BACKGROUND
molecular recognition of pathogenic microorganisms triggers in plants comprehensive transcriptional reprogramming.
a network of defense-regulators transduces information about the attacking microbe into appropriate transcriptional responses.
two functionally distinct classes of pathogen molecules are known to elicit plant immune responses  <cit> .
receptor-mediated recognition of pathogen/microbe associated molecular patterns  activates pamp-triggered immunity , while recognition of pathogen effectors by plant disease resistance  proteins leads to effector-triggered immunity .
eti is a strong immune response that results in incompatible plant pathogen interactions where the plant is resistant and the pathogen is avirulent.
effectors are pathogen-derived molecules  that are secreted into host tissues  <cit> .
some effectors have been shown to suppress pti resulting in a weakened immune response called basal defense which is usually insufficient to halt growth and spread of the respective microbe.
the typical outcome in this case is a compatible interaction, where the plant is susceptible and the pathogen is virulent.
a key player in the regulation of eti as well as basal defense is the hormone salicylic acid , which accumulates in infected tissues of several plant species and triggers downstream defense responses  <cit> .
several other small molecules and multiple proteins involved in defense regulation have been identified by genetic and biochemical studies  <cit> .
in particular the immune system of arabidopsis thaliana  has been extensively examined over the past two to three decades.
while our knowledge about the arabidopsis immune system is far from complete, it has become obvious in recent years, that its individual components are functionally interconnected in a complex manner and constitute a regulatory network rather than simple linear pathways.
crosstalk between distinct branches of this network has been found to be involved in fine-tuning defense outputs and maximizing the effectiveness of immune responses  <cit> .
for example, sophisticated mechanisms mediating crosstalk between sa and jasmonic acid -dependent signaling processes can result in synergistic or antagonistic effects depending on the levels of each of these hormones  <cit> .
in addition, there are extensive interactions of defense signaling with regulatory processes that are not primarily involved in plant immune responses.
for example, crosstalk between defense mechanisms dependent on the defense hormones sa and ja on the one hand and each of the classical growth-controlling phytohormones, auxin, gibberellin, aba, cytokinin or brassinosteroids on the other hand have been reported  <cit> .
details of these interactions are not well understood yet, but it seems that some of them benefit the host by enhancing immune responses, while others are beneficial for the pathogen by suppressing defense reactions.
in addition, several labs reported interferences between light and defense signaling.
depending on the type of light signaling pathway involved, these interactions were found to be synergistic or antagonistic.
for example, phytochrome-mediated light signaling can positively affect sa-dependent defense responses  <cit> , while negative interferences were observed between defense and uv-light signaling  <cit> .
generally, these interactions seem to control resource allocation in the plant ensuring the maintenance of homeostasis and the adjustment of the host's metabolism to a state optimal to cope with the respective environmental challenges.
we are using interactions between the pathogenic oomycete hyaloperonospora parasitica  and arabidopsis to examine regulatory mechanisms operating at the interface between defense signaling and the regulation of the defense transcriptome  <cit> .
several transcriptional regulators have been implicated in these processes, such as npr <dig>  a nuclear transported transcriptional cofactor that acts downstream from sa and interacts with tga-bzip transcription factors  <cit> .
additional transcription factors, including wrkys and erfs, participate in the regulation of the plant defense transcriptome and disease resistance  <cit> .
the dna binding site preferences of these transcription factors have been well characterized.
typically tga-bzips bind to tga boxes , wrkys to w boxes  and erfs to gcc boxes   <cit> .
these promoter sites have been shown in numerous studies to act as pathogen-responsive cis-elements mediating expression of individual defense-associated genes  <cit> .
an enormous amount of global gene expression data from large-scale transcript profiling projects has accumulated during the past decade.
these data allowed for the identification of gene clusters that are co-expressed under certain biological conditions and, hence, are likely to be subject to co-regulation by common mechanisms.
several studies revealed conserved sequence motifs in promoters of such co-regulated genes which may act as cis-elements mediating their coordinated activity  <cit> .
in addition to already known cis-elements, some novel promoter motifs were found to be statistically enriched in co-expressed genes  <cit> .
while in some cases, their function has been experimentally proven  <cit> , reports confirming that such conserved promoter motifs are binding sites of nuclear proteins that can affect gene expression are still rare.
here we report on the identification and functional characterization of a motif conserved in promoters of the forc cluster, a set of arabidopsis genes co-expressed in response to recognition of hp as well as other oomycete and fungal pathogens.
we found this motif, which we termed forca, to exhibit dna sequence specific and differential interactions with nuclear arabidopsis proteins.
furthermore, electrophoretic mobility shift assays as well as reporter gene assays showed that forca integrates stimuli related to defense- and light signaling.
forca acts as a light-responsive enhancer, the activity of which is modulated by pathogen-perception.
under normal light conditions as well as constant darkness, forca activity is reduced by recognition of hp as well as application of sa, while under constant light conditions its activity is enhanced by defense induction.
consistent with the observed functional connection between light and defense signaling, we observed that plants kept under constant light exposure exhibited reduced susceptibility to a virulent hp isolate.
RESULTS
a cluster of arabidopsis genes co-expressed during interactions with oomycete and fungal pathogens is enriched for the forca promoter motif
using affymetrix microarrays we previously identified clusters of genes that are co-expressed during arabidopsis immune responses  <cit> .
two of these clusters, cluster i and the lurp cluster , were defined by distinct patterns of coordinated transcriptional up-regulation during incompatible and compatible interactions with the hp isolates hpemoy <dig>  hphiks <dig> and hpemco <dig>  while lurp transcripts exhibited a kinetic pattern of late and sustained up-regulation accumulating strongly between  <dig> and  <dig> hpi  <cit> , cluster i transcript levels tended to increase earlier and predominantly accumulated within the first  <dig> h after infection  <cit> .
genetic disruption of sa-signaling and r protein-mediated hp recognition strongly attenuated or delayed the response of lurp genes, but had only a minor effect on cluster i responses.
