BACKGROUND
cowpea  is the most important grain legume grown in sub-saharan africa  <cit> .
approximately  <dig>  million tons of cowpea grains are produced worldwide each year, with the majority of the production  taking place on low-input, subsistence farms in west and central africa  <cit> .
in these regions, cowpea is often referred to as “poor man’s meat” because of its high protein content  and good nutritional value  <cit> .
the fruits are consumed at all stages of growth  and the young leaves are often used for soups and stews  <cit> .
in addition to its value as human food, cowpea hay is an important source of animal fodder  <cit> .
two characteristics add to the plant’s agronomic importance: it is generally drought tolerant and it fixes nitrogen symbiotically, thereby enhancing soil fertility, especially when used in rotation with cereals  <cit> .
like most crops, cowpea growth and grain yields are greatly reduced by a variety of biotic pests  and biotic stresses   <cit> .
among the major biotic constraints is parasitism by striga gesnerioides l walp.
, commonly referred to as witch weed.
witch weed is a noxious and persistent pest in farm fields: yield losses due to s. gesnerioides parasitism are extensive in the sudano-sahelian belt of west and central africa  <cit> .
control of the parasite is difficult because it produces thousands of seeds per generation that remain in the seed bank for years and also because most of the damage to its host plant occurs prior to its emergence from the ground  <cit> .
the damaging effects of striga in this region are further compounded by poor soils and drought.
while most cowpea plants are susceptible to striga parasitism, some local landraces and wild accessions have been identified that are resistant and in most reports, resistance is a dominant characteristic, inherited in a monogenic manner  <cit> .
complicating the identification of striga-resistant germplasm is the variable nature of the parasite with at least seven distinct races of s. gesnerioides  now identified throughout west africa  <cit> .
analysis of several advanced populations -- segregating for resistance to one or more of the different races of s. gesnerioides has resulted in the genetic mapping of several race-specific resistance  genes within the cowpea genome and the development of molecular markers linked to these genes  <cit> .
using a positional cloning approach, li and timko  <cit>  isolated and characterized a gene  capable of conferring resistance to s. gesnerioides race  <dig> .
rsg3- <dig> encodes an r protein homolog containing a coiled-coil  protein-protein interaction domain at the n-terminus, a nucleotide binding site , and a leucine-rich repeat domain at the c-terminus.
silencing of rsg3- <dig> expression in the resistant cultivar b <dig> leads to susceptibility to race sg <dig>  but does not affect resistance to other races of the parasite, underscoring the specificity of the resistance response  <cit> .
resistant cowpea genotypes exhibit two different response mechanisms to striga attack.
when challenged by a known race, cultivars carrying the appropriate race-specific resistance gene exhibit a rapid and robust hypersensitive response  typified by browning and necrosis at the site of parasite attachment, and subsequent rapid death of the parasite within 3-4 days  <cit>  .
in host plants lacking the appropriate resistance gene, the parasite rapidly penetrates the host root cortex, forms connections to the host vascular system, swells to form a tubercle, and expands its cotyledons leading to subsequent above ground growth and flowering.
to date, only limited information is available on transcription changes in hosts in response to striga parasitism  <cit> .
to address this gap in our knowledge and to better understand the mechanics of host resistance to parasitic weeds, we have investigated the nature of global gene expression changes that occur during compatible and incompatible s. gesnerioides -cowpea interactions, using a custom designed cowpea nimblegen microarray.
these studies have identified candidate genes and potential pathways involved in resistant and susceptible responses to attempted striga parasitism which can be tested further to gain additional information on this unique plant-plant interaction.
RESULTS
design of cowpea microarray platform
whole genome microarrays provide a means to access gene expression involved in a particular cellular or developmental event on a global scale.
since a microarray platform was not commercially available for cowpea at the time of this study, we designed a  <dig>  feature long-nucleotide custom microarray to interrogate  <dig>  cowpea unigenes of known and unknown function, previously identified in a gene-space sequencing study  <cit> .
on the fabricated microarray, each predicted gene coding region is represented by six to eight 60-mer oligonucleotide probes.
to validate our microarray design, we examined the distribution of mean probe set intensity and probe set variance over all assayed samples  to identify problematic probe sets that failed to hybridize  or hybridized nonspecifically .
the results of this analysis showed that of  <dig>  probe sets on the microarray,  <dig>  probe sets  exhibited intensity profiles consistent with target-specific rna hybridization.
high intra-probe set covariance between individual probe intensities indicate that 75% of probe sets were composed of functional probes whose variation in intensities robustly represented abundance of the intended target rna species .
investigating global changes in gene expression during compatible and incompatible cowpea-striga interactions
to examine the response of cowpea roots to attempted parasitism by s. gesnerioides, we used cowpea cultivar b <dig> as our host plant.
cowpea cultivar b <dig> is a land-race from botswana that was originally identified as resistant to striga in field studies and subsequently shown to be resistant to all races of s. gesnerioides known at the time  <cit> .
however, in the early 1990s, a new hyper virulent race of the parasite , identified in the zakpota region of benin, was discovered to overcome the resistance present in b <dig>  <cit> .
b <dig> still maintains its resistance to all other races outside of this area.
the differential response of b <dig> parasite races provides a good model for examining global changes in gene expression in the same genetic background during compatible/susceptible  and incompatible  interactions .
for our analysis, samples from three biological replicates were collected at two time points .
these time points were chosen on the basis of previous histological studies indicating that the earliest evidence of a hypersensitive response can be found at  <dig> dpi, with complete host cortical root browning and necrosis of the parasite evident by  <dig> dpi  <cit> .
