BACKGROUND
common bean, phaseolus vulgaris, represents a great source of nutrition for millions of people and is the second most important legume crop, after soybean.
it is the target of multiple pests and diseases causing substantial losses.
for example, on susceptible bean cultivars, bean rust, caused by uromyces appendiculatus, may cause yield reduction from  <dig> to 100% with favorable environmental conditions, such as high moisture and temperature between  <dig> and 27°c  <cit> .
among the  <dig> different stages of the bean rust life cycle, basidia, pycnia, aecia, uredinia, and telia, the most devastating on bean is the uredinial stage.
the latent period between the germination of an urediniospore and the formation of a sporulating pustule can be as short as  <dig> days.
signs of infection by uromyces appendiculatus include the presence of uredinia or spore-producing pustules on the surface of the leaf.
the identification of fungal proteins from quiescent and germinating uredospores enhanced the understanding of the infection process of this fungus  <cit> .
based upon mapping and quantitative trait loci  analysis, several genes involved in colletotrichum lindemuthianum resistance and other resistance genes for bean common mosaic virus , bean golden yellow mosaic virus , common bacterial blight, and bean rust are clustered  <cit> .
the large number of resistance  genes for bean rust may correlate with the high pathogen population diversity; with  <dig> different races identified  <cit> .
the locus ur- <dig> confers resistance to  <dig> out of the  <dig> u. appendiculatus races present in the usa  <cit> .
besides the ur- <dig> locus, a number of other r genes were identified in bean; such as locus ur- <dig> for race  <dig>  locus ur- <dig> epistatic to ur- <dig> for race  <dig> or locus ur- <dig> mapped to the linkage group b <dig>  <cit> .
to date, no large scale transcriptomic analysis of bean rust infection has been performed to better understand the mechanism of resistance.
all of these ur genes are effective against one specific rust strain, following the gene-for-gene resistance theory.
consequently, gene pyramiding was used to produce cultivars carrying multiple resistance genes  <cit> .
unfortunately, such resistance may prove to be effective in the field for only a short time due to the adaptation of the fungus to overcome plant defenses  <cit> .
consequently, unraveling and understanding the mechanisms downstream of these r genes is a key research goal to circumvent the adaptation of the fungus to plant resistance.
we investigated the phaseolus vulgaris-uromyces appendiculatus pathosystem at a transcriptional level for a better understanding of the plant response to fungal infection.
in this study, we developed a subtractive suppressive hybridization  library made from the common bean cultivar early gallatin that exhibits susceptibility to u. appendiculatus race  <dig> but resistance to u. appendiculatus race  <dig> .
the resistance to u. appendiculatus is conferred by the presence of the ur- <dig> gene in this cultivar that leads to a hypersensitive response  in presence of the pathogen race  <dig>  <cit> .
this cdna bean library was enriched in expressed sequence tags  that are potentially up-regulated by the compatible and incompatible interactions.
more than  <dig>  clones from the ssh library were sequenced and assembled into contigs.
a total of  <dig>  p. vulgaris sequences and  <dig> u. appendiculatus sequences were added to the ncbi database, significantly increasing the number of ests available for common bean.
the regulation of  <dig> genes was confirmed by quantitative real time polymerase chain reaction  revealing  <dig> main expression patterns and highlighting gene regulation that occurs downstream of r protein activation.
RESULTS
identification of unisequences from tissues infected with virulent or avirulent bean rust
common bean is a diploid  with a small genome size estimated at  <dig> to  <dig> mb  <cit> .
so far, the total number of common bean ests available is  <dig>  .
this number was significantly less before the publication of  <cit>  who added ests from root nodules, phosphorus deficient roots, developing pods, and leaves, and from leaves and shoots with and without c. lindemuthianum inoculation  <cit> .
.
the lack of sufficient p. vulgaris sequences precludes the construction of a useful dna microarray for this plant.
consequently, in order to study the response of bean to u. appendiculatus infection, we created a ssh library and sequenced  <dig>  clones from 3' and 5' ends.
from  <dig>  sequences,  <dig> % were discarded due to the absence of a cloned sequence or low sequence quality.
during cdna generation, sequence tags were incorporated prior to pooling cdnas from different conditions .
the tags identify the treatment and time points used in generating the original mrna.
the distribution of these tags among the library is presented in the figure  <dig>  approximately 17% of the sequences lacked a tag after sequencing, while 31% of the sequences had a "race 49" tag and more than 51% had a "race 41" tag.
it is important to note that the majority of the ests coming from the fungus were tagged "late41", consistent with an effective colonization of the leaf by the virulent fungus .
the lack of tag identification may come from inefficient incorporation of the tag during the library construction or the presence of non-identified nucleotide in the tag sequence making it indiscernible.
the various cdnas in the library could be resolved back to their source tissue by the presence of unique sequence tags.
for example, 51% percent of the est sequences were derived from bean tissue infected with race  <dig> since they had the "race 41" tag.
this likely reflects the compatible interaction between the race  <dig> and its host allowing greater fungal penetration.
biotrophic fungi are known to reprogram the host plant cell to support their growth  <cit>  and plant ests tagged with race  <dig> could be involved in this process.
contig assembly and removal of redundant sequences was performed on  <dig>  sequences using tigr gene indices clustering tools  and cap <dig> software.
two thousand seven hundred twenty one sequences showed no similarity with other sequences and were categorized as singletons.
these sequences had an average length of  <dig> bp.
seven thousand eight hundred sixty contigs were assembled from the remaining  <dig>  sequences.
the average contig length was estimated to be  <dig> kb.
an average contig contains  <dig>  sequences .
ultimately,  <dig>  unisequences were identified and represent genes that are potentially differentially up-regulated during bean infection by virulent or avirulent pathogen isolates.
among these  <dig>  unisequences,  <dig>  were annotated as bean genes and  <dig> were annotated as fungal genes based on best blast hits to the database these  <dig> fungal unisequences included  <dig> singletons and  <dig> contigs .
