Genomic distribution and context dependent functionality of novel WRKY transcription factor binding sites.

BACKGROUND: The WT-boxes NGACTTTN are novel microbe-associated molecular pattern (MAMP)-responsive cis-regulatory sequences. Many of them are uncommon WRKY transcription factor (TF) binding sites. RESULTS: To understand their functional relevance, a genomic distribution analysis of the 16 possible WT-boxes and a functional analysis of a WT-box rich promoter was done. The genomic distribution analysis shows an enrichment of specific WT-boxes within 500 bp upstream of all Arabidopsis thaliana genes. Those that harbour a T 5' to the core sequence GACTTT can also be part of the classic WRKY binding site the W-box TTGACT/C. The MAMP-responsive gene ATEP3, a class IV chitinase, harbours seven WT-boxes within its 1000 bp upstream region. In the context of synthetic promoters, the four proximal WT-boxes confer MAMP responsivity while the three WT-boxes further upstream have no effect. Rendering the nucleotides adjacent and in the vicinity of the WT-box core sequence reveals their functional importance for gene expression. A 158 bp long ATEP3 minimal promoter harbouring the two WT-boxes CGACTTTT, confers WT-box-dependent basal and MAMP-responsive reporter gene expression. The ATEP3 gene is a proposed target of WRKY50 and WRKY70. WRKY50 negatively regulates MAMP responsivity of the two WT-boxes CGACTTTT, while WRKY70 activates gene expression in a WT-box dependent manner. Both WRKY factors bind directly to the WT-box CGACTTTT. CONCLUSION: In summary, WT-boxes are enriched in promoter regions and comprise novel and uncommon WRKY binding sites required for basal and MAMP-induced gene expression. WT-boxes not being part of a W-box may be a missing link for WRKY target gene prediction when these genes do not harbour a W-box.

A dialogue-like cell communication mechanism is conserved in filamentous ascomycete fungi and mediates interspecies interactions.

In many filamentous fungi, germinating spores cooperate by fusing into supracellular structures, which develop into the mycelial colony. In the model fungus Neurospora crassa, this social behavior is mediated by an intriguing mode of communication, in which two fusing cells take turns in signal sending and receiving. Here we show that this dialogue-like cell communication mechanism is highly conserved in distantly related fungal species and mediates interspecies interactions. In mixed populations, cells of N. crassa and the phytopathogenic gray mold Botrytis cinerea coordinate their behavior over a spatial distance and establish physical contact. Subsequent cell­cell fusion is, however, restricted to germlings of the same species, indicating that species specificity of germling fusion has evolved not on the level of the signal/receptor but at subsequent levels of the fusion process. In B. cinerea, fusion and infectious growth are mutually exclusive cellular programs. Remarkably, the presence of N. crassa can reprogram this behavior and induce fusion of the gray mold on plant surfaces, potentially weakening its pathogenic potential. In a third fungal species, the nematode-trapping fungus Arthrobotrys flagrans, the conserved signaling mechanism mediates vegetative fusion within mycelial colonies but has also been repurposed for the formation of nematode-catching traps. In summary, this study identified the cell dialogue mechanism as a conserved complex trait and revealed that even distantly related fungi possess a common molecular language, which promotes cellular contact formation across species borders.

Unusual DNA-binding properties of the Arabidopsis thaliana WRKY50 transcription factor at target gene promoters.

KEY MESSAGE: WRKY50 from A. thaliana requires WT-boxes at target gene promoters for activation and binding. Based on the genome-wide prediction of WRKY50 target genes and the similarity of a WRKY50 binding site to WT-boxes in microbe-associated molecular pattern (MAMP)-responsive cis-regulatory modules (CRM), four WT-box containing CRMs from the promoter region of three WRKY50 target genes were investigated for their interaction with WRKY50. These target genes are DJ1E, WRKY30 and ATBBE4. Two of the four CRMs, one from DJ1E and one from WRKY30, were able to activate reporter gene expression in the presence of WRKY50. Activation requires the WT-boxes GGACTTTT, GGACTTTG from DJ1E and GGACTTTC from WRKY30. WRKY50 does not activate a second CRM from WRKY30 and the CRM from ATBBE4, both containing the WT-box TGACTTTT. In vitro gel-shift assays demonstrate WT-box-specific binding of the WRKY50 DNA-binding domain to all four CRMs. This work shows a high flexibility of WRKY50 binding site recognition beyond the classic W-box TTGACC/T.