inspecting publicly accessible microarray data   <cit>  we found that cluster i genes also exhibit a pronounced pattern of co-activation during interactions of arabidopsis with the fungal pathogen bortytis cinerea as well as a second oomycete, phythophthora infestans .
therefore we renamed cluster i to forc .
although lurp genes are also coordinately activated in response to the oomycete phytohthora infestans, this set does not show an uniform response to fungal pathogens .
tightly co-expressed subsets of both the forc and lurp genes were found to be enriched for distinct promoter motifs, such as potential binding sites of wrky transcription factors  <cit> .
this suggested that members of the forc cluster on the one hand and the lurp cluster on the other hand are controlled by common regulatory mechanisms.
the fact that different motifs are conserved in the promoters of each of these two gene sets suggested that the forc genes are controlled by mechanisms distinct from those controlling the lurp genes.
applying alignace  <cit>  to the  <dig> bp-upstream sequences of a strictly co-expressed subset of forc genes  we found the hexameric motif t/atgggc to be significantly enriched compared to its statistically expected frequency .
we termed this motif forca.
forca specifically interacts with a nuclear dna binding activity that is down-regulated by hp and sa
t/atgggc-containing promoter motifs have not been described as pathogen-response elements.
therefore we tested by electrophoretic mobility shift assays , if forca exhibits hp-dependent differential interactions with nuclear arabidopsis proteins .
using the radioactively labeled forca- <dig> probe consisting of a single copy of the forca consensus sequence with the ttgggc core and  <dig> bp of arbitrary flanking sequence we detected a constitutive dna-binding activity producing a single band shift that is out-competed by unlabeled forca- <dig> probe .
surprisingly, this forca binding activity was down-regulated in response to infections with the avirulent hp isolates hphiks <dig> or hpemoy <dig> as well as exogenously applied sa.
furthermore, we found forca/nuclear protein interactions to be light dependent.
nuclear extracts from plants kept for three days under constant light produced substantially more intense forca-shifts than those from plants kept for the same time in constant darkness .
besides unlabeled forca- <dig> probe, unlabeled competitor probes containing wild-type forca permutations present in several forc promoters  clearly reduced the intensity of forca- <dig> shifts .
mutations in the sequences flanking the invariant tgggc core of forca- <dig> did not result in reduced competition.
however, the forca- <dig> muta competitor probe, that lacks the tgggc core, as well as competitor probes with sequences unrelated to forca  did not efficiently reduce the intensity of forca- <dig> band shifts.
these results showed that the observed forca-binding activity is dna-sequence specific and that only the tgggc sequence of forca and not sequences flanking this invariant core are critical for this interaction.
forca is a light-responsive promoter element whose activity is modulated by defense-related stimuli
to test effects of forca on transcriptional regulation, we fused a forca trimer consisting of three distinct forca permutations to the minimal camv35s promoter followed by gus in pcambia1281x .
untreated nicotiana benthamiana leaves transiently transformed with 3xforca-pcambia by agrobacterium tumefaciens exhibited strong gus activity after histochemical staining or quantitative gus activity assays .
treatment of transformed n. benthamiana leaves with  <dig> mm sa clearly reduced this activity.
with a construct containing a trimer of a motif unrelated to forca we observed no significant reporter gene activity, while a trimer of a second unrelated motif, m <dig>  mediated a strong increase of gus activity upon sa treatment .
formally the sa-triggered reduction of 3xforca-pcambia expression in our agro-transient assays may be due to effects of sa on the viability of agrobacterium or other effects unrelated to forca-mediated gene expression.
however, we believe that this is quite unlikely for the following reasons.
we also observed a sa-triggered reduction of 3xforca-pcambia expression in stably transformed arabidopsis lines ;  motif m <dig> mediated sa-inducibility of gus expression in our agro-transient assays;  treatment with  <dig> mm sa did not reduce gus expression mediated by the constitutive camv35s promoter in previously published agro-transient assays with tobacco leaves  <cit> .
pcambia1281x contains a copy of the full camv35s promoter close to its multiple cloning site.
in order to rule out that this promoter influences the expression pattern observed with 3xforca-pcambia, we constructed a second pair of reporter gene vectors using pbi <dig> , which lacks any functional promoters in the vicinity of its multiple cloning site.
the construct 3xforca-pbi <dig> contains a forca trimer fused to the minimal camv35s promoter followed by gus.
3xforca-mut-pbi <dig> is identical to 3xforca-pbi <dig>  but contains block mutations eliminating the tgggc core motif of each forca copy.
consistent with the results observed with the pcambia versions, 3xforca-pbi <dig> exhibited in agro-transient expression assays a strong constitutive activity that is suppressed by sa, while 3xforca-mut-pbi <dig> did not show any detectable reporter gene activity .
we also generated transgenic arabidopsis lines transformed with 3xforca-pcambia.
t <dig> progeny from two independent primary transformants were selected for the presence of the transgene and examined for gus expression by histochemical staining with x-gluc .
both tested lines exhibited identical gus expression patterns.
four day-old 3xforca-pcambia seedlings showed constitutive gus activity in stems which is most pronounced in vascular tissues .
ten day-old seedlings exhibited 3xforca::gus expression in all plant tissues.
in these plants, gus staining is in particular strongly detectable in vascular tissues and the epidermal tissues surrounding the bases of trichomes .
in mature flowers, gus staining appears in the vascular tissues of sepals and in the anthers, but not in other flower organs .
after fertilization, gus staining is detected at the top of the flower peduncle .
no gus staining was detectable in any of three tested independent t <dig> lines containing only the camv <dig> minimal promoter fused to gus  .
consistent with results from our emsas , seven day-old 3xforca-pcambia plants exhibited stronger gus accumulation in seedlings grown under cl conditions, compared to either nl- or cd-treated seedlings .
application of sa to seedlings grown under nl or cd regime resulted in clearly reduced reporter gene expression compared to the respective mock-treated controls, , while under cl conditions sa treatment resulted in enhanced gus activity .