conversely, by  <dig> dpi of compatible interaction, host-parasite vascular connections have been established, and tubercle swelling and parasite cotyledon expansion have initiated.
differential expression analysis was conducted as described in the materials and methods section, and genes were identified as differentially expressed if the adjusted p value was less than or equal to  <dig>  .
based on these criteria, at  <dig> dpi, the expression of  <dig> genes was significantly altered in b <dig> roots infected with s. gesnerioides race sg <dig>  compared to uninfected control roots .
at  <dig> dpi, the number of significantly altered genes increases dramatically to  <dig>  among which  <dig> are genes whose expression was also altered at the earlier  <dig> dpi time point .
a greater proportion  of the differentially regulated genes were upregulated at both the early and late stages in the incompatible interactions.
the large increase in the number of differentially expressed genes for the late time point of the resistant response is not unexpected since while activation of a defense cascade leading to the resistant phenotype and its associated hypersensitive response  is rapid, propagation of the response and subsequent biosynthetic and physiological changes are broader and more persistent.
given in the table the number of significantly  up- and down-regulated genes and the total number of differentially regulated genes in interactions of cowpea b <dig> roots with striga races sg <dig> and sg4z.
in compatible interactions of b <dig> with sg4z,  <dig> genes had their expression altered compared to control roots at  <dig> dpi .
the expression of the majority  of these genes was down-regulated compared to uninfected control roots.
of the differentially regulated genes found,  <dig> genes were common in both resistant and susceptible interactions at  <dig> dpi .
there were  <dig> and  <dig> genes that were unique in the resistant and susceptible interactions, respectively.
the fold-change ratios, adjusted p values, and most significant genbank annotations for selected genes from the various treatments are presented in tables  <dig>   <dig>  and  <dig>  the genes were chosen based on the assumption that genes with the highest fold-change ratio are most likely to have a direct role in the plant-parasite interaction.
*genes verified by qrt pcr.
*genes verified by qrt pcr.
in the incompatible interaction between b <dig> and sg <dig> , among the most highly up-regulated genes at  <dig> dpi are those encoding various transcription factors and signal transduction components, defense-related proteins , and proteins involved in cell wall biogenesis.
among the most highly down-regulated genes at  <dig> dpi are those encoding cell cycle regulators and cellular transporters.
at the later stages  of incompatible interactions , up-regulated gene expression is dominated by genes encoding various transcription factors, signal transduction components, proteins involved in cellular energy metabolism and proteins associated with developmental regulation.
in contrast, in b <dig> roots being successfully parasitized by sg4z , among the most highly down-regulated genes are those encoding defense-related proteins , plant growth regulators , and proteins involved in cell wall biogenesis .
of the various genes up-regulated in this compatible interaction, of particular note are those encoding various nitrogen and sulfur transporters and permeases.
in order to verify that the changes in gene expression detected in our microarray analysis reflect actual changes in transcript levels, quantitative reverse-transcriptase pcr  was carried out using total rnas samples isolated from plant tissue samples from identical treatments harvested independently.
in most cases for the various genes tested by qrt-pcr, the levels of induction or repression  observed were comparable .
co-regulated genes and pathways in incompatible cowpea b301-s. gesnerioides race sg <dig> interactions
to identify genes and pathways that are co-regulated within the various treatments, differentially expressed genes were clustered based on their intensity values profile across treatments and expressed as a colored grid , with genes ordered by a dendrogram representing covariance across treatments .
several clusters of co-regulated genes can be identified within replicates of early  and late  incompatible interactions, and within compatible b301-sg4z interactions.
representative genes from these various clusters on the heatmap that show a full expression profile at the  <dig> % fdr threshold are depicted in additional file  <dig>
we used gorilla  to identify pathways enriched  with differentially expressed genes in the early  incompatible interaction between b <dig> and sg <dig> .
among the biological processes showing the most significant changes in gene expression were pathways associated with cell death, programmed cell death, and apoptosis, as well as pathways associated with response to stress and biotic stress.
we also see significant enrichment of differentially expressed genes associated with cell wall biogenesis and organization, and genes categorized under molecular processes, such as hydrolase activity and catalytic activity.
enrichment in these categories is not unexpected.
one of the key features of incompatible interactions between cowpea and s. gesnerioides is the elicitation of a hypersensitive response when host roots expressing the appropriate race-specific resistance gene are challenged by a parasite with a recognized avirulence factor, such as in the case of b <dig> parasitized by s. gesnerioides race sg <dig>  <cit> .
during the late stages of the b301-sg <dig> incompatible interaction , defense pathways associated with response to a variety of biotic stresses are among the biological processes showing the most significant changes in gene expression .
significant alteration to gene expression was also seen in pathways involved in response to oxidative stress, signaling pathways for secondary metabolism, and pathways involved in multiple plants’ biosynthetic and chemical detoxification processes.
co-regulated genes and pathways in compatible cowpea b301-s. gesnerioides race sg4z interactions
in striking contrast to the response of b <dig> roots to sg <dig>  b <dig> roots parasitized by sg4z show no phenotypic manifestation during parasite ingress through the cortex.
nonetheless, dramatic alterations in gene expression are elicited with the greatest enrichment of differential expression found in pathways associated with cellular differentiation and growth, cell signaling and metabolism, and defense signaling .
