ests was much higher in the stem tissue than in the leaf tissue, particularly at 8-weeks post inoculation.
two of these ests were annotated as encoding pr proteins, while the other two appeared to be novel .
three transcripts involved in the cell homeostasis, two belonging to the metallothionin family and a sos <dig> protein kinase that is required for sodium and potassium ion homeostasis and salt tolerance in plants, showed a different trend.
expression of both the ests of metallothionin family was down regulated in the stem tissue at  <dig> weeks after inoculation in both the susceptible and resistant genotypes, while the response varied for other stages suggesting different functional roles for these two transcripts.
in contrast, the expression of sos <dig> protein kinase est did not vary significantly in this process .
expression of the l11_67_sh_ct was down regulated  in the resistant 9621- <dig> stem samples collected 24h post inoculation.
this est had sequence similarity to mitogen-activated protein kinase kinase  that was cloned from the control rc shoot library of the same genotype.
discussions and
CONCLUSIONS
this study constitutes the first genome-wide effort to understand the molecular basis of a host-x.
fastidiosa interaction in vitis.
twelve forward and reverse suppression subtractive cdna libraries from two genotypes  for three different tissues and  <dig> different stages of pierce's disease development were constructed to identify spatial and temporal transcriptional changes resulting from x. fastidiosa infection.
because a whole vitis genome sequence is not yet been completed, ests could serve as an efficient alternative approach to the discovery of novel genomic information.
out of the  <dig>  non-redundant ests that were cloned in this study, about 33% were found to be unique, demonstrating the effectiveness of the experimental design and the construction strategy utilized for these ssh libraries.
rt-pcr analysis of seven out of the eight selected ests from ssh confirmed their differential expression under the test conditions.
five out of the six transcripts showed up regulation in the tissue types and condition from which they were cloned.
however, the number of transcripts that were cloned for each of these ests  was several folds lower than their original numbers  in the rna pool, indicating that suppression of the est numbers that appeared in the final pool was effective.
furthermore, more than half  of these sequences did not match the sequences in the genbank and  <dig> were not reported in the vitis est database collection and are therefore unique contributions to the vitis est pool.
a significant difference in the number and diversity of transcripts was observed in response to x. fastidiosa infection in the resistant vs. susceptible genotypes, suggesting host responses to infection are genotype dependent.
the present study identified a group of transcripts that are regulated in response to x. fastidiosa infection and may represent the key elements in development of the defense response.
there was a significant reduction in transcript diversity, particularly in leaf tissues, in both the resistant and susceptible genotypes, after infection with x. fastidiosa .
this transcript variation was supported by the co-expression pattern of the ests with only 28% of the ests overlapping among the four classes and the rest being unique to each of those classes .
the large percentage of transcripts involved in ligand binding, carrier signal transduction, and defense response among the annotated transcripts from the inoculated tissue also supports the presumption that many of these transcripts are specifically involved in the x. fastidiosa resistance response.
these observations are consistent with previously reported studies on host-pathogen interactions  <cit> .
among the three tissue types, comparisons between libraries from resistant and susceptible infected stem tissues produced the most interesting est expression patterns.
the resistant library had ests with primary cell wall modifying and metabolic enzymes and for known pr proteins such as β 1– <dig> glucanase.
plant cell elongation depends on physical properties of the primary cell wall.
the class of enzymes, called alternatively endo-xyloglucan transglycosylase  or xyloglucan endotransglycosylase , modifies xyloglucan  by cleavage and rejoining of the β-xg backbone.
such activity can potentially alter cell size by loosening or tightening of the cell wall.
enzymes with xet activity have been identified in rapidly growing tissues from various plant species  <cit>  and multigene families related to xet have been identified  <cit> .
expression of primary cell wall modifying ests in the ri stem library, suggest active modification and expansion of cell wall tissues.
such cell wall modifications have been hypothesized to be physical barriers to limit further pathogen invasion  <cit> .