this effect was particularly clear in tissues outside the vasculature.
quantitative assays confirmed that in 3xforca-pcambia plants gus expression is down-regulated by sa under normal light conditions as well as after  <dig> days exposure to constant darkness .
however, after constant light treatment, sa triggered a strong increase of reporter gene expression in these lines.
consistent with the sa-induced suppression of forca-mediated gus expression observed in 3xforca-pcambia arabidopsis lines under normal light conditions, gus expression in these lines grown under normal light conditions is also down-regulated after infection with the virulent hp isolate noco <dig> .
in col- <dig> plants, hpnoco <dig> is known to induce a basal defense response that is dependent on sa  <cit> .
in our assays the high stability of the gus enzyme in plant tissue  <cit>  resulted already in strong x-gluc-staining of 3xforca-pcambia seedlings prior to infection with hpnoco <dig> .
to be able to detect a reduction of this background activity, we had to incubate the seedlings for at least  <dig> days after spray-infection with hpnoco <dig> spores.
at this timepoint a dense network of hp hyphae had typically developed in cotyledon and leaf tissues.
to eliminate the possibility that the reduction of gus activity in infected tissues is due to disease associated necrosis, we stained hpnoco2-sprayed seedlings  <dig> dpi with trypan blue, which marks dead plant cells dark blue.
as shown in figure 4d dead plant cells are absent in hpnoco <dig> infected 3xforca-pcambia seedlings  <dig> dpi and only trypan blue-stained hp structures are visible in these samples.
this indicates that the observed reduction of gus expression is a result of down-regulated 3xforca activity.
taken together our reporter gene assays using stably transformed arabidopsis lines, transiently transformed n. benthamiana leaves and emsas with nuclear arabidopsis proteins showed that forca is a constitutive promoter element, the activity of which is modulated by defense- and light-associated signaling processes.
exposure to constant light enhances basal defense to hp
the existence of crosstalk between defense and light signaling suggested that continuous exposure of arabidopsis seedlings to light affects the efficiency of their defense responses.
indeed, we observed that col- <dig> seedlings, when subject to three days of constant light exposure, appear to exhibit reduced susceptibility to hpnoco <dig> .
this effect is more pronounced and highly significant in nahg seedlings, which are compromised in defense-associated sa accumulation and, hence, are hyper-susceptible to hpnoco <dig>  <cit> .
in both col- <dig> and nahg seedlings constant light pre-treatment resulted in the same level of protection to hpnoco <dig>  indicating that this effect does not require sa.
discussion
both pti and eti are associated with massive transcriptional reprogramming  <cit> .
although multiple types of cis-elements and transcription factors controlling defense-related genes have been identified, the important regulatory step of activation of the plant defense transcriptome is still poorly understood.
our current knowledge about defense-associated transcriptional regulation has mainly resulted from the analysis of individual "model genes" such as the arabidopsis pr <dig> and parsley pr <dig> genes  <cit> .
the vast amount of global transcript profiling data that has accumulated for arabidopsis and other model plants now allows us to address the identification of such regulatory mechanisms from a new perspective enabling researchers to directly pursue the discovery of transcriptome-level control elements.
multiple studies reported the existence of statistically conserved promoter motifs in clusters of co-expressed genes  <cit> , but only a few reports provided evidence for their roles as regulatory promoter elements as well as binding sites of nuclear proteins  <cit> .
here we provide another successful demonstration supporting the feasibility of this approach.
forca-related motifs have not been implicated in the control of defense-associated gene expression before.
we found forca to dna-sequence specifically interact with nuclear arabidopsis proteins.
these interactions are differential and suppressed by defense-related stimuli.
furthermore, our data showed forca to function as a constitutive promoter element, whose activity can be modulated by the defense hormone sa or recognition of hp.
the forc cluster constitutes a set of genes that are coordinately up-regulated in response to infections by oomycete and fungal pathogens.
in addition,  <dig> of the  <dig> forc genes represented on the affymetrix ath <dig> array are significantly up-regulated by the sa analog bth  <cit> .
as in arabidopsis plants grown under a normal light regime the constitutive forca activity is suppressed by defense-stimuli, including sa, this element cannot be sufficient to control the defense-associated expression of forc genes.
the forc-type expression pattern can only be the result of the combined activity of forca and additional cis-elements that act as positive pathogen response elements.
we previously reported the enrichment of a w box-like motif and an unknown motif in the promoters of forc genes, which may mediate the pathogen-induced up-regulation of this cluster  <cit> .
indeed, we found these motifs to exhibit hp-inducible band shifts in emsas with nuclear arabidopsis proteins , suggesting a positive contribution to transcriptional activity.
the role of forca may be to limit the amplitude of forc gene induction and/or to mediate the down-regulation of forcs after an early peak of their transcript levels is reached.
future experiments will have to address this possibly interesting interplay between different pathogen-response elements.
both defense-related and light-related stimuli affect the transcriptional output mediated by forca.
under normal light conditions or constant darkness forca exhibits a constitutive activity that is suppressed by defense-related stimuli.
under constant light-exposure forca acts as a positive defense-related element mediating sa-inducible gene expression.
the latter feature of forca, however, is not reflected in our emsas with nuclear arabidopsis proteins.
under constant light conditions no further increase of the intensity of forca band shifts was observed after sa treatment .
this may indicate that under constant light conditions forca-dependent pathogen inducibility is not mediated by enhanced binding of nuclear factors, but rather other mechanisms that cannot be detected under our emsa conditions, such as subtle post-translational modifications of pre-bound factors or transient interactions with co-factors.
to our knowledge forca is unrelated to any described defense-associated promoter elements.
using plant cis-element databases , however, we found several known forca-like promoter motifs that have been implicated in other biological processes .
the bean stem element  <dig> , that appears to act as a strong unspecific enhancer, and the oat light-repressor element  <dig> , which suppresses light-driven reporter gene expression, contain sequences that perfectly match the forca consensus  <cit> .