"unisequence", "singleton", "contig = 2", and "contig>2" columns represent the number of total unisequences, those found once in the ssh library, the contigs made up of only  <dig> sequences and the contigs composed of more than  <dig> sequences, respectively, having a hit with an organism listed in column one.
functional annotation
sequence analysis revealed that  <dig>  ests had significant similarity with sequences in public databases such as dfci or ncbi .
forty-three percent of annotations were based on similarities to sequences in soybean databases and  <dig> % were derived from comparisons with common bean .
these unisequences were grouped into  <dig> different functional categories .
the most abundant category contained the unknown , non-classified , and low or no hit  groups and represents  <dig> % of the entire library.
the remaining  <dig> % of the sequences were grouped into  <dig> categories, such as, protein metabolism and catabolism , nucleotide and nucleic acid metabolism , or stress defense response .
taken together, signal transduction regulation and nucleotide and nucleic acid metabolism represent  <dig> % of the library.
tian et al.
also found that 14% of their est library, made from phosphorus starved bean plants, fell into these two categories  <cit> .
similar observations were made on soybean in response to stresses such as drought, phosphorus starvation or nematode infection  <cit> .
it would seem that under various biotic and abiotic stresses, plants activate several common pathways that alter the expression profile of genes, which allow the plant to response to the specific environmental condition.
the ssh library was normalized to reduce the redundancy of the most highly expressed genes.
however, some genes are very highly expressed and thus remain overrepresented in the normalized library.
as expected, the largest contigs  are involved in basic metabolism processes.
the primary metabolism category comprises the largest component  of the library based upon the number of unisequences and the proportion of contigs composed of a high number of sequences.
for example, cl1contig <dig> is composed of  <dig> aligned sequences and not surprisingly, represents ribulose- <dig> -bisphosphate carboxylase/oxygenase activase .
three of the other  <dig> largest contigs are also found in the primary metabolism category .
* based on the blastx hits  with ests deposited in the dbest at ncbi
this library was constructed to reveal the plant and fungal genes up-regulated during the rust infection process.
contigs correlated with stress response pathways also have a high number of sequences such as the contig cl1contig <dig> with  <dig> sequences encoding an 1-aminocyclopropane-1-carboxylic acid oxidase , the contig cl10contig <dig> with  <dig> sequences encoding for a glucan endo- <dig> -beta-glucosidase, and the contig cl22contig <dig> with  <dig> sequences and encoding an endochitinase.
to determine the proportion of new p. vulgaris unigenes among the library, the sequences were compared with the p. vulgaris ests present in the ncbi database.
by this analysis,  <dig>  sequences, out of the  <dig>  bean ests, can be considered as new p. vulgaris unigenes with the remaining  <dig>  sequences matching known sequences with more than 98% identity over more than  <dig> bp.
the ests present in the ncbi database originate from common bean cultivars such as bat <dig>  negro jamapa  <dig> or g <dig>  facilitating the identification of putative single nucleotide polymorphisms  between these public sequences and the ests derived from this study using cultivar early gallatin.
of the  <dig>  matching sequences,  <dig> sequences present a perfect match,  <dig> sequences have  <dig> mismatch or indel,  <dig> have  <dig> mismatches and/or indels, and  <dig>  have more than  <dig> mismatches and/or indels.
an average of  <dig> snp/indel is putatively identified every  <dig> bp.
however, we were not able to further confirm these snp/indels due to the lack of the sequence trace files for the bean ests present in the ncbi database.
this snp frequency is very similar to that reported previously by ramirez et al.,  who found  <dig> snp every  <dig> bp.
our estimation is based on the comparison of cv early gallatin with  <dig> other cultivars .
when this comparison is made between only  <dig> different cultivars  the snp frequency in the coding sequences decreases to  <dig> snp every  <dig> bp.
for comparison, the snp frequency in the soybean coding sequence was estimated at  <dig> snp/ <dig> bp in exons and  <dig> snp/ <dig> bp in introns  <cit> .
the genes identified by est sequencing represent candidates involved in the plant host's ability to withstand rust infection.
therefore, genetic mapping of these gene candidates is a means to correlate their position with known qtl involved in disease resistance.
the  <dig> fungal sequences represent  <dig> % of the library.
two studies in rice showed that the harvesting time  and the stringency of selection  are very important to accurately sample the abundance of fungal est in infected leaf tissue  <cit> .
in this study, the selected e-value was e- <dig>  greatly reducing the risk of false positive clones.
tissue was sampled after  <dig> days of infection allowing the multiplication of the fungi in the leaf tissue.
at 5dai, the haustoria are already mature and are probably redirecting the nutrient up-taken from the plant based on their genes expression pattern  <cit> .
these genes were mainly annotated predominantly by comparison to ests from germinating uredospores of u. appendiculatus  <cit>  .
two hundred seventy two fungal sequences, representing  <dig> % of the total, were considered identical to ests already present in the ncbi database while  <dig> sequences are new and unique as identified by less than 98% identity over at least  <dig> bp.
interestingly, among the  <dig> fungal ests that showed no similarity with ests from u. appendiculatus germinating uredospores,  <dig> showed similarity with uromyces viciae haustorium-specific cdnas and may be specific to successful infections.
these remaining sequences represent candidates for fungal genes more directly involved in the infection mechanism.
the library was made from tissues infected with a virulent and avirulent rust strain to allow for the identification of genes involved in both pathogen-host compatibility and resistance.
beside, the high similarity of these  <dig> sequences with haustoria-specific ests makes them likely candidates to encode potential effectors or avirulence proteins.
