BACKGROUND
rust fungi are obligate biotrophic pathogens responsible for many economically important plant diseases, particularly on cereals.
puccinia striiformis westend.
f.sp.
tritici eriks.
causes wheat stripe rust, a devastating disease in many wheat growing areas of the world.
yield losses caused by stripe rust over a large area can be up to 50%  <cit> .
chemicals have been used to control the disease, but the most cost-effective strategy to reduce the threat of the disease is through growing resistant wheat cultivars.
however, cultivars with race-specific resistance genes may become susceptible to the disease when new virulent races of the pathogen emerge.
novel methods to control the disease are needed to be developed, which requires a better understanding of the interactions between the host and pathogen, especially genes of the pathogen expressed in the course of the infection process of the compatible interaction to identify new targets for disease control.
traditionally, incompatible interactions have received much more attention than compatible interaction in order to understand the host resistance mechanism as well as to discover and use resistance genes  <cit> .
recently, studies have been reported on pathogen factors that promote compatible interactions and disease development in plant tissue  <cit> .
phenotypically, the compatible and incompatible interactions of wheat-pst are obviously different.
necrotic spots or stripes can be observed after inoculation in the incompatible interaction and the ceasing development of infection hyphae at an early stage can be observed using a microscope  <cit> .
this phenomenon is regarded as hypersensitive response  and also termed programmed cell death   <cit> .
in an incompatible interaction the host tissue sacrifices the infected cell or several cells around the infection site to prevent further growth of the biotrophic pathogen.
in a compatible interaction, the biotrophic fungus pst  <cit>  is armed with a complex strategy to avoid destructive effects in the host tissue during the processes of infection and spread.
when urediniospores land on leaf surfaces of host plants, they germinate under suitable conditions of humidity and temperature, and the germ tubes grow toward stomata.
once germ tubes reach stomatal guard cells, they directly enter into the host leaf tissue through stomata or occasionally form appressoria on stomata  <cit> .
from an appressorium, a penetration peg grows through the stomatal opening and develops into a vesicle in the substomatal cavity.
infection hyphae start growing in the intercellular spaces of the host tissue, and when the tip of an infection hypha reaches the cell wall of its host, a haustorial mother cell is formed.
from the haustorial mother cell, a narrow haustorial neck penetrates the wall of the mesophyll cell and develops into a haustorium, which establishes an obligate biotrophic relationship with the living plant cell by redirecting the host's metabolism to meet nutritional needs of the pathogen without causing immediate death of host cells  <cit> .
the lack of immediate host cell death in plant-pathogen interactions might be achieved by the pathogen through avoiding host recognition or expressing factors suppressing host defense responses.
the pst infection hyphae grow between host cells with haustoria in the pericellular space of host cells.
it seems that specific host genes and proteins are targeted by biotrophic parasites to achieve these goals, and as such they may be considered as compatibility factors that are essential for successful pathogenesis.
in the recent years, the expressed sequence tags  have been studied in many compatible interactions between rust fungi and their hosts.
numerous genes were reported as virulent or pathogenesis factors that may have distinct functions in the compatible plant-pathogen interaction.
hahn and mendgen  <cit>  isolated haustoria from vicia faba leaves infected with uromyces fabae and constructed a haustorium-specific cdna library.
from the library, they identified  <dig> in-planta induced genes  that were specifically expressed in infected plants, but weakly or not expressed urediniospores.
in an effort to interpret the molecular basis of the leaf rust pathogen-wheat compatible interaction, the suppression subtractive hybridization and cdna-aflp techniques were used  <cit> .
the ests originating from infected wheat leaves were obtained and a proportion of them were likely of fungal origin.
the differential expression analysis indicated that most of these genes were specific in rust-infected leaves, and the majority of induced genes showed homology to known pigs or virulence genes in other fungi.
it is predicted that these genes have common functions when the parasites infect their hosts.
recently, an est library was constructed from fully susceptible wheat leaves inoculated with p. graminis f.sp.
tritici  and most ests showed similarities to fungal sequences.
quantitative rt-pcr  was used to test the expression patterns of fungal ests from different organisms including rust-infected leaves  <cit> .
to identify genes expressed at a special physiological stage, a cdna library of germinating phakopsora pachyrhizi urediniospores was constructed and 52% of a total of  <dig> ests were found no significant similarity to the entries in the public protein databases  <cit> .
the result indicates that our knowledge of genes expressed in biotrophic pathogens is still limited.
puccinia striiformis f. sp.
tritici causes severe yield losses in wheat, but the information of gene expressions in infected susceptible wheat cultivars is scanty.
to obtain insights into the molecular basis of the compatible interaction between wheat and pst, we report here an est library constructed from fully susceptible wheat leaves infected with a highly virulent pathotype of pst.
in total,  <dig>  ests and from which  <dig>  unisequences were obtained.
the qrt-pcr analysis of a number of the ests revealed the expression patterns of some important genes involved in the compatible interaction.
the results indicate that the est library is useful for exploring pathogen genes expressed during pathogenesis and plant genes responding to the pathogen in the infection course.