the positive light response element pe <dig> from oat contains a sequence that matches  <dig> of the  <dig> conserved positions of forca  <cit> .
re <dig> and pe <dig> are conserved in the promoters of multiple phya genes that are regulated in a light-dependent manner.
inspecting a data set on light triggered transcriptome changes in arabidopsis  <cit>  we also found forca to be conserved in the promoters of phytochrome a&b-dependently light-induced genes encoding proteins related to photosynthesis or localized to the chloroplast.
in these  <dig> promoters the forca hexamer is significantly enriched compared to its statistically expected frequency .
if forca contributes to the co-regulation of these genes remains to be shown.
in addition, a motif that perfectly matches the forca consensus was found to be conserved in arabidopsis core promoters  <cit> .
a function has not been assigned to this motif, but its preferential location at the distal periphery of core promoter regions may suggest a role distinct from that of conventional core promoter elements.
forca does also perfectly match the recently identified "protein box"  which confers phase-specific diurnal and circadian reporter gene expression in arabidopsis  <cit> .
promoters that contain pbx are enriched for gene ontology annotations related to protein synthesis.
taken together, these reports suggest that forca/pbx-related cis-elements are conserved throughout the plant kingdom and are generally involved in adjusting the transcriptome to daily changes in light-related  environmental conditions.
furthermore, these cis-elements appear to participate in the regulation of a diverse set of genes involved in crucial metabolic processes such as photosynthesis and protein biosynthesis, but also defense.
consistent with such a broad role we observed a  <dig> -fold higher frequency of the forca hexamer in arabidopsis intergenic sequences than statistically expected.
on average, forca is present  <dig>  times per  <dig> kb in all  <dig> kb sequences upstream from the  <dig>  loci of the tair <dig> arabidopsis genome annotation , while only  <dig>  occurrences of this hexamer per  <dig> kb are statistically expected in arabidopsis intergenic regions.
it is presently unclear, what biological purpose it serves that defense-related stimuli reduce forca activity under normal light conditions or constant darkness, but enhance its activity under constant light exposure.
this seemingly unorthodox and contradictory feature of forca may reflect that in natural environments the probability of plant/pathogen encounters can follow complex diurnal patterns which are further influenced by transient environmental factors, such as local light conditions, temperature, humidity and the presence of leaf surface water  <cit> .
hence, forca may act in concert with other regulatory elements to generate fine-tuned gene expression outputs appropriate for a changing array of environmental conditions.
crosstalk between light and defense signaling has been described in several earlier reports and high light fluence rates or long durations of light exposure have been positively correlated with plant defense reactions  <cit> .
for example, genoud et al.
reported interactions between phytochrome-mediated light responses and sa-triggered responses in arabidopsis  <cit> .
sa-dependent responses such as expression of the defense marker pr <dig> and resistance to avirulent pseudomonas syringae bacteria were found to be triggered by exposure to high fluence rates of white light and to require a functional phytochrome signaling apparatus.
it is unclear, however, if the functional characteristics of forca that we observed are related to the effects described by gernoud et al., as different light-related parameters were tested in each study.
while gernound et al.
varied the fluence rate of white light, we varied the length of the photoperiod.
multiple lesion mimic mutants also point to interactions between light and defense signaling, as they are known to exhibit spontaneous defense responses, such as elevated pr gene expression, upon exposure to high light  <cit> .
as a possible connection between light and defense signaling in these mutants, light-driven accumulation of reactive oxygen intermediates , such as superoxide and hydrogenperoxide has been discussed  <cit> .
roi act as an early defense signal and participate in feedback mechanisms modulating sa-dependent defense responses  <cit> .
future experiments will have to address what types of light- and defense signals are important for the crosstalk perceived by forca and if forca-mediated responses are responsible for the reduced susceptibility to hpnoco <dig> we observed in arabidopsis seedlings subject to constant light exposure.
a number of forc members appear to show enhanced transcript levels after exposure to a prolonged photoperiod or high light intensity .
although this would be consistent with a role of forca in light-triggered defense responses, further experimentation will be needed to establish such a functional connection.
moreover, it remains to be examined, if the interference between defense and light signaling we have observed is manifested in signal convergence directly at forca and its cognate transcription factors or in regulatory steps operating further upstream.
alternatively, such crosstalk may not be mediated by discrete signaling steps at all and may be merely a consequence of general disturbances of a highly complex plant signaling network  <cit> .
at this point our knowledge about the architecture and dynamics of regulatory networks in plants is only rudimentary.
emerging new methodologies of computational biology, functional genomics and systems biology will allow us to shift the focus from understanding individual signaling circuits to a more holistic view of regulatory processes.
having promoter elements at hand that integrate signals from multiple input channels will aid in the dissection of such complex regulatory interactions.
CONCLUSIONS
forca  is a hexameric promoter motif that is conserved in clusters of arabidopsis genes co-expressed in response to fungal or oomycete pathogens as well as defined light treatments.
it constitutively interacts in a dna-sequence specific manner with nuclear arabidopsis proteins.
these interactions are suppressed by defense-related stimuli and enhanced by prolonged exposure to constant light.
furthermore forca mediates constitutive reporter gene expression in transiently transformed n. benthamiana leaves as well as in stably transformed arabidopsis plants.
its responsiveness to defense-stimuli is modulated by the duration of light exposure.
we propose that forca integrates light and defense related signals and participates in adjusting the transcriptome to daily changes in light-related environmental conditions.