these observed changes in cellular function are consistent with the dramatic down-regulation of gene expression involved in auxin transport and signaling, both critical for cellular growth and proliferation, and down-regulation of the expression of genes involved in cell wall growth  and reinforcement .
as it might be expected, b <dig> roots that were actively parasitized by sg4z also had gene expression differences enriched in pathways associated with defense signaling and response to biotic and abiotic stress.
the active suppression of defense response cascades by pest- and pathogen-derived effectors that attack host cellular targets is well characterized; the global alterations in signaling and stress response cascades observed in our studies indicate that similar activities are likely in play in these host-parasite associations.
discussion
gene expression in incompatible striga-host interactions
the mechanics of plant host response to attempted parasitism and the nature of the gene expression changes occurring during both compatible and incompatible interactions between s. gesnerioides and cowpea have been investigated using oligonucleotide-based microarrays.
up until now, only limited information was available on global changes in transcription taking place in hosts in response to striga parasitism.
our data show that alterations, both positive and negative, in a wide range of processes occur as a consequence of striga attack.
in cultivars harboring resistance genes capable of interfacing with a defense signal transduction pathway, as in the case of cowpea cultivar b <dig> which contains multirace resistance to s. gesnerioides, a rapid and robust defense response is elicited in the form of hypersensitivity at the site of parasite attachment, under conditions where the invading parasite is recognized.
we found that accompanying the visible hypersensitive response is the up-regulation of genes involved in signal transduction and biosynthetic processes associated with formation barriers to prevent parasite ingress.
these include activation of gene expression associated with cell wall biogenesis and strengthening , as well as processes leading to programmed cell death at the host-parasite interface.
in a study of the interaction of s. hermonthica with its host plant sorghum, hiraoka and sugimoto  <cit>  observed that attempted parasitism induced ja-responsive genes and suppressed sa-responsive genes in the roots of a highly susceptible cultivar, and suggested that susceptible hosts recognize striga parasitism as wounding stress rather than microbial stress.
in contrast, they found that in the roots of a moderately resistant sorghum cultivar, both sa- and ja-responsive genes were induced suggesting that resistance involved pathways associated with both wounding and pathogen challenge.
pretreatment of the roots of the moderately resistant sorghum cultivar with sa led to an inhibition of striga development suggesting that the sa-responsive genes are directly involved in host resistance mechanism.
in a subsequent study, subtractive hybridization was used to examine the interaction of lotus japonicus with p. aegyptiaca  and s. hermonthica   <cit> .
these investigations found little or no overlap among the phelipanche and striga-induced transcripts, suggesting that l. japonicus roots are able to distinguish the compatible parasite from the incompatible one.
among the genes specifically induced by p. aegyptiaca were those encoding components of ja biosynthesis, whereas s. hermonthica parasitism induced genes in phytoalexin biosynthesis.
examination of the differential expression of genes encoding candidate disease resistance and signal transduction components during the interactions of s. gesnerioides with various cowpea cultivars revealed that pr- <dig> transcript levels were dramatically elevated in the roots of cowpea genotypes resistant to striga compared to uninfected roots and roots challenged with a race of striga to which it was susceptible or adapted to another host species  <cit> .
in contrast, transcript levels of coi <dig> and eds <dig> increased during susceptible and non-host interactions but were unchanged during resistance response.
the coi <dig> gene product plays a pivotal role in activation of ja-mediated response cascades and, in some cases, serves as an inhibitor of sa signaling.
eds <dig> encodes lipase-like proteins that control defense activation and programmed cell death in plants.
induction of coi <dig> gene expression in the compatible striga-cowpea interaction suggests that coi <dig> may down-regulate or suppress the resistance response and block sa signaling pathway in cowpea plants, thus allowing striga attachment and further development.
swarbrick et al.
<cit>  characterized gene expression changes in the roots of the s. hermonthica resistant and susceptible rice cultivars nipponbare and iac  <dig> using a rice microarray.
these investigators found that the levels of a large number of transcripts in rice roots were either positively  or negatively  affected by parasitism.
among the genes up-regulated in the striga-resistant cultivar nipponbare were ones encoding hr protein homologs; pr-proteins associated with microbial pathogenesis including endochitinases , glucanases , and thaumatin-like proteins ; pleiotropic drug resistance abc transporters; and enzymes in phenylpropanoid metabolism.
in addition, transcripts encoding several wrky transcription factors , previously implicated in other sa-dependent resistance responses  <cit> , were observed to be more abundant in parasitized roots.
large-scale down-regulation of gene expression was observed in the susceptible cultivar iac  <dig>  particularly transcripts whose encoded products annotate to gene ontology  functional categories of plant growth regulator signaling and metabolism, biogenesis of cellular components, and cell division.
interestingly, a majority of the genes down-regulated following attempted striga parasitism in both iac <dig> and nipponbare roots encodes products that annotate as proteins of unknown function.
a recent study that used whole genome oligonucleotide arrays to examine the transcriptome of a. thaliana roots after inoculation with pre-germinated s. hermonthica seeds found that large numbers of genes involved in cell wall synthesis, defense signaling, regulation of transcription and protein synthesis, oxidative stress, and primary and secondary metabolism were up-regulated at the earliest stages of parasite infection  <cit> .
this included up-regulation of many genes  involved in the sa signaling pathway, as well as the up-regulation of a key wrky transcription factor  that regulates the expression of genes involved in the sa signaling pathway and is thought to have a role in determining the balance between sa and ja signaling.
in addition, there was evidence for the activation of genes involved in the ja and ethylene biosynthetic pathways.