furthermore, expression of ests involved in cell metabolic activities might also reflect the pathogen's minor effect on tissue metabolism in these cells.
the microarray comparative analysis study conducted by bray  <cit>  indicated that the xyloglucan endotransglucosylase/hydrolases  family of genes was down regulated under water deficit conditions in three independent experiments, supporting the non-water stressed nature of the ri plants.
enhanced transcription of β 1– <dig> glucanase activity in grape has been previously associated with exogenous application of ethephon, an ethylene precursor  <cit> .
in a more recent study, kortekamp  <cit>  found that pr- <dig>  expression was associated with responses to pseudoperenospora cubensis infection in the resistant grape rootstock 'gloire de montpellier'  compared to the susceptible cultivar 'riesling' .
est expression in the susceptible stem library involved expression of a different class of pr proteins  and also had different levels of seed storage and proteolytic est expression, compared to their control tissues.
seed storage proteins such as legumins and vicillins are synthesized and accumulated during seed maturation and due to their regulation by agents such as abscisic acid, are associated with developing desiccation tolerance that occurs during seed maturation  <cit> .
small protein ubiquitin  and the 26s proteosome, a 2-mda protease complex, are key components of the proteolytic pathway  <cit> .
in response to pathogen attack, the ub/26s proteosome pathway initiates programmed cell death to localize pathogen spread  <cit> .
activation of proteolysis pathway ests in response to the pathogen attack has been documented previously  <cit> .
some of the pr proteins such as chitinases and  <dig> kda proline-rich protein ests were cloned only from resistant stem libraries.
while ests, such as pr- <dig>  were cloned from infected and control stem libraries of both susceptible and resistant selections.
previous reports in grape on pr- <dig>  expression point to its constitutive pre-infection role in pathogen defense  <cit> .
the previously described proline rich proteins or p-rich proteins in arabidopsis  <cit>  and in drosophila  <cit>  are known antimicrobial compounds.
further functional studies will be required to understand the specific role of these cloned pr proteins in resistant stem tissues during x. fastidiosa infection.
krivanek and walker  <cit>  found that resistant stems host 60-fold fewer x. fastidiosa cells than susceptible stems.
the est profiles produced here found unhindered metabolic activity in the resistant stem tissues and the occurrence of seed storage and proteolytic pathway proteins in the susceptible stem tissues, both suggesting the existence of a response to infection.
although pr protein expression was observed in the susceptible tissues, the nature of this expression was different since few of the pr proteins expressed in the susceptible tissues overlapped with those from resistant tissues.
this finding suggests that even susceptible genotypes have a systemic and broad host defense response mechanism that responds to x. fastidiosa infection, it does not prevent pd and must be augmented to achieve the resistance observed in 9621- <dig>
four-way comparative analysis of the v. arizonica hybrid sequences with three other vitis species contained in the genbank est collections  revealed that 26%  of the v. arizonica sequences were unique.
there are  <dig> ests in common with v. vinifera ,  <dig> ests that were present in this set and the v. shuttleworthii set; and  <dig> ests that were also present in v. aestivalis set, but absent in the other two sets.
in addition, there were  <dig> ests in common with the v. vinifera and v. shuttleworthii sets;  <dig> ests that were also present in v. vinifera and v. aestivalis sets, and  <dig> that were present also in v. shuttleworthii and v. aestivalis sets.
the rest of the ests were found in all four sets.
this is the first study to display the extent of est transcript diversity in grape after infection by x. fastidiosa.
a four-way comparative analysis found that each of the est collections had an independent niche with varying degrees of overlap with the set produced from v. arizonica.
this study has identified likely molecular targets for developing pd resistant varieties and for characterizing their resistance genes.
based on the diversity and specificity of the presented est cloning results, it is clear that stem tissue plays a prominent role in the x. fastidiosa grape interaction, supporting observations by krivanek and walker  <cit> .
the generated ests with its unique collection will serve as an important addition to the grape transcript pool for further large scale expression studies.