 molecular recognition of pathogenic microorganisms triggers in plants comprehensive transcriptional reprogramming. a network of defense-regulators transduces information about the attacking microbe into appropriate transcriptional responses. two functionally distinct classes of pathogen molecules are known to elicit plant immune responses. receptor-mediated recognition of pathogen/microbe associated molecular patterns activates pamp-triggered immunity, while recognition of pathogen effectors by plant disease resistance proteins leads to effector-triggered immunity. eti is a strong immune response that results in incompatible plant pathogen interactions where the plant is resistant and the pathogen is avirulent. effectors are pathogen-derived molecules that are secreted into host tissues. some effectors have been shown to suppress pti resulting in a weakened immune response called basal defense which is usually insufficient to halt growth and spread of the respective microbe. the typical outcome in this case is a compatible interaction, where the plant is susceptible and the pathogen is virulent. a key player in the regulation of eti as well as basal defense is the hormone salicylic acid, which accumulates in infected tissues of several plant species and triggers downstream defense responses. several other small molecules and multiple proteins involved in defense regulation have been identified by genetic and biochemical studies. in particular the immune system of arabidopsis thaliana has been extensively examined over the past two to three decades. while our knowledge about the arabidopsis immune system is far from complete, it has become obvious in recent years, that its individual components are functionally interconnected in a complex manner and constitute a regulatory network rather than simple linear pathways. crosstalk between distinct branches of this network has been found to be involved in fine-tuning defense outputs and maximizing the effectiveness of immune responses. for example, sophisticated mechanisms mediating crosstalk between sa and jasmonic acid -dependent signaling processes can result in synergistic or antagonistic effects depending on the levels of each of these hormones. in addition, there are extensive interactions of defense signaling with regulatory processes that are not primarily involved in plant immune responses. for example, crosstalk between defense mechanisms dependent on the defense hormones sa and ja on the one hand and each of the classical growth-controlling phytohormones, auxin, gibberellin, aba, cytokinin or brassinosteroids on the other hand have been reported. details of these interactions are not well understood yet, but it seems that some of them benefit the host by enhancing immune responses, while others are beneficial for the pathogen by suppressing defense reactions. in addition, several labs reported interferences between light and defense signaling. depending on the type of light signaling pathway involved, these interactions were found to be synergistic or antagonistic. for example, phytochrome-mediated light signaling can positively affect sa-dependent defense responses, while negative interferences were observed between defense and uv-light signaling. generally, these interactions seem to control resource allocation in the plant ensuring the maintenance of homeostasis and the adjustment of the host's metabolism to a state optimal to cope with the respective environmental challenges. we are using interactions between the pathogenic oomycete hyaloperonospora parasitica and arabidopsis to examine regulatory mechanisms operating at the interface between defense signaling and the regulation of the defense transcriptome. several transcriptional regulators have been implicated in these processes, such as npr a nuclear transported transcriptional cofactor that acts downstream from sa and interacts with tga-bzip transcription factors. additional transcription factors, including wrkys and erfs, participate in the regulation of the plant defense transcriptome and disease resistance. the dna binding site preferences of these transcription factors have been well characterized. typically tga-bzips bind to tga boxes, wrkys to w boxes and erfs to gcc boxes. these promoter sites have been shown in numerous studies to act as pathogen-responsive cis-elements mediating expression of individual defense-associated genes. an enormous amount of global gene expression data from large-scale transcript profiling projects has accumulated during the past decade. these data allowed for the identification of gene clusters that are co-expressed under certain biological conditions and, hence, are likely to be subject to co-regulation by common mechanisms. several studies revealed conserved sequence motifs in promoters of such co-regulated genes which may act as cis-elements mediating their coordinated activity. in addition to already known cis-elements, some novel promoter motifs were found to be statistically enriched in co-expressed genes. while in some cases, their function has been experimentally proven, reports confirming that such conserved promoter motifs are binding sites of nuclear proteins that can affect gene expression are still rare. here we report on the identification and functional characterization of a motif conserved in promoters of the forc cluster, a set of arabidopsis genes co-expressed in response to recognition of hp as well as other oomycete and fungal pathogens. we found this motif, which we termed forca, to exhibit dna sequence specific and differential interactions with nuclear arabidopsis proteins. furthermore, electrophoretic mobility shift assays as well as reporter gene assays showed that forca integrates stimuli related to defense- and light signaling. forca acts as a light-responsive enhancer, the activity of which is modulated by pathogen-perception. under normal light conditions as well as constant darkness, forca activity is reduced by recognition of hp as well as application of sa, while under constant light conditions its activity is enhanced by defense induction. consistent with the observed functional connection between light and defense signaling, we observed that plants kept under constant light exposure exhibited reduced susceptibility to a virulent hp isolate.  a cluster of arabidopsis genes co-expressed during interactions with oomycete and fungal pathogens is enriched for the forca promoter motif using affymetrix microarrays we previously identified clusters of genes that are co-expressed during arabidopsis immune responses. two of these clusters, cluster i and the lurp cluster, were defined by distinct patterns of coordinated transcriptional up-regulation during incompatible and compatible interactions with the hp isolates hpemoy hphiks and hpemco while lurp transcripts exhibited a kinetic pattern of late and sustained up-regulation accumulating strongly between and hpi, cluster i transcript levels tended to increase earlier and predominantly accumulated within the first h after infection. genetic disruption of sa-signaling and r protein-mediated hp recognition strongly attenuated or delayed the response of lurp genes, but had only a minor effect on cluster i responses. inspecting publicly accessible microarray data we found that cluster i genes also exhibit a pronounced pattern of co-activation during interactions of arabidopsis with the fungal pathogen bortytis cinerea as well as a second oomycete, phythophthora infestans. therefore we renamed cluster i to forc. although lurp genes are also coordinately activated in response to the oomycete phytohthora infestans, this set does not show an uniform response to fungal pathogens. tightly co-expressed subsets of both the forc and lurp genes were found to be enriched for distinct promoter motifs, such as potential binding sites of wrky transcription