in the present study of cowpea-s. gesnerioides interactions, among the most highly induced genes in the early resistance response  of b <dig> to sg <dig> are those involved in response to biotic stimuli, abiotic stimuli, wounding, oxidative stress and hr, and components of the ja and eth signaling pathways.
among the genes most highly induced were a number of chitinases and chitinase homologs including narbonin, a protein with glycosyl hydrolase activity  <cit>  whose biological function has yet to be elucidated  <cit> .
narbonin has been previously suggested as interfering with parasite growth in the root by altering cell wall extensibility  <cit> .
lignifications and callused deposition have been previously observed at the interface of host plant cells with invading orobanche haustorium  <cit>  and is thought to be correlated with levels of host resistance to orobanche.
we similarly observed an increased expression of genes involved in cell wall biogenesis suggesting that lignifications may be part the mechanism of defense employed by cowpea against s. gesnerioides ingress.
a number of cytochrome p450s, previously implicated in various cellular detoxification pathways in plants, were also found to be induced during the resistance response of b <dig>  increased cytochrome p <dig> gene expression has been previously reported during the resistance reaction of rice infected by s. hermonthica <cit> .
the only other global study of cowpea transcriptional profiling published thus far is the work by das et al.
<cit>  who investigated differences in gene expression in resistant and susceptible cowpea genotypes elicited by feeding of the root-nematode, meloidogyne incognita, using a soybean affymetrix gene chip expression array.
at  <dig> dpi in both compatible and incompatible interactions, more genes were down-regulated than up-regulated.
when expression between infected resistant and susceptible genotypes was compared, a greater number and proportion of genes were down-regulated in the resistant than in the susceptible genotype, whereas more genes were up-regulated in the susceptible than in the resistant genotype in response to nematode infection.
gene ontology based functional categorization revealed that the typical defense response was partially suppressed in resistant roots, allowing nematode juvenile development.
das et al.
<cit>  suggests that suppression of genes in ros formation and other defense related responses might be important negative resistance mechanism.
our findings contrast with this, since we see activation of a wide range of processes as a consequence of striga attack.
direct comparisons of gene lists suggest that in both early stages  of incompatible and compatible interactions there are many induced genes common in both host-parasite interactions suggesting that these may be part of a basal defense response.
among these were genes involved in lignifications and cross-linking.
this response leads to heavy lignifications of cell walls and creates a mechanical barrier for the pathogen.
in the incompatible cowpea-nematode interaction, expansin, thought to be important for maintaining the specialized feeding structures in hosts, was highly down-regulated.
this was not observed in the cowpea-striga interaction but down-regulation of expansins were observed in the response of a resistant rice cultivar to attack by s. hermonthica.
gene expression in compatible striga-host interactions
we know that root parasitic angiosperms secrete pectin methylesterases, polygalacturonase, and other cell wall softening enzymes during attempted penetration of the host root cortex, and that such secretions may in fact assist in overcoming the cell wall reinforcement that occurs in the host root in response to parasite parasitism  <cit> .
it is now evident that many different phytopathogens have also evolved specific effectors and virulence factors that are capable of entering the host cell and suppressing the host resistance machinery or bypassing surveillance  <cit> .
among the most highly down-regulated genes in b <dig> roots during a susceptible interaction with sg4z were genes in the phenylpropanoid and lignin biosynthesis pathways, primary and secondary cell wall biogenesis, and components of the sa and ja signal transduction pathways.
s. gesnerioides-infected cowpea often demonstrated severe reduction in the growth rate and reduction of the host biomass  <cit> .
this was due to two major factors: direct carbon transfer from the host to s. gesnerioides, and reduction of photosynthetic capacity  <cit> .
one of the noticeable changes in gene expression during the susceptible response in b <dig> was the suppression of plant growth regulators, particularly auxin and gibberellins.
some genes, such as cellulose synthase, known to be involved in cell wall development  <cit>  were also observed to be down-regulated.
one of the keys to the success of a parasitic plant-host interaction is the translocation of water and nutrients  from host to parasite through the haustoria  <cit> .
the identification of differentially up-regulated transcripts for transporters responsible for nitrogen, sulfur and amino acids in b <dig> roots during susceptible interactions with invading sg4z suggests that, in addition to suppressing some host functions to facilitate entry, the parasite is also modifying others to provide a source of nutrition.
this is clearly an area that warrants additional study.
CONCLUSIONS
distinct changes in global gene expression profiles occur in host roots following successful and unsuccessful attempted parasitism by striga.
induction of specific defense-related genes and pathways defines components of a unique resistance mechanism.
some genes and pathways up-regulated in the host resistance response to sg <dig> are repressed in the susceptible interactions, suggesting that the parasite is targeting specific components of host defense.
these results add to our understanding of plant-parasite interactions and the evolution of resistance to parasitic weeds.