* based on the blastx hits  with fungal ests
the largest contig has sequence similarity to a putative beta-galactosidase  based on a match to a cdna from germinating p. pachyrhizi uredospores.
comparative analysis with the phaseolus vulgaris bac-end sequences
recently, the university of purdue released the first bean fingerprinted contigs  physical map that contains  <dig>  contigs or singletons and is anchored with  <dig> genetic markers .
the bacterial artificial chromosome-ends  of the clones were sequenced and provide a powerful tool for integrated genomic and genetic analysis.
this recent release of the phaseolus physical map  allowed linkage of some of the  <dig>  unisequences to the physical map by comparison to bac-end sequences.
the number of ests matching a bac-end sequence was assessed using the minimal criteria of 98% sequence identity spanning  <dig> bp.
as a result,  <dig>  unisequences, more than 15% of the total library, could be linked to the physical map .
fourteen of these sequences located to physical contig  <dig> that is composed of  <dig> bacs.
the genetic mapping of these ests might be facilitated by the presence of genetic markers anchoring bacs within the various contigs.
this physical map was made from common bean cultivar g <dig>  the number of putative snp between the bac-end sequences and the ests  was identified among the  <dig>  matching ests.
six hundred seventy ests showed a perfect match with a bac-end sequence,  <dig> ests exhibited only  <dig> mismatch,  <dig> ests contained  <dig> mismatches and  <dig> ests had  <dig> or more mismatches.
this represents an average of  <dig> snp/indel every  <dig> bp.
identification of bean reference genes
among the  <dig>  unisequences, we sought to confirm the expression of  <dig> ests using qrt-pcr.
to normalize gene expression based on qrt-pcr, the identification of constitutively expressed bean reference genes is required.
the use of house keeping genes as reference genes for gene expression normalization can induce some error in the analysis of the data without confirmation of their constitutive expression especially when using qrt-pcr  <cit> .
consequently, three bean genes, tc <dig>  tc <dig>  and tc <dig>  were selected based on their housekeeping function and/or their presence in different bean cdna libraries  <cit> .
additionally, homologs of soybean genes cons <dig>  cons <dig>  and cons <dig> , were chosen since they were recently shown to be expressed constitutively in soybean  <cit> .
preliminary analysis of these putative constitutive genes by qrt-pcr performed on leaf, stem, and pod cdna led to the elimination of tc <dig>  cons <dig> and tc <dig> due to the variability of their expression levels .
the stability of the expression level of the  <dig> remaining genes, tc <dig>  cons <dig> and cons <dig> was evaluated by qrt-pcr on cdnas from bean uninfected or infected with bean rust race  <dig> or  <dig> at  <dig>   <dig>   <dig>   <dig>   <dig>  and  <dig> hours after inoculation .
after analysis of their expression stability using genorm software  <cit> , cons <dig> was the most stably expressed in our experimental conditions .
for this reason, cons <dig> was selected for normalization of the expression data.
it is interesting to note that cons <dig> was also among the most stably expressed constitutive genes in soybean  <cit>  and, therefore, could be a candidate to use for expression normalization in other legumes.
transcriptional analysis of selected ests during the bean rust infection process
in order to compare expression of genes responding to u. appendiculatus race  <dig> to those responding to race  <dig>  during bean infection and colonization, the expression level of six, selected fungal genes was analyzed using qrt-pcr .
during the first  <dig> hours of the infection, the six genes were expressed at comparable levels.
however, by  <dig> hai, the expression of all six genes was significantly higher in tissues infected with the virulent race  <dig> isolate.
this result likely reflects the nature of the compatible, virulent interaction as compared to inhibition of race  <dig> infection by the host defenses.
consistent with this, all six genes used in this analysis came from the ests possessing the tag of the "late41" library.
the est cl3018contig <dig>  encoding for a plant-induced rust protein  <dig>  exhibits significant similarity with nmt <dig> , which is involved in the biosynthesis of the pyrimidine moiety of thiamine .
this gene was strongly expressed only in tissue infected with the virulent fungus race  <dig>  similar observations were made previously using bean plants infected with uromyces fabae  <cit> .
these data also suggest that the haustoria may not only be the site of nutrient uptake from the plant  <cit>  but also the site of metabolite biosynthesis with specific haustorial genes involved in vitamin biosynthesis  <cit> .
ninety bean unisequences were selected  and their regulation was confirmed by qrt-pcr using rna obtained from three independent biological replicates.
unisequences coming from the ests in the "race 49" tagged libraries were desirable due to their potential involvement in a resistance pathway.
the regulation of these genes was evaluated by qrt-pcr using rna from uninoculated leaf tissues or those inoculated with either u. appendiculatus uredospores of race  <dig> or race  <dig> isolates at the time points  <dig>   <dig>   <dig>   <dig>   <dig>   <dig>  or  <dig> hai.
the data obtained were used to compare the ratio of gene expression in tissues infected with race  <dig> or race  <dig> to that in uninoculated bean leaves.
the data also allowed a direct comparison of gene expression induced by either race  <dig> or race  <dig>  the first two comparisons highlight regulation in the infected plants by the rust fungi, while the third comparison highlights gene expression differences between the two types of infection, resistant and susceptible.