factors. this suggested that members of the forc cluster on the one hand and the lurp cluster on the other hand are controlled by common regulatory mechanisms. the fact that different motifs are conserved in the promoters of each of these two gene sets suggested that the forc genes are controlled by mechanisms distinct from those controlling the lurp genes. applying alignace to the bp-upstream sequences of a strictly co-expressed subset of forc genes we found the hexameric motif t/atgggc to be significantly enriched compared to its statistically expected frequency. we termed this motif forca. forca specifically interacts with a nuclear dna binding activity that is down-regulated by hp and sa t/atgggc-containing promoter motifs have not been described as pathogen-response elements. therefore we tested by electrophoretic mobility shift assays, if forca exhibits hp-dependent differential interactions with nuclear arabidopsis proteins. using the radioactively labeled forca- probe consisting of a single copy of the forca consensus sequence with the ttgggc core and bp of arbitrary flanking sequence we detected a constitutive dna-binding activity producing a single band shift that is out-competed by unlabeled forca- probe. surprisingly, this forca binding activity was down-regulated in response to infections with the avirulent hp isolates hphiks or hpemoy as well as exogenously applied sa. furthermore, we found forca/nuclear protein interactions to be light dependent. nuclear extracts from plants kept for three days under constant light produced substantially more intense forca-shifts than those from plants kept for the same time in constant darkness. besides unlabeled forca- probe, unlabeled competitor probes containing wild-type forca permutations present in several forc promoters clearly reduced the intensity of forca- shifts. mutations in the sequences flanking the invariant tgggc core of forca- did not result in reduced competition. however, the forca- muta competitor probe, that lacks the tgggc core, as well as competitor probes with sequences unrelated to forca did not efficiently reduce the intensity of forca- band shifts. these results showed that the observed forca-binding activity is dna-sequence specific and that only the tgggc sequence of forca and not sequences flanking this invariant core are critical for this interaction. forca is a light-responsive promoter element whose activity is modulated by defense-related stimuli to test effects of forca on transcriptional regulation, we fused a forca trimer consisting of three distinct forca permutations to the minimal camv35s promoter followed by gus in pcambia1281x. untreated nicotiana benthamiana leaves transiently transformed with 3xforca-pcambia by agrobacterium tumefaciens exhibited strong gus activity after histochemical staining or quantitative gus activity assays. treatment of transformed n. benthamiana leaves with mm sa clearly reduced this activity. with a construct containing a trimer of a motif unrelated to forca we observed no significant reporter gene activity, while a trimer of a second unrelated motif, m mediated a strong increase of gus activity upon sa treatment. formally the sa-triggered reduction of 3xforca-pcambia expression in our agro-transient assays may be due to effects of sa on the viability of agrobacterium or other effects unrelated to forca-mediated gene expression. however, we believe that this is quite unlikely for the following reasons. we also observed a sa-triggered reduction of 3xforca-pcambia expression in stably transformed arabidopsis lines; motif m mediated sa-inducibility of gus expression in our agro-transient assays; treatment with mm sa did not reduce gus expression mediated by the constitutive camv35s promoter in previously published agro-transient assays with tobacco leaves. pcambia1281x contains a copy of the full camv35s promoter close to its multiple cloning site. in order to rule out that this promoter influences the expression pattern observed with 3xforca-pcambia, we constructed a second pair of reporter gene vectors using pbi, which lacks any functional promoters in the vicinity of its multiple cloning site. the construct 3xforca-pbi contains a forca trimer fused to the minimal camv35s promoter followed by gus. 3xforca-mut-pbi is identical to 3xforca-pbi but contains block mutations eliminating the tgggc core motif of each forca copy. consistent with the results observed with the pcambia versions, 3xforca-pbi exhibited in agro-transient expression assays a strong constitutive activity that is suppressed by sa, while 3xforca-mut-pbi did not show any detectable reporter gene activity. we also generated transgenic arabidopsis lines transformed with 3xforca-pcambia. t progeny from two independent primary transformants were selected for the presence of the transgene and examined for gus expression by histochemical staining with x-gluc. both tested lines exhibited identical gus expression patterns. four day-old 3xforca-pcambia seedlings showed constitutive gus activity in stems which is most pronounced in vascular tissues. ten day-old seedlings exhibited 3xforca::gus expression in all plant tissues. in these plants, gus staining is in particular strongly detectable in vascular tissues and the epidermal tissues surrounding the bases of trichomes. in mature flowers, gus staining appears in the vascular tissues of sepals and in the anthers, but not in other flower organs. after fertilization, gus staining is detected at the top of the flower peduncle. no gus staining was detectable in any of three tested independent t lines containing only the camv minimal promoter fused to gus. consistent with results from our emsas, seven day-old 3xforca-pcambia plants exhibited stronger gus accumulation in seedlings grown under cl conditions, compared to either nl- or cd-treated seedlings. application of sa to seedlings grown under nl or cd regime resulted in clearly reduced reporter gene expression compared to the respective mock-treated controls,, while under cl conditions sa treatment resulted in enhanced gus activity. this effect was particularly clear in tissues outside the vasculature. quantitative assays confirmed that in 3xforca-pcambia plants gus expression is down-regulated by sa under normal light conditions as well as after days exposure to constant darkness. however, after constant light treatment, sa triggered a strong increase of reporter gene expression in these lines. consistent with the sa-induced suppression of forca-mediated gus expression observed in 3xforca-pcambia arabidopsis lines under normal light conditions, gus expression in these lines grown under normal light conditions is also down-regulated after infection with the virulent hp isolate noco. in col- plants, hpnoco is known to induce a basal defense response that is dependent on sa. in our assays the high stability of the gus enzyme in plant tissue resulted already in strong x-gluc-staining of 3xforca-pcambia seedlings prior to infection with hpnoco. to be able to detect a reduction of this background activity, we had to incubate the seedlings for at least days after spray-infection with hpnoco spores. at this timepoint a dense network of hp hyphae had typically developed in cotyledon and leaf tissues. to eliminate the possibility that the reduction of gus activity in infected tissues is due to disease associated necrosis, we stained hpnoco2-sprayed seedlings dpi with trypan blue, which marks dead plant cells dark blue. as shown in figure 4d dead plant cells are absent in hpnoco infected 3xforca-pcambia seedlings dpi and only trypan blue-stained hp structures are visible in these samples. this indicates that the observed reduction of gus expression is a result of down-regulated 3xforca activity. taken together our reporter gene assays using stably transformed arabidopsis lines, transiently transformed