 pierce's disease has been a chronic problem for california's grape industry since the 1880s. the threat from this disease has recently become more severe with the introduction and establishment of a more effective vector, the glassy-winged sharpshooter. the disease is caused by xylella fastidiosa, a xylem-limited, gram negative bacterium that is hosted by a wide range of plant species in and around vineyards in the southern united states and mexico. over the past few years, federal, state governments, and the grape industry have funded pd research. much of this research has focused on means of controlling the vector with insecticides and natural predators as a critical first step in integrated crop management. however, even low populations of the glassy-winged sharpshooter can have severe impact on vineyard health, thus limiting the effectiveness of predators to solve pd. in addition, as pesticide use becomes more restricted and as pesticide resistance develops, it is likely that the ultimate solution to pd will be host resistance. resistance to pd exists in some grape species and cultivars have been bred from these species. for example, accessions of vitis aestivalis, v. arizonica, v. shuttleworthii, and v. smalliana are highly resistant to pd, and breeding programs have utilized these resistant species to develop pd resistant grapes for the southeastern united states. efforts to breed pd resistant grapes for california are underway. the goals of these breeding efforts are to develop durably resistant cultivars, map and identify dna-based markers for resistance to aid in selection, and to identify resistance genes. the introduction of pd resistance genes into wine grapes is complicated by the need for several generations of back-crossing to exclude unfavorable fruit characters associated with the resistant vitis species. once resistance genes are identified it may be possible to directly introduce resistance into elite wine grape cultivars by transgenic technologies. systemic infection studies under greenhouse conditions have shown differential distribution patterns of x. fastidiosa populations between resistant and susceptible genotypes and also among different organs or tissues of resistant genotypes. this study found that x. fastidiosa populations in the tissues of susceptible genotypes did not differ among nodes, internodes, petioles, and leaf blades. however, the resistant genotypes had lower x. fastidiosa population levels, with highest levels in leaf blades, followed by petioles, and lowest levels in stem nodes and internodes. differences between x. fastidiosa populations in the resistant genotypes compared to the susceptible genotypes were greatest in the stem internodes. the inheritance of pd resistance in a v. rupestris × v. arizonica population was also evaluated by quantifying x. fastidiosa levels with elisa and by symptomology, including leaf scorch and a cane maturation index. from genotypic screening and genetic mapping studies, it was concluded that a dominant allele controls pd resistance. more recently, krivanek et al. have identified a locus that is linked to pd resistance and denoted it as'pierce's disease resistance 1'. these studies confirm that there is genetically based pd resistance in grapes. they also found a range of resistance and tolerance to x. fastidiosa, which suggests that host responses to the pathogen are genotype dependent. the results from these studies prompted investigations into molecular basis of these host-pathogen interactions, which are currently poorly understood. functional genomic approaches provide powerful tools for identifying expressed genes. among these techniques, expressed sequence tags,, serial analysis of gene expression, and massively parallel signature sequencing,, have been successfully employed. however due to its relative simplicity and ease, single pass est sequencing has been the most widely used method to characterize genes associated with cellular development, biotic and abiotic stress in plant research. subtractive suppression hybridization est cloning can be used to maximize the identification of genes involved in host responses to pathogen infection and disease development. ssh cloning is also an effective method for cloning differentially regulated genes in cells. this technique has been used to isolate plant genes that are expressed in response to infection. using molecular hybridization and subtraction techniques, the ssh cdna library approach reduces the cloning of abundantly expressed housekeeping genes or genes commonly expressed in both control and treated plants, thereby normalizing expressed cdna profiles during library construction. as a result, it significantly enhances the chances of cloning differentially expressed genes. this is particularly important because many pathogenesis-related genes are expressed at low levels, and can be limited to a particular tissue or cell type. these genes are less likely to be represented in a library if standard est cloning methods are used. recently completed est projects have greatly contributed to the total number of developmentally regulated vitis ests available in the public domain. further, there is information on microarray gene expression associated with