RESULTS
construction of a cdna library from wheat leaves challenged by pst
to expand the knowledge of potential genes involved in the wheat-pst compatible interaction, a cdna library from the wheat genotype suwon  <dig> inoculated with chinese pst race cyr <dig> was constructed.
this interaction generated severe disease symptoms .
three developmental stages of pst in the infected wheat leaves were selected.
1) from  <dig> to  <dig> days post inoculation .
during this early infection stage, pst sequentially forms the infection peg, infection hyphae, haustorial mother cell and primary haustoria that initiate the pathogen biotrophic stage.
from this point, the further life cycle of the pathogen completely depends on the metabolism of the host tissue  <cit> .
2) the growth of pst infectious hyphae takes place inside its host and continues from  <dig> to  <dig> dpi.
based on a study on leaf rust of barley, 81-85% of the volumes of the fungus were mycelium and 15-19% haustoria.
visible symptoms of infection become apparent by  <dig> dpi as macroscopic chlorotic patches  <cit> .
3) in these chlorotic patches the fungus starts sporulating  <dig> dpi.
to representing the important infection stages, infected wheat leaves were harvested at  <dig>   <dig> and  <dig> dpi and used for total rna extraction.
<dig>  ×  <dig> pfuml- <dig> for primary library titer,  <dig>  ×  <dig> pfuml- <dig> for amplified library titer, and 98% recombinants were obtained.
inserted cdna lengths ranged from  <dig>  kb to  <dig>  kb.
cdna sequencing and analyses
from  <dig>  randomly picked clones that were sequenced from 5' end,  <dig>  ests with high quality were obtained and clustered into  <dig> contigs and  <dig>  singletons to give a set of  <dig>  unique sequences  .
est similarity search using the blastx program was carried out by comparing sequences in the non-redundant protein database of the genbank.
about 50% of the ests showed significant matches to genes encoding proteins with known functions; 20% of the ests were similar to genes encoding proteins with unknown functions; and 30% of the ests did not have significant similarities to any sequences in the database.
in the library,  <dig> % ests, which was the largest category, had the highest homology with plant genes   <dig> % ests had significant homology with fungal genes .
of the  <dig> ests in the no-hit category,  <dig> were isolated more than twice.
wric_ <dig> was isolated  <dig> times and wric_ <dig>  <dig> times.
the high frequencies of these genes in the est library might indicate their involvement in pathogenicity and virulence.
in some cases, the lack of similarity to protein database entries could be due to the sequence being derived from the 5' non-translated region of the cdna  <cit> .
functional annotation and analysis
to conduct functional classification of the wheat-pst compatible interaction, each unisequence est was compared with the non-redundant database from the ncbi using the blastx algorithms  <cit> .
the functional classification of all ests was established based on the database entry giving the best e-value and the categories defined by bevan et al.
<cit> .
the unigenes homologous to plant genes were classified into  <dig> functional categories based on the cellular roles of expressed genes .
the largest category counted for 26% of the classified ests with putative functions as enzymes of energy and metabolism.
the second largest was the category of enzymes with unknown and hypothetic protein, counting for 18% of the classified ests.
the third category was disease/defense proteins, counting for 13% of the ests.
genes homologous to those with known functions in other fungal pathogens
among the  <dig>  unisequences,  <dig> showed homologies to fungal genes from model fungal species, such as cryptococcus neoformans, neurospora crassa, aspergillus nidulans, saccharomyces cerevisiae and candida albicans, and plant pathogenic fungi such as ustilago maydis, p. triticina, magnaporthe grisea, eremothecium gossypii, uromyces appendiculatus and p. graminis.
from the fungal ests involved in the wheat-pst compatible interaction, we identified  <dig> genes homologous to other rust fungal genes .
three genes described as pigs as defined by hahn and mendgen  <cit>  were detected.
wric_ <dig> was highly homologous to pig <dig>  which was reported to be continuously up-regulated in infected broad bean leaves from 12- or 18-h infection structures and culminate in haustoria and infected leaves infected with u. fabae.
wric_ <dig>  which was detected  <dig> times in the wheat-pst compatible interaction library, is predicted to encode a protein strongly resembling peptidyl-prolyl cis-trans isomerases  and cyclophilin.
ppiase is considered to catalyze the isomerization of proline-containing peptide bonds and assists in the folding of proteins  <cit> .
the cytoplasmic ppiase also was induced by heat shock in yeast  <cit> .
gene encoding cyclophilins were also specifically expressed in u. fabae and p. triticina  <cit> .
two ests, wris_ <dig> and wris_ <dig>  were similar to genes thi <dig>  and thi <dig>  expressed in haustoria of u. fabae  <cit> .
thi <dig> and thi <dig> were found to be highly expressed in haustoria of u. fabae, representing approximately 5% of the haustorial mrna and homologous to genes involved in thiamine  biosynthesis  <cit> .
this indicates that haustoria not only function in uptaking nutrients from plants but also involved in biosynthesis of fungal metabolites  <cit> , an additional role for haustoria in the biosynthesis of fundamental metabolites that might not be provided in sufficient amounts by the host  <cit> .
the expression patterns of thi <dig> and thi <dig> were very similar.
their transcription levels increased in germinated urediniospores in vitro, and were also accumulated to very high levels in haustoria and rust-infected host leaves.
no expression was observed in healthy plants, urediniospores, and early infection structures of the pathogens.