 cowpea is the most important grain legume grown in sub-saharan africa. approximately million tons of cowpea grains are produced worldwide each year, with the majority of the production taking place on low-input, subsistence farms in west and central africa. in these regions, cowpea is often referred to as “poor man’s meat” because of its high protein content and good nutritional value. the fruits are consumed at all stages of growth and the young leaves are often used for soups and stews. in addition to its value as human food, cowpea hay is an important source of animal fodder. two characteristics add to the plant’s agronomic importance: it is generally drought tolerant and it fixes nitrogen symbiotically, thereby enhancing soil fertility, especially when used in rotation with cereals. like most crops, cowpea growth and grain yields are greatly reduced by a variety of biotic pests and biotic stresses. among the major biotic constraints is parasitism by striga gesnerioides l walp., commonly referred to as witch weed. witch weed is a noxious and persistent pest in farm fields: yield losses due to s. gesnerioides parasitism are extensive in the sudano-sahelian belt of west and central africa. control of the parasite is difficult because it produces thousands of seeds per generation that remain in the seed bank for years and also because most of the damage to its host plant occurs prior to its emergence from the ground. the damaging effects of striga in this region are further compounded by poor soils and drought. while most cowpea plants are susceptible to striga parasitism, some local landraces and wild accessions have been identified that are resistant and in most reports, resistance is a dominant characteristic, inherited in a monogenic manner. complicating the identification of striga-resistant germplasm is the variable nature of the parasite with at least seven distinct races of s. gesnerioides now identified throughout west africa. analysis of several advanced populations -- segregating for resistance to one or more of the different races of s. gesnerioides has resulted in the genetic mapping of several race-specific resistance genes within the cowpea genome and the development of molecular markers linked to these genes. using a positional cloning approach, li and timko isolated and characterized a gene capable of conferring resistance to s. gesnerioides race. rsg3- encodes an r protein homolog containing a coiled-coil protein-protein interaction domain at the n-terminus, a nucleotide binding site, and a leucine-rich repeat domain at the c-terminus. silencing of rsg3- expression in the resistant cultivar b leads to susceptibility to race sg but does not affect resistance to other races of the parasite, underscoring the specificity of the resistance response. resistant cowpea genotypes exhibit two different response mechanisms to striga attack. when challenged by a known race, cultivars carrying the appropriate race-specific resistance gene exhibit a rapid and robust hypersensitive response typified by browning and necrosis at the site of parasite attachment, and subsequent rapid death of the parasite within 3-4 days. in host plants lacking the appropriate resistance gene, the parasite rapidly penetrates the host root cortex, forms connections to the host vascular system, swells to form a tubercle, and expands its cotyledons leading to subsequent above ground growth and flowering. to date, only limited information is available on transcription changes in hosts in response to striga parasitism. to address this gap in our knowledge and to better understand the mechanics of host resistance to parasitic weeds, we have investigated the nature of global gene expression changes that occur during compatible and incompatible s. gesnerioides -cowpea interactions, using a custom designed cowpea nimblegen microarray. these studies have identified candidate genes and potential pathways involved in resistant and susceptible responses to attempted striga parasitism which can be tested further to gain additional information on this unique plant-plant interaction.  design of cowpea microarray platform whole genome microarrays provide a means to access gene expression involved in a particular cellular or developmental event on a global scale. since a microarray platform was not commercially available for cowpea at the time of this study, we designed a feature long-nucleotide custom microarray to interrogate cowpea unigenes of known and unknown function, previously identified in a gene-space sequencing study. on the fabricated microarray, each predicted gene coding region is represented by six to eight 60-mer oligonucleotide probes. to validate our microarray design, we examined the distribution of mean probe set intensity and probe set variance over all assayed samples to identify problematic probe sets that failed to hybridize or hybridized nonspecifically. the results of this analysis showed that of probe sets on the microarray, probe sets exhibited intensity profiles consistent with target-specific rna hybridization. high intra-probe set covariance between individual probe intensities indicate that 75% of probe sets were composed of functional probes whose variation in intensities robustly represented abundance of the intended target rna species. investigating global changes in gene expression during compatible and incompatible cowpea-striga interactions to examine the response of cowpea roots to attempted parasitism by s. gesnerioides, we used cowpea cultivar b as our host plant. cowpea cultivar b is a land-race from botswana that was originally identified as resistant to striga in field studies and subsequently shown to be resistant to all races of s. gesnerioides known at the time. however, in the early 1990s, a new hyper virulent race of the parasite, identified in the zakpota region of benin, was discovered to overcome the resistance present in b. b still maintains its resistance to all other races outside of this area. the differential response of b parasite races provides a good model for examining global changes in gene expression in the same genetic background during compatible/susceptible and incompatible interactions. for our analysis, samples from three biological replicates were collected at two time points. these time points were chosen on the basis of previous histological studies indicating that the earliest evidence of a hypersensitive response can be found at dpi, with complete host cortical root browning and necrosis of the parasite evident by dpi. conversely, by dpi of compatible interaction, host-parasite vascular connections have been established, and tubercle swelling and parasite cotyledon expansion have initiated. differential expression analysis was conducted as described in the materials and methods section, and genes were identified as differentially expressed if the adjusted p value was less than or equal to. based on these criteria, at dpi, the expression of genes was significantly altered in b roots infected with s. gesnerioides race sg compared to uninfected control roots. at dpi, the number of significantly altered genes increases dramatically to among which are genes whose expression was also altered at the earlier dpi time point. a greater proportion of the differentially regulated genes were upregulated at both the early and late stages in the incompatible interactions. the large increase in the number