viral infection and on individual ests involved in host defense such as nonspecific lipid-transfer proteins and phytoalexin. however, to date information on ests expressed in response to the x. fastidiosa challenge is lacking. the goal of this study was to characterize the molecular events in the grape/x. fastidiosa interaction using the ssh technique to compare populations of mrna from highly resistant and susceptible grape genotypes from a grape mapping population being used to characterize pd resistance derived from a v. arizonica × v. candicans hybrid. for instance, the identified putative genes that are associated with host defense and/or resistance responses in this study can be used to develop molecular markers for pd resistance genetic mapping project. they are also useful for molecular-assistant-selection if they are found to be tightly linked to the pd resistance genes. to maximize cloning expression profiles associated with the host-pathogen interaction, a time course sampling scheme was designed tissue specific cdna libraries were constructed from stem, leaf and shoot tissues. this report provides the transcriptome analysis of contrasting genotypes in response to x. fastidiosa infection among different tissues and provides ests associated with this host-pathogen interaction.  sequencing and assembly a total of ests with an average of ests per library were sequenced from the ssh libraries. the average size of the est was bp with sequences of bp or more. the number of ests sequenced from each library varied from to sequences. transcript redundancy in the est collection was reduced by first comparing clusters within each library and then among all libraries. these comparisons resulted in the assembling of unique sequences including clusters and singleton ests. the percentage of unique sequences in each library varied from to %. in the resistant genotype 9621- transcript diversity from leaf and shoot tissues was reduced from to % and from to %, respectively, after infection by x. fastidiosa. however the opposite results were observed in the stem tissue where transcript diversity increased from to %. in the susceptible genotype, on the other hand, transcript diversity was reduced in infected leaf and stem tissues and also in the non-infected shoot tissue. in order to assess the number of unique and overlapping transcripts among the libraries, four comparisons were made: those derived from resistant infected -libraries; those derived from resistant control -libraries; those derived from susceptible infected -libraries; and those from susceptible control -libraries. there were a total of contigs and that were further assembled into the four respective classes,, and. these sequences were later used to construct the non-redundant contigs for all libraries. singletons were not included in this analysis. contigs were grouped as present in one, two, three or all the four classes. the number of non-overlapping sequences in the above four classes was,, and, respectively. only sequences were common among all four classes; contigs had ests that were expressed in the two control classes and had ests common between the two infected classes. the distribution also included contigs that were made from si and rc classes and contigs that were made from ri and sc classes. after this analysis, 72% of the unique contigs belonged to one of the above four class, while the remaining 28% were overlaps. functional annotation of the ests and comparative expression analysis comparison of the non-redundant sequences from the ssh libraries against the non-redundant protein database of the ncbi revealed that sequences have significant similarity to existing sequences and were unique. only two ests showed significant similarity to x. fastidiosa. complete details of the blast results are available through our website. when these sequences were passed through the ht-go-fat toolkit and blast searched against the supplied database, sequences generated a hit, out of which had at least one go term. based on the generated go information, these sequences were divided in to the three principal go categories: molecular function, cellular component and biological process. under the molecular function category, ligand binding and carrier protein contributed for 27% of the total contigs followed by the ribosomal coding transcripts 15%. transport sequences 24% followed by signal transduction and defense response sequences 19% accounted for the majority of those in the biological process category, while many of the sequences in the cellular component category were in the chloroplast 30%, membrane and nucleus subsections 26%. more than half of the sequences did not match sequences in the existing databases and other sequences were divided among the three principle categories. the full list of gene annotation along with the corresponding go terms can be queried through our website. non-redundant sequences from each individual library were analyzed using the go classification. in order to address the issue of uneven est numbers from each library, we compared relative abundance of the gene function categories based on their relative proportions from different library types. among the leaf tissue libraries, the non-infected