also the isolation of such genes from the wheat leaves infected by p. triticina has been reported  <cit> .
our results together with the previous reports indicate that the functions of these genes are essential in obligate biotrophic rust fungi.
wris_ <dig> and wris_ <dig> were predicted to encode chitinase because of their best homology to the fungal chitinase gene of p. triticina.
both genes are up-regulated from  <dig> dpi  <cit> .
since chitin represents a main component of the cell wall of pst, it may be assumed that chitinases play an essential role in cell wall synthesis in the tips of intercellularly spreading hyphae.
wric_ <dig> was assembled with two ests and predicted to be a rust transferred protein  because of its similarity to genes in uromyces striatus.
rtp was originally described as pig <dig> among the  <dig> pigs isolated from the broad bean leaves infected by u. fabae  <cit> .
an immuno-fluorescence staining test provided evidence that rtp protein represents a secreted protein which is transferred from the rust haustoria into the nucleus of the infected cells of v. faba.
this was the first report demonstrating that a fungal protein was transferred to the nucleus of its host.
it has been suggested that the rtp protein plays an important role in the biotrophic interaction between the parasite and its host  <cit> .
this finding is similar to the bacterial type iii secretion system which is well-known for the transfer of virulence proteins into its host cells  <cit> .
the sequence of wris_ <dig> is similar to the gene encoding a cyclase-associated protein  <dig>  from cryptococcus gattii.
cap <dig> has three domains.
the n-terminal domain binds to adenylyl cyclase and is involved in ras-responsiveness  <cit> .
the c-terminal domain binds to g-actin and strongly inhibits actin polymerization  <cit> .
the function of the internal, proline-rich domain of cap is still unclear.
over-expression of an arabidopsis cap homologue  in transgenic tobacco plants reduced expansion of epidermal and mesophyll cells in leaf tissues  <cit> .
a cap <dig> gene of candida albicans was identified as a virulence factor when it interacts with its host, and was required for bud-hypha transitions, filamentous growth, and regulation of camp levels.
c. albicans strains with the cap <dig> gene were virulent when they infect their mice host, but the cap <dig> mutant was avirulent  <cit> .
wric_ <dig> was homologous to the chitin deacetylase, which catalyzes deacetylation of chitin to yield chitosan.
the chitin deacetylase was specifically regulated in infection structures of u. viciae-fabae and seems to help to counter hyphal degradation by plant chitinases  <cit> .
more importantly, chitosan is present in hyphal cell walls of pst immediately at the beginning of the plant-pathogen interaction  <cit> .
this indicates that the gene of chitin deacetylase is transcribed specifically at the intimate contact stage of the pathogen with its host tissue.
thus, deacetylation of chitin may protect hyphal cell walls or other infection structures from degrading by plant chitinases  <cit> .
atp-binding cassette  transporters are membrane proteins which drive the transport of compounds over the biological membranes.
wris_ <dig> and wris_ <dig> are two genes homologous to abc transporters of candida albicans and venturia inaequalis, respectively.
abc transporters are known to be involved in drug resistance which has been described in filamentous fungi  <cit> .
in plant pathogenic fungi abc transporters may act as virulence factors which mediate secretion of pathogenicity factors or protecting the fungi against defense compounds produced by host plants  <cit> .
thus, abc transporters may play a dual role in pathogenic fungi resulting in enhancing their virulence.
these results strongly suggest that pst requires the up-regulation of specific abc transporters for pathogenesis and protection against plant defense mechanisms.
recently, the cloned lr34/yr <dig> for durable resistance to leaf rust and stripe rust in wheat was found to be a putative abc transporter  <cit> .
thus, wris_ <dig> and wris_ <dig> are also likely wheat genes involved in the basal defense during the compatible interaction.
in our cdna library, wris_ <dig> had a similar sequence to that of a cryptococcus neoformans transketolase gene, which is an enzyme involved in primary carbohydrate metabolism.
the finding of wris_ <dig> may provide evidence for the normal biosynthetic capabilities of rust fungi when it intrudes into the plant tissue as previously hypothesized  <cit> .
actin is an important framework compound and ubiquitous in various organisms.
we identified the high occurrence of wris_ <dig> and wris_ <dig>  which were predicted to encode actin proteins based on their high sequence similarities  to those of p. graminis.
this provided evidence that both pst and p. graminis have developed common structures in susceptible wheat cultivars.
quantitative real-time pcr  analysis of candidate pathogenicity-related factors
seven genes were selected to conduct the qrt-pcr analysis for transcription levels at different developmental and infection stages .
four of these genes were found from wheat and three from pst based on the results of the blast search.
the four wheat genes, wris_ <dig> , wris_ <dig> , wris_ <dig>  and wric_ <dig>  were predicted to be related to defense against cell death at different infection stages of wheat-pst interaction based on the expression patterns .
bax inhibitor  <dig>  was reported as the cell death suppressor of bax-induced cell death in mammals and plants  <cit> .
the expression of bi- <dig> gene was rapidly up-regulated during abiotic and biotic stresses  <cit> .
the over-expression and knock-down of the bi- <dig> in arabidopsis plants did not affect the plant growth under general growth conditions  <cit> .