the  <dig> genes showed significant expression differences in at least one of the  <dig> comparisons .
the transcriptional response was profiled in relation to the time after inoculation .
for example,  <dig> and  <dig> genes showed differential regulation within  <dig> and  <dig> hai, respectively, in tissue inoculated with race  <dig>  at these same time points  <dig> and  <dig> genes, respectively, were differentially regulated in tissues infected with race  <dig>  at the latest time points,  <dig> and  <dig> hai,  <dig> and  <dig> genes, respectively, for race  <dig> and  <dig> and  <dig> genes, respectively, for race  <dig> were differentially regulated.
it is interesting to note that the regulation occurring at the early time points appeared to be independent of the fungal race used for inoculation.
at the early time points , only  <dig> genes  showed a difference in expression in tissues inoculated with the two fungal races.
however, at the later time points, this number increased to  <dig> genes with  <dig>  and  <dig>  at  <dig> and  <dig> hai, respectively.
these results suggested that during the beginning of the infection most of the bean gene regulation is independent of the fungal race, but differences due to fungal race occur as the infection progresses.
it is possible that fungal-pathogen associated molecular pattern  elicitors  induce the same response from the plant at the beginning of the infection.
subsequently, the avr <dig> protein in the race  <dig> is recognized after a couple of days leading to the induction of defense-related genes.
however, in bean infected by race  <dig>  no plant defense is activated and gene expression may reflect the reprogramming of the plant host by the fungus especially at the haustorial site.
a key finding of the van de mortel et al.
study on glycine max – phakopsora pachyrhizi was that most genes were regulated early during infection  and at the latest time points tested .
however, at the intermediate time points , few genes were regulated; this phenomenon was called a "dip" by van de mortel et al.
.
this same expression profile was also observed in bean upon rust infection by both races.
at  <dig> and  <dig> hai few genes were regulated in comparison to 6–12– <dig> and  <dig> hai.
in common bean, those genes regulated at  <dig> hai were very different from those expressed at 72– <dig> hai .
therefore, the dip pattern of gene expression upon rust infection appears to occur in both bean and soybean.
furthermore, this biphasic regulation seems to be shared not only by rust fungi but by other biotrophic fungi.
for example barley infected by blumeria graminis , also showed a biphasic gene response, the first set of genes responded in the first  <dig> hours of the infection in the epidermis whereas the second set responded after 72– <dig> hours of infection in the mesophyll cells  <cit> .
in contrast, soybean plants infected with phytophthora sojae, a hemibiotrophic oomycete, did not show a biphasic pattern of gene response  <cit> .
based on these examples, this biphasic pattern might be specific to the biotrophic rust fungi.
further comparisons need to be made to establish the specificity of this "dip" pattern of gene expression in response to biotrophic fungal infection.
a more detailed analysis was performed on the expression ratio of transcripts in bean leaves inoculated with the fungus race  <dig> or race  <dig> versus uninoculated bean leaves.
these analyses are presented in a hierarchical cluster based on euclidian distance .
this cluster can be divided into five main groups.
the first, group a , is composed of  <dig> genes up-regulated by both fungal races in the first  <dig> hours of the infection but enhanced expression is subsequently maintained only in the plants infected by race  <dig> at the later time points .
genes in this group include those annotated as plant defense  containing pr <dig>  wound induced protein  <dig>  genes, cell-wall related , and signal transduction regulation category with a g-box binding protein pg <dig> or sensory transduction histidine kinase genes.
these genes are likely involved in the defense pathways induced by a fungal-pamp since they have the same expression pattern during the first  <dig> hours of infection independent of the fungal race used.
for example, the wound-induced protein win <dig> protein has anti-fungal activity  <cit>  and possesses a domain that can bind a well known pamp, chitin  <cit> .
the formation of haustoria by the fungus in the plant can occur within hours of infection  <cit> .
after successful colonization of the bean cell, rust race  <dig> likely secretes effector proteins that can suppress the plant defense pathway induced by pamps.
the initial induction of genes such as win <dig> by race  <dig> and their subsequent reduction in expression may be associated with this suppression of defense by the virulent pathogen only.
the second group, group b, is composed of  <dig> genes that were induced at the beginning of the infection but were slightly down-regulated at the later time points independent of the fungal race.
this group is rich in genes categorized as plant defense representing 56% of this group.
the third group, group c , is composed of  <dig> genes that appeared to be repressed by inoculation.
in group c <dig>  the genes were repressed during the first  <dig> hours by both races but this repression was only maintained at later time points  in tissues infected with race  <dig>  in contrast to group c <dig>  the apparent repression of genes occurred only after  <dig> hai with both races.
the fourth group, group d, consists of  <dig> genes that were repressed in the first  <dig> hours by both races and subsequently expressed at levels comparable to the uninfected tissue.
the genes repressed specifically at the early time points could also be involved in the basal defense response.
this pool is composed of ests known to be involved in plant defense pathways .
finally, the fifth group represents  <dig> genes that gave no discernable pattern of expression.
these  <dig> representative genes mainly identified genes involved in the early responses of the bean under rust infection .
these genes share different expression patterns but are likely involved in the basal defense response, which is induced by pamps.
these genes were induced by both races at the early time points but their regulation was often maintained only in plants infected by the fungus race  <dig>  this may be due to the inability of this avirulent pathogen to suppress the plant defense system.
the same observation was made also by lee et al.
at the protein level.
lee et al.
proposed a new model for plant disease resistance where r-gene mediated resistance is integrated into the basal immunity system of the plant and functions primarily to restore the innate immunity response that is actively suppressed by virulent pathogens <cit> .
similar patterns of expression, independent of the pathogen virulence, were observed in arabidopsis  <cit>  and barley  <cit> .
another category of genes  involved in the plant defense system are likely involved in the hr and were regulated only at the later time points in plants infected by the race  <dig> fungus.
the expression of these genes may be the result of recognition of avrur- <dig> by the ur- <dig> resistance protein and lead to the presence of hr ten days after infection with this isolates.