A strong NF-κB p65 responsive cis-regulatory sequence from Arabidopsis thaliana interacts with WRKY40.

KEY MESSAGE: Transcription factors from mammals and plants, which play a role in innate immunity, interact with the same microbe-associated molecular pattern (MAMP)-responsive sequences from Arabidopsis thaliana. The interaction of mouse NF-κB p65 with MAMP-responsive sequences containing the core motif GACTTT of the WT-box was investigated. This revealed one sequence, derived from the promoter of the A. thaliana gene At1g76960, a gene with unknown function, to activate NF-κB p65 dependent reporter gene expression in plant cells very strongly. A bioinformatic analysis predicts three putative NF-κB p65 binding sites in this sequence and all three sites are required for reporter gene activation and binding. The sequence is one of the weakest MAMP-responsive sequences previously isolated, but the introduction of a GCC-box increases its MAMP responsivity in combination with upstream WT-box sequences. Although a bioinformatic analysis of the unmutated cis-sequence only predicts NF-κB p65 binding, A. thaliana WRKY40 was selected in a yeast one-hybrid screen. WRKY40, which is a transcriptional repressor, requires the sequence TTTTCTA for direct binding. This sequence is similar to the WK-box TTTTCCAC, previously shown to interact with tobacco NtWRKY12. In summary, this work supports the similarity in binding site recognition between NF-κB and WRKY factors.

Transcription factors involved in basal immunity in mammals and plants interact with the same MAMP-responsive cis-sequence from Arabidopsis thaliana.

KEY MESSAGE: WRKY and NF-κB transcription factors, involved in innate immunity in plants and mammals, interact with the same cis-sequence. Novel microbe-associated molecular pattern (MAMP)-responsive cis-sequences, designated type II WT-boxes, are required for flg22-responsive gene expression in Arabidopsis thaliana protoplasts. While type I WT-boxes like TGACTTTT and CGACTTTT interact with WRKY transcription factors (TFs), the question remained which TFs bind to the type II WT-boxes GGACTTTC, GGACTTTT, and GGACTTTG. Surprisingly, a bioinformatic analysis predicts mouse (Mus musculus) NF-κB p65 as a TF interacting with type II WT-boxes. NF-κB p65, like WRKY factors in plants, plays a role in innate immunity in mammals. Therefore, the interaction of NF-κB p65 with type II WT-boxes was tested experimentally. NF-κB p65 requires the WT-boxes GGACTTTC, GGACTTTT, and GGACTTTG for activating reporter gene expression in plant cells. NF-κB p65 directly binds to WT-box containing synthetic promoters in vitro and requires the WT-box for binding. Earlier studies indicate that the sequence GGACTTTC is also required for WRKY26 mediated reporter gene activation. Here it is shown that WRKY26, like NF-κB p65, binds to the sequence GGACTTTC. Consistent with other recent studies, type II WT boxes are WRKY binding sites and the distinction between type I and type II no longer applies.

From experiment-driven database analyses to database-driven experiments in Arabidopsis thaliana transcription factor research.

Experiment-driven database analysis is employed in forward genetics to predict the function of genes assocíated with a mutant phenotype. These analyses subsequently lead to database-driven experiments involving reverse genetics to verify functional predictions based on bioinformatic analyses. Genomic transcription factors (TFs) are key regulators of gene expression by binding to short regulatory sequences and by interacting with other TFs. Currently more than 2400 TFs are predicted for A. thaliana. As DNA-binding proteins they are particularly amenable to database-driven experiments, especially when their binding site specificities are known. Databases are available for predicting binding sites for specific TFs in regulatory sequences. Since most of these bioinformatically identified binding sites may not be functional, additional experiments for identifying the actual in vivo binding sites for TFs are required. Recently, large scale approaches were employed to determine binding sites for many A. thaliana TFs. With these approaches binding sites for 984 unique TFs were determined experimentally. An area deserving further research is proposed for interacting TFs. Most of the A. thaliana genes are under combinatorial control, and in vivo interacting TFs, similar to mammalian TFs, may bind to combinatorial elements in which the binding sites vary from those detected with the single TFs.

Combinatorial requirement of W- and WT-boxes in microbe-associated molecular pattern-responsive synthetic promoters.