these results demonstrate that bi- <dig> is specifically regulated by some environmental factors.
in the compatible and incompatible interactions of wheat and pst, bi- <dig> had a similar transcription expression pattern.
it was up-regulated at  <dig> hpi after inoculation with nearly equal transcription levels and then slightly decreased at  <dig> hpi and reached the lowest levels by  <dig> hpi in the compatible interaction, but decreased drastically at  <dig> hpi in the incompatible interaction.
however, it was again up-regulated abruptly  <dig> hpi in the incompatible interaction.
in the compatible interaction, wris_ <dig>  was up-regulated slightly at  <dig> hpi, but subsequently down-regulated, while in the incompatible interaction, the gene was drastically up-regulated at  <dig> and  <dig> hpi.
these results suggest that cyclophilin has more functional activities in the incompatible interaction.
in contrast, in the compatible interaction, cyclophilin was down-regulated at  <dig> hpi.
wris_ <dig> is a defender against cell death  <dig> , which is different from dad <dig> at seven amino acid residues.
dad <dig> is a negative regulator of programmed cell death  identified as a part of the oligo saccharyltransferase  complex.
it has been reported also as a cell death suppressor protein, whose defect at restrictive temperature causes pcd in a hamster mutant cell line  <cit> .
pcd results in a hypersensitive response of plants in which cells invaded by the pathogen and surrounding cells die in order to limit pathogen spread  <cit> .
pcd is one of the vital characters of incompatible plant-pathogen interactions.
dad <dig> was highly expressed in the wheat-pst interaction as two peaks during the early infection stage at  <dig> and  <dig> hpi.
in the compatible interaction, it dropped to the lowest level at  <dig> hpi.
it is unclear whether dad modulates the process of pcd in the wheat-pst compatible interaction.
however, dad <dig> expression at high levels at the infection stages indicates that it plays likely a crucial but so far an unknown role.
another gene  was labeled as putative cell death suppressor, which doesn't have a clear function annotation.
it was up-regulated at  <dig> and  <dig> hpi at high levels and down-regulated at  <dig> and  <dig> hpi in the compatible interaction, but up-regulated only at  <dig> hpi in the incompatible interaction.
cell death suppressor was an anti-apoptosis protein in transgenic plants and was demonstrated as bcl- <dig>  bcl-xl and ced- <dig> to provide protection from pathogens  <cit> .
as a biotrophic pathogen, pst intrudes into the plant tissue by avoiding damage to plant tissue in the compatible interaction, a voluntary cell death can restrict establishing infection and spreading of the pathogen.
the expression patterns of the three pst genes are shown in fig.
<dig>  the sequences of wris_ <dig> and wric_ <dig> were similar to the pathogenesis related proteins pig <dig>  and pig <dig>  from u. fabae, respectively.
two ests were homologous to genes involved in thiamine  biosynthesis in yeast and have a very similar transcriptional pattern.
these genes were reported to be mainly transcribed in haustoria and rust-infected broad bean leaves, but were not detected in urediniospores and in infection structures differentiating in vitro until  <dig> hpi  <cit> .
pig <dig> and pig <dig> primers were designed according to the est sequences generated from the cdna library from wheat-pst compatible interaction and the mrna expression pattern of these two ests tested by qrt-pcr.
both genes were remarkably up-regulated at the later stage of pst development, but no transcripts were detected in the early stage post inoculation and in urediniospores.
according to the results of a previous study  <cit>  and our findings, pig <dig> and pig <dig> have similar transcription patterns in the compatible rust-plant interaction and their expression begin at the stage of haustorial formation.
wric_ <dig> is homologue to a chitin deacetylase gene from flammulina velutipes.
the enzyme plays a role in the formation of a protection compound against fungal infection s  <cit> .
in the wheat-pst compatible interaction, it was highly transcribed at the later stage of pst development in wheat tissue, but no transcripts were detected in healthy plants, early stages after inoculation and in urediniospores .
according to the character of obligate biotrophic fungi  <cit> , the reason might be that in the initial phase rust infection structures are not in close contact to the plant cells and expression is not triggered.
when more infection hyphae and the rust haustoria are formed, a tight contact to plant cells is established and the real biotrophic phases operate.
the host secrets higher activities of chitinases to degrade the chitin in fungal cell walls, however, synthesis of fungal chitin deacetylase is elicited to evade the action of host defense  <cit> .
CONCLUSIONS
from a total of  <dig>  ests of high quality,  <dig>  unisequences were obtained from a cdna library of a compatible wheat-pst interaction.
the library is useful for identifying the functional genes involved in the wheat-pst compatible interaction, and has established a new database for studying pst pathogenesis genes and wheat defense genes.
about 50% of the ests were significantly homologous to genes encoding proteins with known functions; 20% were similar to genes encoding proteins with unknown functions; and 30% did not have significant homology to any sequence in the database.
<dig> % of the ests had the highest homology with plant genes and  <dig> % had significant homology to fungal genes.
the transcription patterns of seven genes were determined by qrt-pcr assays, revealing their differential expression patterns in wheat-pst compatible and incompatible interaction.