of differentially expressed genes for the late time point of the resistant response is not unexpected since while activation of a defense cascade leading to the resistant phenotype and its associated hypersensitive response is rapid, propagation of the response and subsequent biosynthetic and physiological changes are broader and more persistent. given in the table the number of significantly up- and down-regulated genes and the total number of differentially regulated genes in interactions of cowpea b roots with striga races sg and sg4z. in compatible interactions of b with sg4z, genes had their expression altered compared to control roots at dpi. the expression of the majority of these genes was down-regulated compared to uninfected control roots. of the differentially regulated genes found, genes were common in both resistant and susceptible interactions at dpi. there were and genes that were unique in the resistant and susceptible interactions, respectively. the fold-change ratios, adjusted p values, and most significant genbank annotations for selected genes from the various treatments are presented in tables  and the genes were chosen based on the assumption that genes with the highest fold-change ratio are most likely to have a direct role in the plant-parasite interaction. *genes verified by qrt pcr. *genes verified by qrt pcr. in the incompatible interaction between b and sg, among the most highly up-regulated genes at dpi are those encoding various transcription factors and signal transduction components, defense-related proteins, and proteins involved in cell wall biogenesis. among the most highly down-regulated genes at dpi are those encoding cell cycle regulators and cellular transporters. at the later stages of incompatible interactions, up-regulated gene expression is dominated by genes encoding various transcription factors, signal transduction components, proteins involved in cellular energy metabolism and proteins associated with developmental regulation. in contrast, in b roots being successfully parasitized by sg4z, among the most highly down-regulated genes are those encoding defense-related proteins, plant growth regulators, and proteins involved in cell wall biogenesis. of the various genes up-regulated in this compatible interaction, of particular note are those encoding various nitrogen and sulfur transporters and permeases. in order to verify that the changes in gene expression detected in our microarray analysis reflect actual changes in transcript levels, quantitative reverse-transcriptase pcr was carried out using total rnas samples isolated from plant tissue samples from identical treatments harvested independently. in most cases for the various genes tested by qrt-pcr, the levels of induction or repression observed were comparable. co-regulated genes and pathways in incompatible cowpea b301-s. gesnerioides race sg interactions to identify genes and pathways that are co-regulated within the various treatments, differentially expressed genes were clustered based on their intensity values profile across treatments and expressed as a colored grid, with genes ordered by a dendrogram representing covariance across treatments. several clusters of co-regulated genes can be identified within replicates of early and late incompatible interactions, and within compatible b301-sg4z interactions. representative genes from these various clusters on the heatmap that show a full expression profile at the % fdr threshold are depicted in additional file we used gorilla to identify pathways enriched with differentially expressed genes in the early incompatible interaction between b and sg. among the biological processes showing the most significant changes in gene expression were pathways associated with cell death, programmed cell death, and apoptosis, as well as pathways associated with response to stress and biotic stress. we also see significant enrichment of differentially expressed genes associated with cell wall biogenesis and organization, and genes categorized under molecular processes, such as hydrolase activity and catalytic activity. enrichment in these categories is not unexpected. one of the key features of incompatible interactions between cowpea and s. gesnerioides is the elicitation of a hypersensitive response when host roots expressing the appropriate race-specific resistance gene are challenged by a parasite with a recognized avirulence factor, such as in the case of b parasitized by s. gesnerioides race sg. during the late stages of the b301-sg incompatible interaction, defense pathways associated with response to a variety of biotic stresses are among the biological processes showing the most significant changes in gene expression. significant alteration to gene expression was also seen in pathways involved in response to oxidative stress, signaling pathways for secondary metabolism, and pathways involved in multiple plants’ biosynthetic and chemical detoxification processes. co-regulated genes and pathways in compatible cowpea b301-s. gesnerioides race sg4z interactions in striking contrast to the response of b roots to sg b roots parasitized by sg4z show no phenotypic manifestation during parasite ingress through the cortex. nonetheless, dramatic alterations in gene expression are elicited with the greatest enrichment of differential expression found in pathways associated with cellular differentiation and growth, cell signaling and metabolism, and defense signaling. these observed changes in cellular function are consistent with the dramatic down-regulation of gene expression involved in auxin transport and signaling, both critical for cellular growth and proliferation, and down-regulation of the expression of genes involved in cell wall growth and reinforcement. as it might be expected, b roots that were actively parasitized by sg4z also had gene expression differences enriched in pathways associated with defense signaling and response to biotic and abiotic stress. the active suppression of defense response cascades by pest- and pathogen-derived effectors that attack host cellular targets is well characterized; the global alterations in signaling and stress response cascades observed in our studies indicate that similar activities are likely in play in these host-parasite associations. discussion gene expression in incompatible striga-host interactions the mechanics of plant host response to attempted parasitism and the nature of the gene expression changes occurring during both compatible and incompatible interactions between s. gesnerioides and cowpea have been investigated using oligonucleotide-based microarrays. up until now, only limited information was available on global changes in transcription taking place in hosts in response to striga parasitism. our data show that alterations, both positive and negative, in a wide range of processes occur as a consequence of striga attack. in cultivars harboring resistance genes capable of interfacing with a defense signal transduction pathway, as in the case of cowpea cultivar b which contains multirace resistance to s. gesnerioides, a rapid and robust defense response is elicited in the form of hypersensitivity at the site of parasite attachment, under conditions where the invading parasite is recognized. we found that accompanying the visible hypersensitive response is the up-regulation of genes involved in signal transduction and biosynthetic