KEY MESSAGE: The WT-box GGACTTTC belongs to a novel class of MAMP-responsive cis-regulatory sequences that are part of combinatorial elements. Microbe-associated molecular pattern (MAMP)-responsive synthetic promoters were generated with two cis-regulatory modules (CRM1 and CRM2) from the Arabidopsis thaliana WRKY30 promoter. Both modules harbour two W-boxes and one WT-box. Mutation analysis of the synthetic promoters and transient gene expression analysis in parsley protoplasts underline the importance of the W- and WT-boxes for MAMP-responsive gene expression and reveal the combinatorial requirement of at least two boxes for full MAMP responsivity. In the context of the native promoter, CRM1 is required for MAMP responsivity, while CRM2 alone is not sufficient. Yeast one-hybrid screenings using CRM1 with a transcription factor (TF) only prey library select only WRKY factors. Selection of WRKY26, 40, 41, and 70 requires the W-boxes. The WT-box is also required for selection of WRKY26 and 41 in yeast. In plant cells, WRKY26, 40, and 41 act as repressors of MAMP-responsive gene expression, whereas WRKY70 is an activator. To investigate whether the WT-box is also required for WRKY26 and 41 mediated gene expression in plant cells, both were converted into transcriptional activators by adding the GAL4 activating domain (AD). In contrast to yeast, transient gene expression in parsley protoplasts shows that only the W-boxes from CRM1 are required for WRKY41AD-activated reporter gene activity but not the WT-box. In addition, WRKY70-activated reporter gene activity in parsley cells does not require the WT-box of CRM1. The results demonstrate the importance of the WT-box as a new cis-regulatory sequence for MAMP-responsive gene expression. Based on these and earlier results, two types of WT-boxes are proposed.

Analysis of Microbe-Associated Molecular Pattern-Responsive Synthetic Promoters with the Parsley Protoplast System.

Plants recognize pathogens by microbe-associated molecular patterns (MAMPs) and subsequently induce an immune response. The regulation of gene expression during the immune response depends largely on cis-sequences conserved in promoters of MAMP-responsive genes. These cis-sequences can be analyzed by constructing synthetic promoters linked to a reporter gene and by testing these constructs in transient expression systems. Here, the use of the parsley (Petroselinum crispum) protoplast system for analyzing MAMP-responsive synthetic promoters is described. The synthetic promoter consists of four copies of a potential MAMP-responsive cis-sequence cloned upstream of a minimal promoter and the uidA reporter gene. The reporter plasmid contains a second reporter gene, which is constitutively expressed and hence eliminates the requirement of a second plasmid used as a transformation control. The reporter plasmid is transformed into parsley protoplasts that are elicited by the MAMP Pep25. The MAMP responsiveness is validated by comparing the reporter gene activity from MAMP-treated and untreated cells and by normalizing reporter gene activity using the constitutively expressed reporter gene.

Bioinformatic Identification of Conserved Cis-Sequences in Coregulated Genes.

Bioinformatics tools can be employed to identify conserved cis-sequences in sets of coregulated plant genes because more and more gene expression and genomic sequence data become available. Knowledge on the specific cis-sequences, their enrichment and arrangement within promoters, facilitates the design of functional synthetic plant promoters that are responsive to specific stresses. The present chapter illustrates an example for the bioinformatic identification of conserved Arabidopsis thaliana cis-sequences enriched in drought stress-responsive genes. This workflow can be applied for the identification of cis-sequences in any sets of coregulated genes. The workflow includes detailed protocols to determine sets of coregulated genes, to extract the corresponding promoter sequences, and how to install and run a software package to identify overrepresented motifs. Further bioinformatic analyses that can be performed with the results are discussed.

In Silico Expression Analysis.

Information on the specificity of cis-sequences enables the design of functional synthetic plant promoters that are responsive to specific stresses. Potential cis-sequences may be experimentally tested, however, correlation of genomic sequence with gene expression data enables an in silico expression analysis approach to bioinformatically assess the stress specificity of candidate cis-sequences prior to experimental verification. The present chapter demonstrates an example for the in silico validation of a potential cis-regulatory sequence responsive to cold stress. The described online tool can be applied for the bioinformatic assessment of cis-sequences responsive to most abiotic and biotic stresses of plants. Furthermore, a method is presented based on a reverted in silico expression analysis approach that predicts highly specific potentially functional cis-regulatory elements for a given stress.