 rust fungi are obligate biotrophic pathogens responsible for many economically important plant diseases, particularly on cereals. puccinia striiformis westend. f.sp. tritici eriks. causes wheat stripe rust, a devastating disease in many wheat growing areas of the world. yield losses caused by stripe rust over a large area can be up to 50%. chemicals have been used to control the disease, but the most cost-effective strategy to reduce the threat of the disease is through growing resistant wheat cultivars. however, cultivars with race-specific resistance genes may become susceptible to the disease when new virulent races of the pathogen emerge. novel methods to control the disease are needed to be developed, which requires a better understanding of the interactions between the host and pathogen, especially genes of the pathogen expressed in the course of the infection process of the compatible interaction to identify new targets for disease control. traditionally, incompatible interactions have received much more attention than compatible interaction in order to understand the host resistance mechanism as well as to discover and use resistance genes. recently, studies have been reported on pathogen factors that promote compatible interactions and disease development in plant tissue. phenotypically, the compatible and incompatible interactions of wheat-pst are obviously different. necrotic spots or stripes can be observed after inoculation in the incompatible interaction and the ceasing development of infection hyphae at an early stage can be observed using a microscope. this phenomenon is regarded as hypersensitive response and also termed programmed cell death. in an incompatible interaction the host tissue sacrifices the infected cell or several cells around the infection site to prevent further growth of the biotrophic pathogen. in a compatible interaction, the biotrophic fungus pst is armed with a complex strategy to avoid destructive effects in the host tissue during the processes of infection and spread. when urediniospores land on leaf surfaces of host plants, they germinate under suitable conditions of humidity and temperature, and the germ tubes grow toward stomata. once germ tubes reach stomatal guard cells, they directly enter into the host leaf tissue through stomata or occasionally form appressoria on stomata. from an appressorium, a penetration peg grows through the stomatal opening and develops into a vesicle in the substomatal cavity. infection hyphae start growing in the intercellular spaces of the host tissue, and when the tip of an infection hypha reaches the cell wall of its host, a haustorial mother cell is formed. from the haustorial mother cell, a narrow haustorial neck penetrates the wall of the mesophyll cell and develops into a haustorium, which establishes an obligate biotrophic relationship with the living plant cell by redirecting the host's metabolism to meet nutritional needs of the pathogen without causing immediate death of host cells. the lack of immediate host cell death in plant-pathogen interactions might be achieved by the pathogen through avoiding host recognition or expressing factors suppressing host defense responses. the pst infection hyphae grow between host cells with haustoria in the pericellular space of host cells. it seems that specific host genes and proteins are targeted by biotrophic parasites to achieve these goals, and as such they may be considered as compatibility factors that are essential for successful pathogenesis. in the recent years, the expressed sequence tags have been studied in many compatible interactions between rust fungi and their hosts. numerous genes were reported as virulent or pathogenesis factors that may have distinct functions in the compatible plant-pathogen interaction. hahn and mendgen isolated haustoria from vicia faba leaves infected with uromyces fabae and constructed a haustorium-specific cdna library. from the library, they identified in-planta induced genes that were specifically expressed in infected plants, but weakly or not expressed urediniospores. in an effort to interpret the molecular basis of the leaf rust pathogen-wheat compatible interaction, the suppression subtractive hybridization and cdna-aflp techniques were used. the ests originating from infected wheat leaves were obtained and a proportion of them were likely of fungal origin. the differential expression analysis indicated that most of these genes were specific in rust-infected leaves, and the majority of induced genes showed homology to known pigs or virulence genes in other fungi. it is predicted that these genes have common functions when the parasites infect their hosts. recently, an est library was constructed from fully susceptible wheat leaves inoculated with p. graminis f.sp. tritici and most ests showed similarities to fungal sequences. quantitative rt-pcr was used to test the expression patterns of fungal ests from different organisms including rust-infected leaves. to identify genes expressed at a special physiological stage, a cdna library of germinating phakopsora pachyrhizi urediniospores was constructed and 52% of a total of ests were found no significant similarity to the entries in the public protein databases. the result indicates that our knowledge of genes expressed in biotrophic pathogens is still limited. puccinia striiformis f. sp. tritici causes severe yield losses in wheat, but the information of gene expressions in infected susceptible wheat cultivars is scanty. to obtain insights into the molecular basis of the compatible interaction between wheat and pst, we report here an est library constructed from fully susceptible wheat leaves infected with a highly virulent pathotype of pst. in total, ests and from which unisequences were obtained. the qrt-pcr analysis of a number of the ests revealed the expression patterns of some important genes involved in the compatible interaction. the results indicate that the est library is useful for exploring pathogen genes expressed during pathogenesis and plant genes responding to the pathogen in the infection course.  construction of a cdna library from wheat leaves challenged by pst to expand the knowledge of potential genes involved in the wheat-pst compatible interaction, a cdna library from the wheat genotype suwon inoculated with chinese pst race cyr was constructed. this interaction generated severe disease symptoms. three developmental stages of pst in the infected wheat leaves were selected. 