leaf rc library was significantly different from the other leaf libraries because of the higher percentage of ests representing signal transduction and defense response, xenobiotic metabolism, nutrient reservoir activity, hydrolase activity and hydrolyzing o-glycosyl compounds. infected leaf libraries showed multi-fold over expression of the monoxygenase/oxydoreductase activity related ests compared to the non-infected leaf libraries. comparison of the four stem libraries showed that the si library differed significantly for ests related to xenobiotic metabolism, nutrient reservoir activity and monooxygenase/oxidoreductase activity. in contrast, ligand binding/carrier est category was markedly lower than for that of the other three libraries. control libraries from both the genotypes had a higher percentage of the transport related ests compared to the infected libraries. among the four shoot libraries, the ri was significantly different for ests of protein modification and targeting and monooxygenase/oxidoreductase activity in comparison to the other three libraries while the si shoot library differed for protein kinase and phosphatase activity ests compared to the other three libraries. interestingly, stem libraries showed a higher percentage of the signal transduction and defense-related response ests than the leaf and shoot libraries. with the exception of the rc leaf library, chloroplast related ests were abundant in the leaf libraries. in order to evaluate the diversity and specificity of the transcripts that were specific to a physiological condition, individual library specific ests were studied. there were singleton ests and contigs that fell into this category. the stem libraries showed reduced transcript diversity following x. fastidiosa infection in both the resistant and susceptible selections. while the control libraries had a wide range of functional ests including pathogen related proteins in both the selections, infected libraries were enriched with pr proteins. the resistant infected libraries also were more diverse than the susceptible infected libraries. the resistant stem infected library had transcripts encoding pr protein such as β 1– glucanase and kda proline-rich protein, primary cell wall modifying proteins such as, xyloglucan endotransglycosylase, endoxyloglucan transferase, and metabolic enzymes such as cinnamoyl-coa reductase, isopropylmalate dehydrogenase, glutamate decarboxylase, 3-hydroxybutyryl-coa dehydrogenase, pep carboxylase, quinine reductase, and auxin responsive factor that appeared following x. fastidiosa infection. on the other hand, the susceptible infected stem library was over represented by transcripts encoding pr proteins such as pr-23s np protein precursor and osmotin-like protein tpm- glucan -beta-glucosidase, seed storage legumin like protein and proteolytic pathway proteins such as aspartic protease, beta proteasome subunit and 20s proteasome beta subunit that were absent in the control library. the infected leaf libraries were free of any known transcripts of pr proteins, with control libraries having a greater percentage of transcripts encoding unknown proteins compared to the infected libraries. only the si shoot library had pathogen responsive ests and an f-box/lrr-repeat protein-20). the ri shoot library did not have any of the above transcripts in this given transcriptome set. real-time quantitative rt-pcr analysis of the differential expression rt-pcr analysis of out of the selected ests confirmed differential expression under the conditions studied. four out of the eight ests had greater expression in the susceptible variety, with gradual accumulation of the transcript as the disease progressed. expression of these ests was much higher in the stem tissue than in the leaf tissue, particularly at 8-weeks post inoculation. two of these ests were annotated as encoding pr proteins, while the other two appeared to be novel. three transcripts involved in the cell homeostasis, two belonging to the metallothionin family and a sos protein kinase that is required for sodium and potassium ion homeostasis and salt tolerance in plants, showed a different trend. expression of both the ests of metallothionin family was down regulated in the stem tissue at weeks after inoculation in both the susceptible and resistant genotypes, while the response varied for other stages suggesting different functional roles for these two transcripts. in contrast, the expression of sos protein kinase est did not vary significantly in this process. expression of the l11_67_sh_ct was down regulated in the resistant 9621- stem samples collected 24h post inoculation. this est had sequence similarity to mitogen-activated protein kinase kinase that was cloned from the control rc shoot library of the same genotype. discussions and  this study constitutes the first genome-wide effort to understand the molecular basis of a host-x. fastidiosa interaction in vitis. twelve forward and reverse suppression subtractive cdna libraries from two genotypes for three different tissues and different stages of pierce's disease development were constructed to identify spatial and temporal transcriptional changes resulting from x. fastidiosa infection. because a whole vitis