A cis-regulatory sequence from a short intergenic region gives rise to a strong microbe-associated molecular pattern-responsive synthetic promoter.

The high gene density in Arabidopsis thaliana leaves only relatively short intergenic regions for potential cis-regulatory sequences. To learn more about the regulation of genes harbouring only very short upstream intergenic regions, this study investigates a recently identified novel microbe-associated molecular pattern (MAMP)-responsive cis-sequence located within the 101 bp long intergenic region upstream of the At1g13990 gene. It is shown that the cis-regulatory sequence is sufficient for MAMP-responsive reporter gene activity in the context of its native promoter. The 3' UTR of the upstream gene has a quantitative effect on gene expression. In context of a synthetic promoter, the cis-sequence is shown to achieve a strong increase in reporter gene activity as a monomer, dimer and tetramer. Mutation analysis of the cis-sequence determined the specific nucleotides required for gene expression activation. In transgenic A. thaliana the synthetic promoter harbouring a tetramer of the cis-sequence not only drives strong pathogen-responsive reporter gene expression but also shows a high background activity. The results of this study contribute to our understanding how genes with very short upstream intergenic regions are regulated and how these regions can serve as a source for MAMP-responsive cis-sequences for synthetic promoter design.

Functional dissection of a strong and specific microbe-associated molecular pattern-responsive synthetic promoter.

Synthetic promoters are important for temporal and spatial gene expression in transgenic plants. To identify novel microbe-associated molecular pattern (MAMP)-responsive cis-regulatory sequences for synthetic promoter design, a combination of bioinformatics and experimental approaches was employed. One cis-sequence was identified which confers strong MAMP-responsive reporter gene activity with low background activity. The 35-bp-long cis-sequence was identified in the promoter of the Arabidopsis thaliana DJ1E gene, a homologue of the human oncogene DJ1. In this study, this cis-sequence is shown to be a tripartite cis-regulatory module (CRM). A synthetic promoter with four copies of the CRM linked to a minimal promoter increases MAMP-responsive reporter gene expression compared to the wild-type DJ1E promoter. The CRM consists of two WT-boxes (GGACTTTT and GGACTTTG) and a variant of the GCC-box (GCCACC), all required for MAMP and salicylic acid (SA) responsivity. Yeast one-hybrid screenings using a transcription factor (TF)-only prey library identified two AP2/ERFs, ORA59 and ERF10, interacting antagonistically with the CRM. ORA59 activates reporter gene activity and requires the consensus core sequence GCCNCC for gene expression activation. ERF10 down-regulates MAMP-responsive gene expression. No TFs interacting with the WT-boxes GGACTTTT and GGACTTTG were selected in yeast one-hybrid screenings with the TF-only prey library. In transgenic Arabidopsis, the synthetic promoter confers strong and specific reporter gene activity in response to biotrophs and necrotrophs as well as SA.

Boosting AthaMap Database Content with Data from Protein Binding Microarrays.

The AthaMap database generates a map of predicted transcription factor binding sites (TFBS) and small RNA target sites for the whole Arabidopsis thaliana genome. With the advent of protein binding microarrays (PBM), the number of known TFBS for A. thaliana transcription factors (TFs) has increased dramatically. Using 113 positional weight matrices (PWMs) generated from a single PBM study and representing a total number of 68 different TFs, the number of predicted TFBS in AthaMap was now increased by about 3.8 × 10(7) to 4.9 × 10(7). The number of TFs with PWM-predicted TFBS annotated in AthaMap has increased to 126, representing a total of 29 TF families and newly including ARF, AT-Hook, YABBY, LOB/AS2 and SRS. Furthermore, links from all Arabidopsis TFs and genes to the newly established Arabidopsis Information Portal (AIP) have been implemented. With this qualitative and quantitative update, the improved AthaMap increases its value as a resource for the analysis of A. thaliana gene expression regulation at www.athamap.de.

AthaMap web tools for the analysis of transcriptional and posttranscriptional regulation of gene expression in Arabidopsis thaliana.

The AthaMap database provides a map of verified and predicted transcription factor (TF) and small RNA-binding sites for the A. thaliana genome. The database can be used for bioinformatic predictions of putative regulatory sites. Several online web tools are available that address specific questions. Starting with the identification of transcription factor-binding sites (TFBS) in any gene of interest, colocalizing TFBS can be identified as well as common TFBS in a set of user-provided genes. Furthermore, genes can be identified that are potentially targeted by specific transcription factors or small inhibitory RNAs. This chapter provides detailed information on how each AthaMap web tool can be used online. Examples on how this database is used to address questions in circadian and diurnal regulation are given. Furthermore, complementary databases and databases that go beyond questions addressed with AthaMap are discussed.