1) from to days post inoculation. during this early infection stage, pst sequentially forms the infection peg, infection hyphae, haustorial mother cell and primary haustoria that initiate the pathogen biotrophic stage. from this point, the further life cycle of the pathogen completely depends on the metabolism of the host tissue. 2) the growth of pst infectious hyphae takes place inside its host and continues from to dpi. based on a study on leaf rust of barley, 81-85% of the volumes of the fungus were mycelium and 15-19% haustoria. visible symptoms of infection become apparent by dpi as macroscopic chlorotic patches. 3) in these chlorotic patches the fungus starts sporulating dpi. to representing the important infection stages, infected wheat leaves were harvested at and dpi and used for total rna extraction. × pfuml- for primary library titer, × pfuml- for amplified library titer, and 98% recombinants were obtained. inserted cdna lengths ranged from kb to kb. cdna sequencing and analyses from randomly picked clones that were sequenced from 5' end, ests with high quality were obtained and clustered into contigs and singletons to give a set of unique sequences. est similarity search using the blastx program was carried out by comparing sequences in the non-redundant protein database of the genbank. about 50% of the ests showed significant matches to genes encoding proteins with known functions; 20% of the ests were similar to genes encoding proteins with unknown functions; and 30% of the ests did not have significant similarities to any sequences in the database. in the library, % ests, which was the largest category, had the highest homology with plant genes % ests had significant homology with fungal genes. of the ests in the no-hit category, were isolated more than twice. wric_ was isolated times and wric_ times. the high frequencies of these genes in the est library might indicate their involvement in pathogenicity and virulence. in some cases, the lack of similarity to protein database entries could be due to the sequence being derived from the 5' non-translated region of the cdna. functional annotation and analysis to conduct functional classification of the wheat-pst compatible interaction, each unisequence est was compared with the non-redundant database from the ncbi using the blastx algorithms. the functional classification of all ests was established based on the database entry giving the best e-value and the categories defined by bevan et al.. the unigenes homologous to plant genes were classified into functional categories based on the cellular roles of expressed genes. the largest category counted for 26% of the classified ests with putative functions as enzymes of energy and metabolism. the second largest was the category of enzymes with unknown and hypothetic protein, counting for 18% of the classified ests. the third category was disease/defense proteins, counting for 13% of the ests. genes homologous to those with known functions in other fungal pathogens among the unisequences, showed homologies to fungal genes from model fungal species, such as cryptococcus neoformans, neurospora crassa, aspergillus nidulans, saccharomyces cerevisiae and candida albicans, and plant pathogenic fungi such as ustilago maydis, p. triticina, magnaporthe grisea, eremothecium gossypii, uromyces appendiculatus and p. graminis. from the fungal ests involved in the wheat-pst compatible interaction, we identified genes homologous to other rust fungal genes. three genes described as pigs as defined by hahn and mendgen were detected. wric_ was highly homologous to pig which was reported to be continuously up-regulated in infected broad bean leaves from 12- or 18-h infection structures and culminate in haustoria and infected leaves infected with u. fabae. wric_ which was detected times in the wheat-pst compatible interaction library, is predicted to encode a protein strongly resembling peptidyl-prolyl cis-trans isomerases and cyclophilin. ppiase is considered to catalyze the isomerization of proline-containing peptide bonds and assists in the folding of proteins. the cytoplasmic ppiase also was induced by heat shock in yeast. gene encoding cyclophilins were also specifically expressed in u. fabae and p. triticina. two ests, wris_ and wris_ were similar to genes thi and thi expressed in haustoria of u. fabae. thi and thi were found to be highly expressed in haustoria of u. fabae, representing approximately 5% of the haustorial mrna and homologous to genes involved in thiamine biosynthesis. this indicates that haustoria not only function in uptaking nutrients from plants but also involved in biosynthesis of fungal metabolites, an additional role for haustoria in the biosynthesis of fundamental metabolites that might not be provided in sufficient amounts by the host. the expression patterns of thi and thi were very similar. their transcription levels increased in germinated urediniospores in vitro, and were also accumulated to very high levels in haustoria and rust-infected host leaves. no expression was observed in healthy plants, urediniospores, and early infection structures of the pathogens. also the isolation of such genes from the wheat leaves infected by p. triticina has been reported. our results together with the previous reports indicate that the functions of these genes are essential in obligate biotrophic rust fungi. wris_ and wris_ were predicted to encode chitinase because of their best homology to the fungal chitinase gene of p. triticina. both genes are up-regulated from dpi. since chitin represents a main component of the cell wall of pst, it may be assumed that chitinases play an essential role in cell wall synthesis in the tips of intercellularly spreading hyphae. wric_ was assembled with two ests and predicted to be a rust transferred protein because of its similarity to genes in uromyces striatus. rtp was originally described as pig among the pigs isolated from the broad bean leaves infected by u. fabae. an immuno-fluorescence staining test provided evidence that rtp protein represents a secreted protein which is transferred from the rust haustoria into the nucleus of the infected cells of v. faba. this was the first report demonstrating that a fungal protein was transferred to the nucleus of its host. it has been suggested that the rtp protein plays an important role in the biotrophic interaction between the parasite and its host. this finding is similar to the bacterial type iii secretion system which is well-known for the transfer of virulence proteins into its host cells. the sequence of wris_ is similar to the gene encoding a cyclase-associated protein from cryptococcus gattii. cap has three domains. the n-terminal domain binds to adenylyl cyclase and is involved