n. benthamiana leaves and emsas with nuclear arabidopsis proteins showed that forca is a constitutive promoter element, the activity of which is modulated by defense- and light-associated signaling processes. exposure to constant light enhances basal defense to hp the existence of crosstalk between defense and light signaling suggested that continuous exposure of arabidopsis seedlings to light affects the efficiency of their defense responses. indeed, we observed that col- seedlings, when subject to three days of constant light exposure, appear to exhibit reduced susceptibility to hpnoco. this effect is more pronounced and highly significant in nahg seedlings, which are compromised in defense-associated sa accumulation and, hence, are hyper-susceptible to hpnoco. in both col- and nahg seedlings constant light pre-treatment resulted in the same level of protection to hpnoco indicating that this effect does not require sa. discussion both pti and eti are associated with massive transcriptional reprogramming. although multiple types of cis-elements and transcription factors controlling defense-related genes have been identified, the important regulatory step of activation of the plant defense transcriptome is still poorly understood. our current knowledge about defense-associated transcriptional regulation has mainly resulted from the analysis of individual "model genes" such as the arabidopsis pr and parsley pr genes. the vast amount of global transcript profiling data that has accumulated for arabidopsis and other model plants now allows us to address the identification of such regulatory mechanisms from a new perspective enabling researchers to directly pursue the discovery of transcriptome-level control elements. multiple studies reported the existence of statistically conserved promoter motifs in clusters of co-expressed genes, but only a few reports provided evidence for their roles as regulatory promoter elements as well as binding sites of nuclear proteins. here we provide another successful demonstration supporting the feasibility of this approach. forca-related motifs have not been implicated in the control of defense-associated gene expression before. we found forca to dna-sequence specifically interact with nuclear arabidopsis proteins. these interactions are differential and suppressed by defense-related stimuli. furthermore, our data showed forca to function as a constitutive promoter element, whose activity can be modulated by the defense hormone sa or recognition of hp. the forc cluster constitutes a set of genes that are coordinately up-regulated in response to infections by oomycete and fungal pathogens. in addition, of the forc genes represented on the affymetrix ath array are significantly up-regulated by the sa analog bth. as in arabidopsis plants grown under a normal light regime the constitutive forca activity is suppressed by defense-stimuli, including sa, this element cannot be sufficient to control the defense-associated expression of forc genes. the forc-type expression pattern can only be the result of the combined activity of forca and additional cis-elements that act as positive pathogen response elements. we previously reported the enrichment of a w box-like motif and an unknown motif in the promoters of forc genes, which may mediate the pathogen-induced up-regulation of this cluster. indeed, we found these motifs to exhibit hp-inducible band shifts in emsas with nuclear arabidopsis proteins, suggesting a positive contribution to transcriptional activity. the role of forca may be to limit the amplitude of forc gene induction and/or to mediate the down-regulation of forcs after an early peak of their transcript levels is reached. future experiments will have to address this possibly interesting interplay between different pathogen-response elements. both defense-related and light-related stimuli affect the transcriptional output mediated by forca. under normal light conditions or constant darkness forca exhibits a constitutive activity that is suppressed by defense-related stimuli. under constant light-exposure forca acts as a positive defense-related element mediating sa-inducible gene expression. the latter feature of forca, however, is not reflected in our emsas with nuclear arabidopsis proteins. under constant light conditions no further increase of the intensity of forca band shifts was observed after sa treatment. this may indicate that under constant light conditions forca-dependent pathogen inducibility is not mediated by enhanced binding of nuclear factors, but rather other mechanisms that cannot be detected under our emsa conditions, such as subtle post-translational modifications of pre-bound factors or transient interactions with co-factors. to our knowledge forca is unrelated to any described defense-associated promoter elements. using plant cis-element databases, however, we found several known forca-like promoter motifs that have been implicated in other biological processes. the bean stem element, that appears to act as a strong unspecific enhancer, and the oat light-repressor element, which suppresses light-driven reporter gene expression, contain sequences that perfectly match the forca consensus. the positive light response element pe from oat contains a sequence that matches of the conserved positions of forca. re and pe are conserved in the promoters of multiple phya genes that are regulated in a light-dependent manner. inspecting a data set on light triggered transcriptome changes in arabidopsis we also found forca to be conserved in the promoters of phytochrome a&b-dependently light-induced genes encoding proteins related to photosynthesis or localized to the chloroplast. in these promoters the forca hexamer is significantly enriched compared to its statistically expected frequency. if forca contributes to the co-regulation of these genes remains to be shown. in addition, a motif that perfectly matches the forca consensus was found to be conserved in arabidopsis core promoters. a function has not been assigned to this motif, but its preferential location at the distal periphery of core promoter regions may suggest a role distinct from that of conventional core promoter elements. forca does also perfectly match the recently identified "protein box" which confers phase-specific diurnal and circadian reporter gene expression in arabidopsis. promoters that contain pbx are enriched for gene ontology annotations related to protein synthesis. taken together, these reports suggest that forca/pbx-related cis-elements are conserved throughout the plant kingdom and are generally involved in adjusting the transcriptome to daily changes in light-related environmental conditions. furthermore, these cis-elements appear to participate in the regulation of a diverse set of genes involved in crucial metabolic processes such as photosynthesis and protein biosynthesis, but also defense. consistent with such a broad role we observed a -fold higher frequency of the forca hexamer in arabidopsis intergenic sequences than statistically expected. on average, forca is present times per kb in all kb sequences upstream from the loci of the tair arabidopsis genome annotation, while only occurrences of this hexamer per kb are statistically expected in arabidopsis intergenic regions. it is presently unclear, what biological purpose it serves that defense-related stimuli reduce forca activity under normal light conditions or constant darkness, but enhance its activity under constant light exposure. this seemingly unorthodox and contradictory feature of forca may reflect that in natural environments the probability of plant/pathogen encounters can follow complex diurnal patterns which are further influenced by transient environmental factors, such as local light conditions, temperature, humidity and the presence of leaf surface water. hence, forca