'In silico expression analysis', a novel PathoPlant web tool to identify abiotic and biotic stress conditions associated with specific cis-regulatory sequences.

Using bioinformatics, putative cis-regulatory sequences can be easily identified using pattern recognition programs on promoters of specific gene sets. The abundance of predicted cis-sequences is a major challenge to associate these sequences with a possible function in gene expression regulation. To identify a possible function of the predicted cis-sequences, a novel web tool designated 'in silico expression analysis' was developed that correlates submitted cis-sequences with gene expression data from Arabidopsis thaliana. The web tool identifies the A. thaliana genes harbouring the sequence in a defined promoter region and compares the expression of these genes with microarray data. The result is a hierarchy of abiotic and biotic stress conditions to which these genes are most likely responsive. When testing the performance of the web tool, known cis-regulatory sequences were submitted to the 'in silico expression analysis' resulting in the correct identification of the associated stress conditions. When using a recently identified novel elicitor-responsive sequence, a WT-box (CGACTTTT), the 'in silico expression analysis' predicts that genes harbouring this sequence in their promoter are most likely Botrytis cinerea induced. Consistent with this prediction, the strongest induction of a reporter gene harbouring this sequence in the promoter is observed with B. cinerea in transgenic A. thaliana. DATABASE URL: http://www.pathoplant.de/expression_analysis.php.

Integrating bioinformatic resources to predict transcription factors interacting with cis-sequences conserved in co-regulated genes.

BACKGROUND: Using motif detection programs it is fairly straightforward to identify conserved cis-sequences in promoters of co-regulated genes. In contrast, the identification of the transcription factors (TFs) interacting with these cis-sequences is much more elaborate. To facilitate this, we explore the possibility of using several bioinformatic and experimental approaches for TF identification. This starts with the selection of co-regulated gene sets and leads first to the prediction and then to the experimental validation of TFs interacting with cis-sequences conserved in the promoters of these co-regulated genes. RESULTS: Using the PathoPlant database, 32 up-regulated gene groups were identified with microarray data for drought-responsive gene expression from Arabidopsis thaliana. Application of the binding site estimation suite of tools (BEST) discovered 179 conserved sequence motifs within the corresponding promoters. Using the STAMP web-server, 49 sequence motifs were classified into 7 motif families for which similarities with known cis-regulatory sequences were identified. All motifs were subjected to a footprintDB analysis to predict interacting DNA binding domains from plant TF families. Predictions were confirmed by using a yeast-one-hybrid approach to select interacting TFs belonging to the predicted TF families. TF-DNA interactions were further experimentally validated in yeast and with a Physcomitrella patens transient expression system, leading to the discovery of several novel TF-DNA interactions. CONCLUSIONS: The present work demonstrates the successful integration of several bioinformatic resources with experimental approaches to predict and validate TFs interacting with conserved sequence motifs in co-regulated genes.

Identification of a novel type of WRKY transcription factor binding site in elicitor-responsive cis-sequences from Arabidopsis thaliana.

Using a combination of bioinformatics and synthetic promoters, novel elicitor-responsive cis-sequences were discovered in promoters of pathogen-upregulated genes from Arabidopsis thaliana. One group of functional sequences contains the conserved core sequence GACTTTT. This core sequence and adjacent nucleotides are essential for elicitor-responsive gene expression in a parsley protoplast system. By yeast one-hybrid screening, WRKY70 was selected with a cis-sequence harbouring the core sequence GACTTTT but no known WRKY binding site (W-box). Transactivation experiments, mutation analyses, and electrophoretic mobility shift assays demonstrate that the sequence CGACTTTT is the binding site for WRKY70 in the investigated cis-sequence and is required for WRKY70-activated gene expression. Using several cis-sequences in transactivation experiments and binding studies, the CGACTTTT sequence can be extended to propose YGACTTTT as WRKY70 binding site. This binding site, designated WT-box, is enriched in promoters of genes upregulated in a WRKY70 overexpressing line. Interestingly, functional WRKY70 binding sites are present in the promoter of WRKY30, supporting recent evidence that both factors play a role in the same regulatory network.