in ras-responsiveness. the c-terminal domain binds to g-actin and strongly inhibits actin polymerization. the function of the internal, proline-rich domain of cap is still unclear. over-expression of an arabidopsis cap homologue in transgenic tobacco plants reduced expansion of epidermal and mesophyll cells in leaf tissues. a cap gene of candida albicans was identified as a virulence factor when it interacts with its host, and was required for bud-hypha transitions, filamentous growth, and regulation of camp levels. c. albicans strains with the cap gene were virulent when they infect their mice host, but the cap mutant was avirulent. wric_ was homologous to the chitin deacetylase, which catalyzes deacetylation of chitin to yield chitosan. the chitin deacetylase was specifically regulated in infection structures of u. viciae-fabae and seems to help to counter hyphal degradation by plant chitinases. more importantly, chitosan is present in hyphal cell walls of pst immediately at the beginning of the plant-pathogen interaction. this indicates that the gene of chitin deacetylase is transcribed specifically at the intimate contact stage of the pathogen with its host tissue. thus, deacetylation of chitin may protect hyphal cell walls or other infection structures from degrading by plant chitinases. atp-binding cassette transporters are membrane proteins which drive the transport of compounds over the biological membranes. wris_ and wris_ are two genes homologous to abc transporters of candida albicans and venturia inaequalis, respectively. abc transporters are known to be involved in drug resistance which has been described in filamentous fungi. in plant pathogenic fungi abc transporters may act as virulence factors which mediate secretion of pathogenicity factors or protecting the fungi against defense compounds produced by host plants. thus, abc transporters may play a dual role in pathogenic fungi resulting in enhancing their virulence. these results strongly suggest that pst requires the up-regulation of specific abc transporters for pathogenesis and protection against plant defense mechanisms. recently, the cloned lr34/yr for durable resistance to leaf rust and stripe rust in wheat was found to be a putative abc transporter. thus, wris_ and wris_ are also likely wheat genes involved in the basal defense during the compatible interaction. in our cdna library, wris_ had a similar sequence to that of a cryptococcus neoformans transketolase gene, which is an enzyme involved in primary carbohydrate metabolism. the finding of wris_ may provide evidence for the normal biosynthetic capabilities of rust fungi when it intrudes into the plant tissue as previously hypothesized. actin is an important framework compound and ubiquitous in various organisms. we identified the high occurrence of wris_ and wris_ which were predicted to encode actin proteins based on their high sequence similarities to those of p. graminis. this provided evidence that both pst and p. graminis have developed common structures in susceptible wheat cultivars. quantitative real-time pcr analysis of candidate pathogenicity-related factors seven genes were selected to conduct the qrt-pcr analysis for transcription levels at different developmental and infection stages. four of these genes were found from wheat and three from pst based on the results of the blast search. the four wheat genes, wris_, wris_, wris_ and wric_ were predicted to be related to defense against cell death at different infection stages of wheat-pst interaction based on the expression patterns. bax inhibitor was reported as the cell death suppressor of bax-induced cell death in mammals and plants. the expression of bi- gene was rapidly up-regulated during abiotic and biotic stresses. the over-expression and knock-down of the bi- in arabidopsis plants did not affect the plant growth under general growth conditions. these results demonstrate that bi- is specifically regulated by some environmental factors. in the compatible and incompatible interactions of wheat and pst, bi- had a similar transcription expression pattern. it was up-regulated at hpi after inoculation with nearly equal transcription levels and then slightly decreased at hpi and reached the lowest levels by hpi in the compatible interaction, but decreased drastically at hpi in the incompatible interaction. however, it was again up-regulated abruptly hpi in the incompatible interaction. in the compatible interaction, wris_ was up-regulated slightly at hpi, but subsequently down-regulated, while in the incompatible interaction, the gene was drastically up-regulated at and hpi. these results suggest that cyclophilin has more functional activities in the incompatible interaction. in contrast, in the compatible interaction, cyclophilin was down-regulated at hpi. wris_ is a defender against cell death, which is different from dad at seven amino acid residues. dad is a negative regulator of programmed cell death identified as a part of the oligo saccharyltransferase complex. it has been reported also as a cell death suppressor protein, whose defect at restrictive temperature causes pcd in a hamster mutant cell line. pcd results in a hypersensitive response of plants in which cells invaded by the pathogen and surrounding cells die in order to limit pathogen spread. pcd is one of the vital characters of incompatible plant-pathogen interactions. dad was highly expressed in the wheat-pst interaction as two peaks during the early infection stage at and hpi. in the compatible interaction, it dropped to the lowest level at hpi. it is unclear whether dad modulates the process of pcd in the wheat-pst compatible interaction. however, dad expression at high levels at the infection stages indicates that it plays likely a crucial but so far an unknown role. another gene was labeled as putative cell death suppressor, which doesn't have a clear function annotation. it was up-regulated at and hpi at high levels and down-regulated at and hpi in the compatible interaction, but up-regulated only at hpi in the incompatible interaction. cell death suppressor was an anti-apoptosis protein in transgenic plants and was demonstrated as bcl- bcl-xl and ced- to provide protection from pathogens. as a biotrophic pathogen, pst intrudes into the plant tissue by avoiding damage to plant tissue in the compatible interaction, a voluntary cell death can restrict establishing infection and spreading of the pathogen. the expression patterns of the three pst genes are shown in fig. the sequences of wris_ and wric_ were similar to the pathogenesis related proteins pig and pig from