CONCLUSIONS
in summary, we identified  <dig>  p. vulgaris unisequences and confirmed the regulation of  <dig> plant genes by rust infection in common bean.
these data have added significantly to the genomic resources available for common bean, while also providing insight into how this plant responds to fungal infection.
as part of this study, we identified constitutively expressed bean genes that can be used for normalization in gene expression studies.
the data also suggest that a biphasic gene expression pattern may be a common feature in plants infected by biotrophic fungi.

 common bean, phaseolus vulgaris, represents a great source of nutrition for millions of people and is the second most important legume crop, after soybean. it is the target of multiple pests and diseases causing substantial losses. for example, on susceptible bean cultivars, bean rust, caused by uromyces appendiculatus, may cause yield reduction from to 100% with favorable environmental conditions, such as high moisture and temperature between and 27°c. among the different stages of the bean rust life cycle, basidia, pycnia, aecia, uredinia, and telia, the most devastating on bean is the uredinial stage. the latent period between the germination of an urediniospore and the formation of a sporulating pustule can be as short as days. signs of infection by uromyces appendiculatus include the presence of uredinia or spore-producing pustules on the surface of the leaf. the identification of fungal proteins from quiescent and germinating uredospores enhanced the understanding of the infection process of this fungus. based upon mapping and quantitative trait loci analysis, several genes involved in colletotrichum lindemuthianum resistance and other resistance genes for bean common mosaic virus, bean golden yellow mosaic virus, common bacterial blight, and bean rust are clustered. the large number of resistance genes for bean rust may correlate with the high pathogen population diversity; with different races identified. the locus ur- confers resistance to out of the u. appendiculatus races present in the usa. besides the ur- locus, a number of other r genes were identified in bean; such as locus ur- for race locus ur- epistatic to ur- for race or locus ur- mapped to the linkage group b. to date, no large scale transcriptomic analysis of bean rust infection has been performed to better understand the mechanism of resistance. all of these ur genes are effective against one specific rust strain, following the gene-for-gene resistance theory. consequently, gene pyramiding was used to produce cultivars carrying multiple resistance genes. unfortunately, such resistance may prove to be effective in the field for only a short time due to the adaptation of the fungus to overcome plant defenses. consequently, unraveling and understanding the mechanisms downstream of these r genes is a key research goal to circumvent the adaptation of the fungus to plant resistance. we investigated the phaseolus vulgaris-uromyces appendiculatus pathosystem at a transcriptional level for a better understanding of the plant response to fungal infection. in this study, we developed a subtractive suppressive hybridization library made from the common bean cultivar early gallatin that exhibits susceptibility to u. appendiculatus race but resistance to u. appendiculatus race. the resistance to u. appendiculatus is conferred by the presence of the ur- gene in this cultivar that leads to a hypersensitive response in presence of the pathogen race. this cdna bean library was enriched in expressed sequence tags that are potentially up-regulated by the compatible and incompatible interactions. more than clones from the ssh library were sequenced and assembled into contigs. a total of p. vulgaris sequences and u. appendiculatus sequences were added to the ncbi database, significantly increasing the number of ests available for common bean. the regulation of genes was confirmed by quantitative real time polymerase chain reaction revealing main expression patterns and highlighting gene regulation that occurs downstream of r protein activation.  identification of unisequences from tissues infected with virulent or avirulent bean rust common bean is a diploid with a small genome size estimated at to mb. so far, the total number of common bean ests available is. this number was significantly less before the publication of who added ests from root nodules, phosphorus deficient roots, developing pods, and leaves, and from leaves and shoots with and without c. lindemuthianum inoculation.. the lack of sufficient p. vulgaris sequences precludes the construction of a useful dna microarray for this plant. consequently, in order to study the response of bean to u. appendiculatus infection, we created a ssh library and sequenced clones from 3' and 5' ends. from sequences, % were discarded due to the absence of a cloned sequence or low sequence quality. during cdna generation, sequence tags were incorporated prior to pooling cdnas from different conditions. the tags identify the treatment and time points used in generating the original mrna. the distribution of these tags among the library is presented in the figure approximately 17% of the sequences lacked a tag after sequencing, while 31% of the sequences had a "race 49" tag and more than 51% had a "race 41" tag. it is important to note that the majority of the ests coming from the fungus were tagged "late41", consistent with an effective colonization of the leaf by the virulent fungus. the lack of tag identification may come from inefficient incorporation of the tag during the library construction or the presence of non-identified nucleotide in the tag sequence making it indiscernible. the various cdnas in the library could be resolved back to their source tissue by the presence of unique sequence tags. for example, 51% percent of the est sequences were derived from bean tissue infected with race since they had the "race 41" tag. this likely reflects the compatible interaction between the race and its host allowing greater fungal penetration. biotrophic fungi are known to reprogram the host plant cell to support their growth and plant ests tagged with race could be involved in this process. contig assembly and removal of redundant sequences was performed on sequences using tigr gene indices clustering tools and cap software. two thousand seven hundred twenty one sequences showed no similarity with other sequences and were categorized as singletons. these sequences had an average length of bp. seven thousand eight hundred sixty contigs were assembled from the remaining sequences. the average contig length was estimated to be kb. an average contig contains sequences. ultimately, unisequences were identified and represent genes that are potentially differentially up-regulated during bean infection by virulent or avirulent pathogen isolates. among these unisequences, were annotated as bean genes and were annotated as fungal genes based on best blast hits to the database these fungal unisequences included singletons and contigs. "unisequence", "singleton", "contig = 2", and "contig>2" columns represent the number of total unisequences, those found once in the ssh library, the contigs made