genome sequence is not yet been completed, ests could serve as an efficient alternative approach to the discovery of novel genomic information. out of the non-redundant ests that were cloned in this study, about 33% were found to be unique, demonstrating the effectiveness of the experimental design and the construction strategy utilized for these ssh libraries. rt-pcr analysis of seven out of the eight selected ests from ssh confirmed their differential expression under the test conditions. five out of the six transcripts showed up regulation in the tissue types and condition from which they were cloned. however, the number of transcripts that were cloned for each of these ests was several folds lower than their original numbers in the rna pool, indicating that suppression of the est numbers that appeared in the final pool was effective. furthermore, more than half of these sequences did not match the sequences in the genbank and were not reported in the vitis est database collection and are therefore unique contributions to the vitis est pool. a significant difference in the number and diversity of transcripts was observed in response to x. fastidiosa infection in the resistant vs. susceptible genotypes, suggesting host responses to infection are genotype dependent. the present study identified a group of transcripts that are regulated in response to x. fastidiosa infection and may represent the key elements in development of the defense response. there was a significant reduction in transcript diversity, particularly in leaf tissues, in both the resistant and susceptible genotypes, after infection with x. fastidiosa. this transcript variation was supported by the co-expression pattern of the ests with only 28% of the ests overlapping among the four classes and the rest being unique to each of those classes. the large percentage of transcripts involved in ligand binding, carrier signal transduction, and defense response among the annotated transcripts from the inoculated tissue also supports the presumption that many of these transcripts are specifically involved in the x. fastidiosa resistance response. these observations are consistent with previously reported studies on host-pathogen interactions. among the three tissue types, comparisons between libraries from resistant and susceptible infected stem tissues produced the most interesting est expression patterns. the resistant library had ests with primary cell wall modifying and metabolic enzymes and for known pr proteins such as β 1– glucanase. plant cell elongation depends on physical properties of the primary cell wall. the class of enzymes, called alternatively endo-xyloglucan transglycosylase or xyloglucan endotransglycosylase, modifies xyloglucan by cleavage and rejoining of the β-xg backbone. such activity can potentially alter cell size by loosening or tightening of the cell wall. enzymes with xet activity have been identified in rapidly growing tissues from various plant species and multigene families related to xet have been identified. expression of primary cell wall modifying ests in the ri stem library, suggest active modification and expansion of cell wall tissues. such cell wall modifications have been hypothesized to be physical barriers to limit further pathogen invasion. furthermore, expression of ests involved in cell metabolic activities might also reflect the pathogen's minor effect on tissue metabolism in these cells. the microarray comparative analysis study conducted by bray indicated that the xyloglucan endotransglucosylase/hydrolases family of genes was down regulated under water deficit conditions in three independent experiments, supporting the non-water stressed nature of the ri plants. enhanced transcription of β 1– glucanase activity in grape has been previously associated with exogenous application of ethephon, an ethylene precursor. in a more recent study, kortekamp found that pr- expression was associated with responses to pseudoperenospora cubensis infection in the resistant grape rootstock'gloire de montpellier' compared to the susceptible cultivar'riesling'. est expression in the susceptible stem library involved expression of a different class of pr proteins and also had different levels of seed storage and proteolytic est expression, compared to their control tissues. seed storage proteins such as legumins and vicillins are synthesized and accumulated during seed maturation and due to their regulation by agents such as abscisic acid, are associated with developing desiccation tolerance that occurs during seed maturation. small protein ubiquitin and the 26s proteosome, a 2-mda protease complex, are key components of the proteolytic pathway. in response to pathogen attack, the ub/26s proteosome pathway initiates programmed cell death to localize pathogen spread. activation of proteolysis pathway ests in response to the pathogen attack has been documented previously. some of the pr proteins such as chitinases and kda proline-rich protein ests were cloned only from resistant stem libraries. while ests, such as pr- were cloned from infected and control stem libraries of both susceptible and resistant selections. previous reports in grape on pr- expression point to its constitutive pre-infection