processes associated with formation barriers to prevent parasite ingress. these include activation of gene expression associated with cell wall biogenesis and strengthening, as well as processes leading to programmed cell death at the host-parasite interface. in a study of the interaction of s. hermonthica with its host plant sorghum, hiraoka and sugimoto observed that attempted parasitism induced ja-responsive genes and suppressed sa-responsive genes in the roots of a highly susceptible cultivar, and suggested that susceptible hosts recognize striga parasitism as wounding stress rather than microbial stress. in contrast, they found that in the roots of a moderately resistant sorghum cultivar, both sa- and ja-responsive genes were induced suggesting that resistance involved pathways associated with both wounding and pathogen challenge. pretreatment of the roots of the moderately resistant sorghum cultivar with sa led to an inhibition of striga development suggesting that the sa-responsive genes are directly involved in host resistance mechanism. in a subsequent study, subtractive hybridization was used to examine the interaction of lotus japonicus with p. aegyptiaca and s. hermonthica. these investigations found little or no overlap among the phelipanche and striga-induced transcripts, suggesting that l. japonicus roots are able to distinguish the compatible parasite from the incompatible one. among the genes specifically induced by p. aegyptiaca were those encoding components of ja biosynthesis, whereas s. hermonthica parasitism induced genes in phytoalexin biosynthesis. examination of the differential expression of genes encoding candidate disease resistance and signal transduction components during the interactions of s. gesnerioides with various cowpea cultivars revealed that pr- transcript levels were dramatically elevated in the roots of cowpea genotypes resistant to striga compared to uninfected roots and roots challenged with a race of striga to which it was susceptible or adapted to another host species. in contrast, transcript levels of coi and eds increased during susceptible and non-host interactions but were unchanged during resistance response. the coi gene product plays a pivotal role in activation of ja-mediated response cascades and, in some cases, serves as an inhibitor of sa signaling. eds encodes lipase-like proteins that control defense activation and programmed cell death in plants. induction of coi gene expression in the compatible striga-cowpea interaction suggests that coi may down-regulate or suppress the resistance response and block sa signaling pathway in cowpea plants, thus allowing striga attachment and further development. swarbrick et al. characterized gene expression changes in the roots of the s. hermonthica resistant and susceptible rice cultivars nipponbare and iac using a rice microarray. these investigators found that the levels of a large number of transcripts in rice roots were either positively or negatively affected by parasitism. among the genes up-regulated in the striga-resistant cultivar nipponbare were ones encoding hr protein homologs; pr-proteins associated with microbial pathogenesis including endochitinases, glucanases, and thaumatin-like proteins; pleiotropic drug resistance abc transporters; and enzymes in phenylpropanoid metabolism. in addition, transcripts encoding several wrky transcription factors, previously implicated in other sa-dependent resistance responses, were observed to be more abundant in parasitized roots. large-scale down-regulation of gene expression was observed in the susceptible cultivar iac particularly transcripts whose encoded products annotate to gene ontology functional categories of plant growth regulator signaling and metabolism, biogenesis of cellular components, and cell division. interestingly, a majority of the genes down-regulated following attempted striga parasitism in both iac and nipponbare roots encodes products that annotate as proteins of unknown function. a recent study that used whole genome oligonucleotide arrays to examine the transcriptome of a. thaliana roots after inoculation with pre-germinated s. hermonthica seeds found that large numbers of genes involved in cell wall synthesis, defense signaling, regulation of transcription and protein synthesis, oxidative stress, and primary and secondary metabolism were up-regulated at the earliest stages of parasite infection. this included up-regulation of many genes involved in the sa signaling pathway, as well as the up-regulation of a key wrky transcription factor that regulates the expression of genes involved in the sa signaling pathway and is thought to have a role in determining the balance between sa and ja signaling. in addition, there was evidence for the activation of genes involved in the ja and ethylene biosynthetic pathways. in the present study of cowpea-s. gesnerioides interactions, among the most highly induced genes in the early resistance response of b to sg are those involved in response to biotic stimuli, abiotic stimuli, wounding, oxidative stress and hr, and components of the ja and eth signaling pathways. among the genes most highly induced were a number of chitinases and chitinase homologs including narbonin, a protein with glycosyl hydrolase activity whose biological function has yet to be elucidated. narbonin has been previously suggested as interfering with parasite growth in the root by altering cell wall extensibility. lignifications and callused deposition have been previously observed at the interface of host plant cells with invading orobanche haustorium and is thought to be correlated with levels of host resistance to orobanche. we similarly observed an increased expression of genes involved in cell wall biogenesis suggesting that lignifications may be part the mechanism of defense employed by cowpea against s. gesnerioides ingress. a number of cytochrome p450s, previously implicated in various cellular detoxification pathways in plants, were also found to be induced during the resistance response of b increased cytochrome p gene expression has been previously reported during the resistance reaction of rice infected by s. hermonthica. the only other global study of cowpea transcriptional profiling published thus far is the work by das et al. who investigated differences in gene expression in resistant and susceptible cowpea genotypes elicited by feeding of the root-nematode, meloidogyne incognita, using a soybean affymetrix gene chip expression array. at dpi in both compatible and incompatible interactions, more genes were down-regulated than up-regulated. when expression between infected resistant and susceptible genotypes was compared, a greater number and proportion of genes were down-regulated in the resistant than in the susceptible genotype, whereas more genes were up-regulated in the susceptible than in the resistant genotype in response to nematode infection. gene ontology based functional categorization revealed that the typical defense response was partially suppressed in resistant roots, allowing nematode juvenile development. das et al. suggests that suppression of genes in ros formation and other defense related responses might be important negative resistance mechanism. our findings contrast with this, since we see activation of a wide range of processes as a consequence of striga attack. direct comparisons of gene lists suggest that