u. fabae, respectively. two ests were homologous to genes involved in thiamine biosynthesis in yeast and have a very similar transcriptional pattern. these genes were reported to be mainly transcribed in haustoria and rust-infected broad bean leaves, but were not detected in urediniospores and in infection structures differentiating in vitro until hpi. pig and pig primers were designed according to the est sequences generated from the cdna library from wheat-pst compatible interaction and the mrna expression pattern of these two ests tested by qrt-pcr. both genes were remarkably up-regulated at the later stage of pst development, but no transcripts were detected in the early stage post inoculation and in urediniospores. according to the results of a previous study and our findings, pig and pig have similar transcription patterns in the compatible rust-plant interaction and their expression begin at the stage of haustorial formation. wric_ is homologue to a chitin deacetylase gene from flammulina velutipes. the enzyme plays a role in the formation of a protection compound against fungal infection s. in the wheat-pst compatible interaction, it was highly transcribed at the later stage of pst development in wheat tissue, but no transcripts were detected in healthy plants, early stages after inoculation and in urediniospores. according to the character of obligate biotrophic fungi, the reason might be that in the initial phase rust infection structures are not in close contact to the plant cells and expression is not triggered. when more infection hyphae and the rust haustoria are formed, a tight contact to plant cells is established and the real biotrophic phases operate. the host secrets higher activities of chitinases to degrade the chitin in fungal cell walls, however, synthesis of fungal chitin deacetylase is elicited to evade the action of host defense.  from a total of ests of high quality, unisequences were obtained from a cdna library of a compatible wheat-pst interaction. the library is useful for identifying the functional genes involved in the wheat-pst compatible interaction, and has established a new database for studying pst pathogenesis genes and wheat defense genes. about 50% of the ests were significantly homologous to genes encoding proteins with known functions; 20% were similar to genes encoding proteins with unknown functions; and 30% did not have significant homology to any sequence in the database. % of the ests had the highest homology with plant genes and % had significant homology to fungal genes. the transcription patterns of seven genes were determined by qrt-pcr assays, revealing their differential expression patterns in wheat-pst compatible and incompatible interaction. 
about 50% of the ests were significantly homologous to genes encoding proteins with known functions; 20% were similar to genes encoding proteins with unknown functions and 30% did not have significant homology to any sequence in the database. the cdna library is useful for identifying the functional genes involved in the wheat-pst compatible interaction, and established a new database for studying pst pathogenesis genes and wheat defense genes. the transcription patterns of seven genes were confirmed by the qrt-pcr assay to be differentially expressed in wheat-pst compatible and incompatible interaction. total rna, extracted from susceptible infected wheat leaves harvested at and days post inoculation, was used to create a cdna library, from which ests with high quality were obtained and clustered into contigs and singletons to give a set of unisequences. the aim of this study was to develop an est database of wheat infected by pst in order to determine transcription profiles of genes involved in compatible wheat-pst interaction. wheat stripe rust, caused by puccinia striiformis f. sp. although compatible interaction between wheat and pst has been studied using various approaches, research on molecular mechanisms of the interaction is limited. the functional classification of all ests was established based on the database entry giving the best e-value using the bevan's classification categories. the quantitative real-time pcr analysis determined the transcription profiles and their involvement in the wheat-pst interaction for seven of the gene. of the unisequences, % were highly homologous to plant genes; % to fungal genes and 30% of no-hit. the blastx program was used to search for homologous genes of the unisequences in the genbank non-redundant protein database. to establish compatibility with the host, pst forms special infection structures to invade the plant with minimal damage to host cells.
 wheat stripe rust, caused by puccinia striiformis f. sp. tritici, is one of the most destructive diseases of wheat worldwide. to establish compatibility with the host, pst forms special infection structures to invade the plant with minimal damage to host cells. although compatible interaction between wheat and pst has been studied using various approaches, research on molecular mechanisms of the interaction is limited. the aim of this study was to develop an est database of wheat infected by pst in order to determine transcription profiles of genes involved in compatible wheat-pst interaction.  total rna, extracted from susceptible infected wheat leaves harvested at and days post inoculation, was used to create a cdna library, from which ests with high quality were obtained and clustered into contigs and singletons to give a set of unisequences. the blastx program was used to search for homologous genes of the unisequences in the genbank non-redundant protein database. of the unisequences, % were highly homologous to plant genes; % to fungal genes and 30% of no-hit. the functional classification of all ests was established based on the database entry giving the best e-value using the bevan's classification categories. about 50% of the ests were significantly homologous to genes encoding proteins with known functions; 20% were similar to genes encoding proteins with unknown functions and 30% did not have significant homology to any sequence in the database. the quantitative real-time pcr analysis determined the transcription profiles and their involvement in the wheat-pst interaction for seven of the gene.  the cdna library is useful for identifying the functional genes involved in the wheat-pst compatible interaction, and established a new database for studying pst pathogenesis genes and wheat defense genes. the transcription patterns of seven genes were confirmed by the qrt-pcr assay to be differentially expressed in wheat-pst compatible and incompatible interaction. 