role in pathogen defense. the previously described proline rich proteins or p-rich proteins in arabidopsis and in drosophila are known antimicrobial compounds. further functional studies will be required to understand the specific role of these cloned pr proteins in resistant stem tissues during x. fastidiosa infection. krivanek and walker found that resistant stems host 60-fold fewer x. fastidiosa cells than susceptible stems. the est profiles produced here found unhindered metabolic activity in the resistant stem tissues and the occurrence of seed storage and proteolytic pathway proteins in the susceptible stem tissues, both suggesting the existence of a response to infection. although pr protein expression was observed in the susceptible tissues, the nature of this expression was different since few of the pr proteins expressed in the susceptible tissues overlapped with those from resistant tissues. this finding suggests that even susceptible genotypes have a systemic and broad host defense response mechanism that responds to x. fastidiosa infection, it does not prevent pd and must be augmented to achieve the resistance observed in 9621- four-way comparative analysis of the v. arizonica hybrid sequences with three other vitis species contained in the genbank est collections revealed that 26% of the v. arizonica sequences were unique. there are ests in common with v. vinifera, ests that were present in this set and the v. shuttleworthii set; and ests that were also present in v. aestivalis set, but absent in the other two sets. in addition, there were ests in common with the v. vinifera and v. shuttleworthii sets; ests that were also present in v. vinifera and v. aestivalis sets, and that were present also in v. shuttleworthii and v. aestivalis sets. the rest of the ests were found in all four sets. this is the first study to display the extent of est transcript diversity in grape after infection by x. fastidiosa. a four-way comparative analysis found that each of the est collections had an independent niche with varying degrees of overlap with the set produced from v. arizonica. this study has identified likely molecular targets for developing pd resistant varieties and for characterizing their resistance genes. based on the diversity and specificity of the presented est cloning results, it is clear that stem tissue plays a prominent role in the x. fastidiosa grape interaction, supporting observations by krivanek and walker. the generated ests with its unique collection will serve as an important addition to the grape transcript pool for further large scale expression studies. 
this study constitutes the first attempt to characterize the vitis differential transcriptome associated with host-pathogen interactions from different explants and genotypes. using gene ontology hierarchy, the non-redundant sequences were classified into the three principal categories: molecular function, cellular components and biological processes. all the generated ests have been submitted to genbank and are also available through our website for further functional studies. comparative analysis found variations in est expression pattern between infected and non-infected pd resistant and pd susceptible grape genotypes. twelve tissue specific subtractive suppression hybridization cdna libraries derived from a time course sampling scheme were constructed from stems, leaves and shoots of pd resistant and susceptible sibling genotypes in response to xf infection. the current molecular breeding efforts have identified genetic basis of pd resistance in grapes. among the three tissues, libraries from stem tissues showed significant differences in transcript quality suggesting their important role in grape-xylella interaction.
 the gram-negative bacterium xylella fastidiosa is the causal agent of pierce's disease in grape as well as diseases of many fruit and ornamental plants. the current molecular breeding efforts have identified genetic basis of pd resistance in grapes. however, the transcriptome level characterization of the host response to this pathogen is lacking.  twelve tissue specific subtractive suppression hybridization cdna libraries derived from a time course sampling scheme were constructed from stems, leaves and shoots of pd resistant and susceptible sibling genotypes in response to xf infection. a total of sequences were obtained from these cdna libraries from which contigs and singletons were derived. using gene ontology hierarchy, the non-redundant sequences were classified into the three principal categories: molecular function, cellular components and biological processes. comparative analysis found variations in est expression pattern between infected and non-infected pd resistant and pd susceptible grape genotypes. among the three tissues, libraries from stem tissues showed significant differences in transcript quality suggesting their important role in grape-xylella interaction.  this study constitutes the first attempt to characterize the vitis differential transcriptome associated with host-pathogen interactions from different explants and genotypes. all the generated ests have been submitted to genbank and are also available through our website for further functional studies. 