up of only sequences and the contigs composed of more than sequences, respectively, having a hit with an organism listed in column one. functional annotation sequence analysis revealed that ests had significant similarity with sequences in public databases such as dfci or ncbi. forty-three percent of annotations were based on similarities to sequences in soybean databases and % were derived from comparisons with common bean. these unisequences were grouped into different functional categories. the most abundant category contained the unknown, non-classified, and low or no hit groups and represents % of the entire library. the remaining % of the sequences were grouped into categories, such as, protein metabolism and catabolism, nucleotide and nucleic acid metabolism, or stress defense response. taken together, signal transduction regulation and nucleotide and nucleic acid metabolism represent % of the library. tian et al. also found that 14% of their est library, made from phosphorus starved bean plants, fell into these two categories. similar observations were made on soybean in response to stresses such as drought, phosphorus starvation or nematode infection. it would seem that under various biotic and abiotic stresses, plants activate several common pathways that alter the expression profile of genes, which allow the plant to response to the specific environmental condition. the ssh library was normalized to reduce the redundancy of the most highly expressed genes. however, some genes are very highly expressed and thus remain overrepresented in the normalized library. as expected, the largest contigs are involved in basic metabolism processes. the primary metabolism category comprises the largest component of the library based upon the number of unisequences and the proportion of contigs composed of a high number of sequences. for example, cl1contig is composed of aligned sequences and not surprisingly, represents ribulose- -bisphosphate carboxylase/oxygenase activase. three of the other largest contigs are also found in the primary metabolism category. * based on the blastx hits with ests deposited in the dbest at ncbi this library was constructed to reveal the plant and fungal genes up-regulated during the rust infection process. contigs correlated with stress response pathways also have a high number of sequences such as the contig cl1contig with sequences encoding an 1-aminocyclopropane-1-carboxylic acid oxidase, the contig cl10contig with sequences encoding for a glucan endo- -beta-glucosidase, and the contig cl22contig with sequences and encoding an endochitinase. to determine the proportion of new p. vulgaris unigenes among the library, the sequences were compared with the p. vulgaris ests present in the ncbi database. by this analysis, sequences, out of the bean ests, can be considered as new p. vulgaris unigenes with the remaining sequences matching known sequences with more than 98% identity over more than bp. the ests present in the ncbi database originate from common bean cultivars such as bat negro jamapa or g facilitating the identification of putative single nucleotide polymorphisms between these public sequences and the ests derived from this study using cultivar early gallatin. of the matching sequences, sequences present a perfect match, sequences have mismatch or indel, have mismatches and/or indels, and have more than mismatches and/or indels. an average of snp/indel is putatively identified every bp. however, we were not able to further confirm these snp/indels due to the lack of the sequence trace files for the bean ests present in the ncbi database. this snp frequency is very similar to that reported previously by ramirez et al., who found snp every bp. our estimation is based on the comparison of cv early gallatin with other cultivars. when this comparison is made between only different cultivars the snp frequency in the coding sequences decreases to snp every bp. for comparison, the snp frequency in the soybean coding sequence was estimated at snp/ bp in exons and snp/ bp in introns. the genes identified by est sequencing represent candidates involved in the plant host's ability to withstand rust infection. therefore, genetic mapping of these gene candidates is a means to correlate their position with known qtl involved in disease resistance. the fungal sequences represent % of the library. two studies in rice showed that the harvesting time and the stringency of selection are very important to accurately sample the abundance of fungal est in infected leaf tissue. in this study, the selected e-value was e- greatly reducing the risk of false positive clones. tissue was sampled after days of infection allowing the multiplication of the fungi in the leaf tissue. at 5dai, the haustoria are already mature and are probably redirecting the nutrient up-taken from the plant based on their genes expression pattern. these genes were mainly annotated predominantly by comparison to ests from germinating uredospores of u. appendiculatus. two hundred seventy two fungal sequences, representing % of the total, were considered identical to ests already present in the ncbi database while sequences are new and unique as identified by less than 98% identity over at least bp. interestingly, among the fungal ests that showed no similarity with ests from u. appendiculatus germinating uredospores, showed similarity with uromyces viciae haustorium-specific cdnas and may be specific to successful infections. these remaining sequences represent candidates for fungal genes more directly involved in the infection mechanism. the library was made from tissues infected with a virulent and avirulent rust strain to allow for the identification of genes involved in both pathogen-host compatibility and resistance. beside, the high similarity of these sequences with haustoria-specific ests makes them likely candidates to encode potential effectors or avirulence proteins. * based on the blastx hits with fungal ests the largest contig has sequence similarity to a putative beta-galactosidase based on a match to a cdna from germinating p. pachyrhizi uredospores. comparative analysis with the phaseolus vulgaris bac-end sequences recently, the university of purdue released the first bean fingerprinted contigs physical map that contains contigs or singletons and is anchored with genetic markers. the bacterial artificial chromosome-ends of the clones were sequenced and provide a powerful tool for integrated genomic and genetic analysis. this recent release of the phaseolus physical map allowed linkage of some of the unisequences to the physical map by comparison to bac-end sequences. the number of ests matching a bac-end sequence was assessed using the minimal criteria of 98% sequence identity spanning bp. as a result, unisequences, more than 15% of the total library, could be linked to the physical map. fourteen of these sequences located to physical contig that is composed of bacs. the genetic mapping of these ests might be facilitated by the presence of genetic markers anchoring bacs within the various contigs. this physical map was made from common bean cultivar g the number of putative snp between the bac-end sequences and the ests was identified among the matching