in both early stages of incompatible and compatible interactions there are many induced genes common in both host-parasite interactions suggesting that these may be part of a basal defense response. among these were genes involved in lignifications and cross-linking. this response leads to heavy lignifications of cell walls and creates a mechanical barrier for the pathogen. in the incompatible cowpea-nematode interaction, expansin, thought to be important for maintaining the specialized feeding structures in hosts, was highly down-regulated. this was not observed in the cowpea-striga interaction but down-regulation of expansins were observed in the response of a resistant rice cultivar to attack by s. hermonthica. gene expression in compatible striga-host interactions we know that root parasitic angiosperms secrete pectin methylesterases, polygalacturonase, and other cell wall softening enzymes during attempted penetration of the host root cortex, and that such secretions may in fact assist in overcoming the cell wall reinforcement that occurs in the host root in response to parasite parasitism. it is now evident that many different phytopathogens have also evolved specific effectors and virulence factors that are capable of entering the host cell and suppressing the host resistance machinery or bypassing surveillance. among the most highly down-regulated genes in b roots during a susceptible interaction with sg4z were genes in the phenylpropanoid and lignin biosynthesis pathways, primary and secondary cell wall biogenesis, and components of the sa and ja signal transduction pathways. s. gesnerioides-infected cowpea often demonstrated severe reduction in the growth rate and reduction of the host biomass. this was due to two major factors: direct carbon transfer from the host to s. gesnerioides, and reduction of photosynthetic capacity. one of the noticeable changes in gene expression during the susceptible response in b was the suppression of plant growth regulators, particularly auxin and gibberellins. some genes, such as cellulose synthase, known to be involved in cell wall development were also observed to be down-regulated. one of the keys to the success of a parasitic plant-host interaction is the translocation of water and nutrients from host to parasite through the haustoria. the identification of differentially up-regulated transcripts for transporters responsible for nitrogen, sulfur and amino acids in b roots during susceptible interactions with invading sg4z suggests that, in addition to suppressing some host functions to facilitate entry, the parasite is also modifying others to provide a source of nutrition. this is clearly an area that warrants additional study.  distinct changes in global gene expression profiles occur in host roots following successful and unsuccessful attempted parasitism by striga. induction of specific defense-related genes and pathways defines components of a unique resistance mechanism. some genes and pathways up-regulated in the host resistance response to sg are repressed in the susceptible interactions, suggesting that the parasite is targeting specific components of host defense. these results add to our understanding of plant-parasite interactions and the evolution of resistance to parasitic weeds. 
cowpea cultivar b contains the rsg3- gene for resistance to s. gesnerioides race sg but is susceptible to race sg4z. gene expression in the roots of the cowpea cultivar b during compatible and incompatible interactions with s. gesnerioides races sg4z and sg respectively, were investigated at the early ) and late stages of the resistance response using a nimblegen custom design cowpea microarray. induction of specific defense related genes and pathways defines components of a unique resistance mechanism. when challenged by sg roots of cultivar b develop a strong resistance response characterized by a hypersensitive reaction and cell death at the site of parasite attachment. genes and pathways involved in signal transduction, programmed cell death and apoptosis, and defense response to biotic and abiotic stress were differentially expressed in the early resistance response; at the later time point, enrichment was primarily for defense-related gene expression, and genes encoding components of lignifications and secondary wall formation. some genes and pathways up-regulated in the host resistance response to sg are repressed in the susceptible interactions, suggesting that the parasite is targeting specific components of the host’s defense. these results add to our understanding of plant-parasite interactions and the evolution of resistance to parasitic weeds. distinct changes in global gene expression profiles occur in host roots following successful and unsuccessful attempted parasitism by striga. in compatible interactions, multiple defense pathways were repressed, including those involved in lignin biosynthesis and secondary cell wall modifications, while cellular transport processes for nitrogen and sulfur were increased.
 cowpea, vigna unguiculata l. walp., is one of the most important food and forage legumes in the semi-arid tropics. while most domesticated forms of cowpea are susceptible to the root parasitic weed striga gesnerioides, several cultivars have been identified that show race-specific resistance. cowpea cultivar b contains the rsg3- gene for resistance to s. gesnerioides race sg but is susceptible to race sg4z. when challenged by sg roots of cultivar b develop a strong resistance response characterized by a hypersensitive reaction and cell death at the site of parasite attachment. in contrast, no visible response occurs in b roots parasitized by sg4z.  gene expression in the roots of the cowpea cultivar b during compatible and incompatible interactions with s. gesnerioides races sg4z and sg respectively, were investigated at the early ) and late stages of the resistance response using a nimblegen custom design cowpea microarray. a total of genes were differentially expressed in b roots at dpi; this number increased to genes at dpi. at dpi, a total of genes were differentially expressed during compatible interactions of b with sg4z. genes and pathways involved in signal transduction, programmed cell death and apoptosis, and defense response to biotic and abiotic stress were differentially expressed in the early resistance response; at the later time point, enrichment was primarily for defense-related gene expression, and genes encoding components of lignifications and secondary wall formation. in compatible interactions, multiple defense pathways were repressed, including those involved in lignin biosynthesis and secondary cell wall modifications, while cellular transport processes for nitrogen and sulfur were increased.  distinct changes in global gene expression profiles occur in host roots following successful and unsuccessful attempted parasitism by striga. induction of specific defense related genes and pathways defines components of a unique resistance mechanism. some genes and pathways up-regulated in the host resistance response to sg are repressed in the susceptible interactions, suggesting that the parasite is targeting specific components of the host’s defense. these results add to our understanding of plant-parasite interactions and the evolution of resistance to parasitic weeds. cowpeadefenseresistancestriga gesnerioidestranscriptionvigna unguiculatawitchweed 