may act in concert with other regulatory elements to generate fine-tuned gene expression outputs appropriate for a changing array of environmental conditions. crosstalk between light and defense signaling has been described in several earlier reports and high light fluence rates or long durations of light exposure have been positively correlated with plant defense reactions. for example, genoud et al. reported interactions between phytochrome-mediated light responses and sa-triggered responses in arabidopsis. sa-dependent responses such as expression of the defense marker pr and resistance to avirulent pseudomonas syringae bacteria were found to be triggered by exposure to high fluence rates of white light and to require a functional phytochrome signaling apparatus. it is unclear, however, if the functional characteristics of forca that we observed are related to the effects described by gernoud et al., as different light-related parameters were tested in each study. while gernound et al. varied the fluence rate of white light, we varied the length of the photoperiod. multiple lesion mimic mutants also point to interactions between light and defense signaling, as they are known to exhibit spontaneous defense responses, such as elevated pr gene expression, upon exposure to high light. as a possible connection between light and defense signaling in these mutants, light-driven accumulation of reactive oxygen intermediates, such as superoxide and hydrogenperoxide has been discussed. roi act as an early defense signal and participate in feedback mechanisms modulating sa-dependent defense responses. future experiments will have to address what types of light- and defense signals are important for the crosstalk perceived by forca and if forca-mediated responses are responsible for the reduced susceptibility to hpnoco we observed in arabidopsis seedlings subject to constant light exposure. a number of forc members appear to show enhanced transcript levels after exposure to a prolonged photoperiod or high light intensity. although this would be consistent with a role of forca in light-triggered defense responses, further experimentation will be needed to establish such a functional connection. moreover, it remains to be examined, if the interference between defense and light signaling we have observed is manifested in signal convergence directly at forca and its cognate transcription factors or in regulatory steps operating further upstream. alternatively, such crosstalk may not be mediated by discrete signaling steps at all and may be merely a consequence of general disturbances of a highly complex plant signaling network. at this point our knowledge about the architecture and dynamics of regulatory networks in plants is only rudimentary. emerging new methodologies of computational biology, functional genomics and systems biology will allow us to shift the focus from understanding individual signaling circuits to a more holistic view of regulatory processes. having promoter elements at hand that integrate signals from multiple input channels will aid in the dissection of such complex regulatory interactions.  forca is a hexameric promoter motif that is conserved in clusters of arabidopsis genes co-expressed in response to fungal or oomycete pathogens as well as defined light treatments. it constitutively interacts in a dna-sequence specific manner with nuclear arabidopsis proteins. these interactions are suppressed by defense-related stimuli and enhanced by prolonged exposure to constant light. furthermore forca mediates constitutive reporter gene expression in transiently transformed n. benthamiana leaves as well as in stably transformed arabidopsis plants. its responsiveness to defense-stimuli is modulated by the duration of light exposure. we propose that forca integrates light and defense related signals and participates in adjusting the transcriptome to daily changes in light-related environmental conditions. 
in plants grown under normal light conditions or constant darkness defense-related stimuli result in suppression of forca-mediated reporter gene expression, while in plants grown under constant light exposure, defense-induction results in enhanced forca-mediated expression. in addition, we found plants subjected to constant light exposure to exhibit reduced susceptibility to virulent h. parasitica. recognition of pathogenic microorganisms triggers in plants comprehensive transcriptional reprogramming. forca is a hexameric promoter motif that is conserved in clusters of arabidopsis genes co-expressed in response to fungal or oomycete pathogens as well as defined light treatments. furthermore forca mediates constitutive reporter gene expression in transiently transformed nicotiana benthamiana leaves as well as in stably transformed arabidopsis plants. it integrates light- and defense-related signals and participates in adjusting the transcriptome to changes in environmental conditions. these interactions are suppressed by defense-related stimuli and enhanced by prolonged exposure to constant light. promoter motifs conserved in clusters of co-expressed genes may be involved in mediating coordinated gene activity patterns. although numerous studies identified such conserved promoter motifs in co-expressed gene sets, reports confirming their function as regulatory elements are rare. it constitutively interacts in a dna-sequence specific manner with nuclear arabidopsis proteins. in order to identify transcriptome-level control elements required for plant immune responses we are examining several sets of genes found by microarray experiments to be co-activated in arabidopsis thaliana seedlings infected with the oomycete hyaloperonospora parasitica. its responsiveness to defense-stimuli is modulated by the duration of light exposure.
 recognition of pathogenic microorganisms triggers in plants comprehensive transcriptional reprogramming. in order to identify transcriptome-level control elements required for plant immune responses we are examining several sets of genes found by microarray experiments to be co-activated in arabidopsis thaliana seedlings infected with the oomycete hyaloperonospora parasitica. promoter motifs conserved in clusters of co-expressed genes may be involved in mediating coordinated gene activity patterns. although numerous studies identified such conserved promoter motifs in co-expressed gene sets, reports confirming their function as regulatory elements are rare.  forca is a hexameric promoter motif that is conserved in clusters of arabidopsis genes co-expressed in response to fungal or oomycete pathogens as well as defined light treatments. forca is generally more frequently present in arabidopsis promoter regions than statistically expected. it constitutively interacts in a dna-sequence specific manner with nuclear arabidopsis proteins. these interactions are suppressed by defense-related stimuli and enhanced by prolonged exposure to constant light. furthermore forca mediates constitutive reporter gene expression in transiently transformed nicotiana benthamiana leaves as well as in stably transformed arabidopsis plants. its responsiveness to defense-stimuli is modulated by the duration of light exposure. in plants grown under normal light conditions or constant darkness defense-related stimuli result in suppression of forca-mediated reporter gene expression, while in plants grown under constant light exposure, defense-induction results in enhanced forca-mediated expression. in addition, we found plants subjected to constant light exposure to exhibit reduced susceptibility to virulent h. parasitica.  we propose that forca is a regulatory cis-element that is present in a wide variety of arabidopsis promoters. it integrates light- and defense-related signals and participates in adjusting the transcriptome to changes in environmental conditions. 