ests. six hundred seventy ests showed a perfect match with a bac-end sequence, ests exhibited only mismatch, ests contained mismatches and ests had or more mismatches. this represents an average of snp/indel every bp. identification of bean reference genes among the unisequences, we sought to confirm the expression of ests using qrt-pcr. to normalize gene expression based on qrt-pcr, the identification of constitutively expressed bean reference genes is required. the use of house keeping genes as reference genes for gene expression normalization can induce some error in the analysis of the data without confirmation of their constitutive expression especially when using qrt-pcr. consequently, three bean genes, tc tc and tc were selected based on their housekeeping function and/or their presence in different bean cdna libraries. additionally, homologs of soybean genes cons cons and cons, were chosen since they were recently shown to be expressed constitutively in soybean. preliminary analysis of these putative constitutive genes by qrt-pcr performed on leaf, stem, and pod cdna led to the elimination of tc cons and tc due to the variability of their expression levels. the stability of the expression level of the remaining genes, tc cons and cons was evaluated by qrt-pcr on cdnas from bean uninfected or infected with bean rust race or at and hours after inoculation. after analysis of their expression stability using genorm software, cons was the most stably expressed in our experimental conditions. for this reason, cons was selected for normalization of the expression data. it is interesting to note that cons was also among the most stably expressed constitutive genes in soybean and, therefore, could be a candidate to use for expression normalization in other legumes. transcriptional analysis of selected ests during the bean rust infection process in order to compare expression of genes responding to u. appendiculatus race to those responding to race during bean infection and colonization, the expression level of six, selected fungal genes was analyzed using qrt-pcr. during the first hours of the infection, the six genes were expressed at comparable levels. however, by hai, the expression of all six genes was significantly higher in tissues infected with the virulent race isolate. this result likely reflects the nature of the compatible, virulent interaction as compared to inhibition of race infection by the host defenses. consistent with this, all six genes used in this analysis came from the ests possessing the tag of the "late41" library. the est cl3018contig encoding for a plant-induced rust protein exhibits significant similarity with nmt, which is involved in the biosynthesis of the pyrimidine moiety of thiamine. this gene was strongly expressed only in tissue infected with the virulent fungus race similar observations were made previously using bean plants infected with uromyces fabae. these data also suggest that the haustoria may not only be the site of nutrient uptake from the plant but also the site of metabolite biosynthesis with specific haustorial genes involved in vitamin biosynthesis. ninety bean unisequences were selected and their regulation was confirmed by qrt-pcr using rna obtained from three independent biological replicates. unisequences coming from the ests in the "race 49" tagged libraries were desirable due to their potential involvement in a resistance pathway. the regulation of these genes was evaluated by qrt-pcr using rna from uninoculated leaf tissues or those inoculated with either u. appendiculatus uredospores of race or race isolates at the time points or hai. the data obtained were used to compare the ratio of gene expression in tissues infected with race or race to that in uninoculated bean leaves. the data also allowed a direct comparison of gene expression induced by either race or race the first two comparisons highlight regulation in the infected plants by the rust fungi, while the third comparison highlights gene expression differences between the two types of infection, resistant and susceptible. the genes showed significant expression differences in at least one of the comparisons. the transcriptional response was profiled in relation to the time after inoculation. for example, and genes showed differential regulation within and hai, respectively, in tissue inoculated with race at these same time points and genes, respectively, were differentially regulated in tissues infected with race at the latest time points, and hai, and genes, respectively, for race and and genes, respectively, for race were differentially regulated. it is interesting to note that the regulation occurring at the early time points appeared to be independent of the fungal race used for inoculation. at the early time points, only genes showed a difference in expression in tissues inoculated with the two fungal races. however, at the later time points, this number increased to genes with and at and hai, respectively. these results suggested that during the beginning of the infection most of the bean gene regulation is independent of the fungal race, but differences due to fungal race occur as the infection progresses. it is possible that fungal-pathogen associated molecular pattern elicitors induce the same response from the plant at the beginning of the infection. subsequently, the avr protein in the race is recognized after a couple of days leading to the induction of defense-related genes. however, in bean infected by race no plant defense is activated and gene expression may reflect the reprogramming of the plant host by the fungus especially at the haustorial site. a key finding of the van de mortel et al. study on glycine max – phakopsora pachyrhizi was that most genes were regulated early during infection and at the latest time points tested. however, at the intermediate time points, few genes were regulated; this phenomenon was called a "dip" by van de mortel et al.. this same expression profile was also observed in bean upon rust infection by both races. at and hai few genes were regulated in comparison to 6–12– and hai. in common bean, those genes regulated at hai were very different from those expressed at 72– hai. therefore, the dip pattern of gene expression upon rust infection appears to occur in both bean and soybean. furthermore, this biphasic regulation seems to be shared not only by rust fungi but by other biotrophic fungi. for example barley infected by blumeria graminis, also showed a biphasic gene response, the first set of genes responded in the first hours of the infection in the epidermis whereas the second set responded after 72– hours of infection in the mesophyll cells. in contrast, soybean plants infected with phytophthora sojae, a hemibiotrophic oomycete, did not show a biphasic pattern of gene response. based on these examples, this biphasic pattern might be specific to the biotrophic rust fungi. further comparisons need to be made to establish the specificity of this "dip" pattern of gene expression in response to biotrophic fungal infection. a more detailed analysis was performed on the expression ratio of transcripts in bean leaves inoculated with the fungus race or race versus uninoculated bean leaves. these analyses are presented in a hierarchical cluster based on euclidian distance. this cluster can be divided into five main groups. the first, group a, is composed of genes up-regulated by both fungal races in the first hours of the infection but enhanced expression is subsequently maintained only in the plants infected by race at the later time points. genes in this group include those annotated as plant defense containing pr wound induced protein genes, cell-wall related, and signal transduction regulation category with a g-box binding protein pg or sensory transduction histidine kinase genes. these genes are likely involved in the defense pathways induced by a fungal-pamp since they have the same expression pattern during the first hours of infection independent of the fungal race used. for example, the wound-induced protein win protein has anti-fungal activity and possesses a domain that can bind a well known pamp, chitin. the formation of haustoria by the fungus in the plant can occur within hours of infection. after successful colonization of the bean cell, rust race likely secretes effector proteins that can suppress the plant defense pathway induced by pamps. the initial induction of genes such as win by race and their subsequent reduction in expression may be associated with this suppression of defense by the virulent pathogen only. the second group, group b, is composed of genes that were induced at the beginning of the infection but were slightly down-regulated at the later time points independent of the fungal race. this group is rich in genes categorized as plant defense representing 56% of this group. the third group, group c, is composed of genes that appeared to be repressed by inoculation. in group c the genes were repressed during the first hours by both races but this repression was only maintained at later time points in tissues infected with race in contrast to group c the apparent repression of genes occurred only after hai with both races. the fourth group, group d, consists of genes that were repressed in the first hours by both races and subsequently expressed at levels comparable to the uninfected tissue. the genes repressed specifically at the early time points could also be involved in the basal defense response. this pool is composed of ests known to be involved in plant defense pathways. finally, the fifth group represents genes that gave no discernable pattern of expression. these representative genes mainly identified genes involved in the early responses of the bean under rust infection. these genes share different expression patterns but are likely involved in the basal defense response, which is induced by pamps. these genes were induced by both races at the early time points but their regulation was often maintained only in plants infected by the fungus race this may be due to the inability of this avirulent pathogen to suppress the plant defense system. the same observation was made also by lee et al. at the protein level. lee et al. proposed a new model for plant disease resistance where r-gene mediated resistance is integrated into the basal immunity system of the plant and functions primarily to restore the innate immunity response that is actively suppressed by virulent pathogens. similar patterns of expression, independent of the pathogen virulence, were observed in arabidopsis and barley. another category of genes involved in the plant defense system are likely involved in the hr and were regulated only at the later time points in plants infected by the race fungus. the expression of these genes may be the result of recognition of avrur- by the ur- resistance protein and lead to the presence of hr ten days after infection with this isolates.  in summary, we identified p. vulgaris unisequences and confirmed the regulation of plant genes by rust infection in common bean. these data have added significantly to the genomic resources available for common bean, while also providing insight into how this plant responds to fungal infection. as part of this study, we identified constitutively expressed bean genes that can be used for normalization in gene expression studies. the data also suggest that a biphasic gene expression pattern may be a common feature in plants infected by biotrophic fungi. 
phaseolus vulgaris is the second most important legume crop in the world after soybean. a biphasic pattern of gene expression was observed for several genes regulated in response to fungal infection. the enrichment of the public database with over bean ests significantly adds to the genomic resources available for this important crop plant. regulation of selected bean genes in response to bean rust infection was confirmed by qrt-pcr. the expression pattern of bean genes upon rust infection shares several features with other legumes infected by biotrophic fungi. plant gene expression was similar for both race and during the first hours of the infection process but varied significantly at the later time points mainly due to the presence of the avr gene in the race leading to a hypersensitive response in the bean plants. consequently, yield losses due to fungal infection, like uromyces appendiculatus, have strong consequences. several resistant genes were identified that confer resistance to bean rust infection. the analysis of these genes in response to bean rust infection provides a foundation for further studies of the mechanism of fungal disease resistance.
 phaseolus vulgaris is the second most important legume crop in the world after soybean. consequently, yield losses due to fungal infection, like uromyces appendiculatus, have strong consequences. several resistant genes were identified that confer resistance to bean rust infection. however, the downstream genes and mechanisms involved in bean resistance to infection are poorly characterized.  a subtractive bean cdna library composed of unisequences was constructed and enriched in sequences regulated by either bean rust race a virulent strain, or race an avirulent strain on cultivar early gallatin carrying the resistance gene ur- the construction of this library allowed the identification of new bean ests, significantly adding to the available sequences for this plant. regulation of selected bean genes in response to bean rust infection was confirmed by qrt-pcr. plant gene expression was similar for both race and during the first hours of the infection process but varied significantly at the later time points mainly due to the presence of the avr gene in the race leading to a hypersensitive response in the bean plants. a biphasic pattern of gene expression was observed for several genes regulated in response to fungal infection.  the enrichment of the public database with over bean ests significantly adds to the genomic resources available for this important crop plant. the analysis of these genes in response to bean rust infection provides a foundation for further studies of the mechanism of fungal disease resistance. the expression pattern of bean genes upon rust infection shares several features with other legumes infected by biotrophic fungi. this finding suggests that the p. vulgaris-u. appendiculatus pathosystem could serve as a model to explore legume-rust interaction. 