Inducible expression of p50 from TMV for increased resistance to bacterial crown gall disease in tobacco.

The dominant tobacco mosaic virus (TMV) resistance gene N induces a hypersensitive response upon TMV infection and protects tobacco against systemic spread of the virus. It has been proposed to change disease resistance specificity by reprogramming the expression of resistance genes or their corresponding avirulence genes. To reprogramme the resistance response of N towards bacterial pathogens, the helicase domain (p50) of the TMV replicase, the avirulence gene of N, was linked to synthetic promoters 4D and 2S2D harbouring elicitor-responsive cis-elements. These promoter::p50 constructs induce local necrotic lesions on NN tobacco plants in an Agrobacterium tumefaciens infiltration assay. A tobacco genotype void of N (nn) was transformed with the promoter::p50 constructs and subsequently crossed to NN plants. Nn F1 offspring selected for the T-DNA develop normally under sterile conditions. After transfer to soil, some of the F1 plants expressing the 2S2D::p50 constructs develop spontaneous necrosis. Transgenic Nn F1 plants with 4D::p50 and 2S2D::p50 expressing constructs upregulate p50 transcription and induce local necrotic lesions in an A. tumefaciens infiltration assay. When leaves and stems of Nn F1 offspring harbouring promoter::p50 constructs are infected with oncogenic A. tumefaciens C58, transgenic lines harbouring the 2S2D::p50 construct induce necrosis and completely lack tumor development. These results demonstrate a successful reprogramming of the viral N gene response against bacterial crown gall disease and highlight the importance of achieving tight regulation of avirulence gene expression and the control of necrosis in the presence of the corresponding resistance gene.

Differential expression of the TMV resistance gene N prevents a hypersensitive response in seeds and during germination.

The dominant tobacco mosaic virus (TMV) resistance gene N confers a hypersensitive response (HR) at the site of TMV infection and protects tobacco against systemic spread of the virus. To study N gene activity in seeds and early seedling development, the avirulence gene of N, the helicase domain (p50) of the TMV replicase, was constitutively expressed in a tobacco genotype without N (nn). Transgenic F1 expressing N and p50 were generated by crossing with an NN genotype. Surprisingly, Nn F1 seeds expressing p50 are viable and germinate. Only about 5 days after sowing, seedlings started to show an HR. This paralleled the upregulation of several pathogenesis-related and HR genes. The timing of the HR is consistent with the upregulation of N gene transcript 4-6 days after sowing. The expression of p50 has a stimulating effect on the N gene transcript level during germination. These results show that tobacco seeds and very young seedlings do not express a functional N gene product.

'MicroRNA Targets', a new AthaMap web-tool for genome-wide identification of miRNA targets in Arabidopsis thaliana.

BACKGROUND: The AthaMap database generates a genome-wide map for putative transcription factor binding sites for A. thaliana. When analyzing transcriptional regulation using AthaMap it may be important to learn which genes are also post-transcriptionally regulated by inhibitory RNAs. Therefore, a unified database for transcriptional and post-transcriptional regulation will be highly useful for the analysis of gene expression regulation. METHODS: To identify putative microRNA target sites in the genome of A. thaliana, processed mature miRNAs from 243 annotated miRNA genes were used for screening with the psRNATarget web server. Positional information, target genes and the psRNATarget score for each target site were annotated to the AthaMap database. Furthermore, putative target sites for small RNAs from seven small RNA transcriptome datasets were used to determine small RNA target sites within the A. thaliana genome. RESULTS: Putative 41,965 genome wide miRNA target sites and 10,442 miRNA target genes were identified in the A. thaliana genome. Taken together with genes targeted by small RNAs from small RNA transcriptome datasets, a total of 16,600 A. thaliana genes are putatively regulated by inhibitory RNAs. A novel web-tool, 'MicroRNA Targets', was integrated into AthaMap which permits the identification of genes predicted to be regulated by selected miRNAs. The predicted target genes are displayed with positional information and the psRNATarget score of the target site. Furthermore, putative target sites of small RNAs from selected tissue datasets can be identified with the new 'Small RNA Targets' web-tool. CONCLUSIONS: The integration of predicted miRNA and small RNA target sites with transcription factor binding sites will be useful for AthaMap-assisted gene expression analysis. URL: http://www.athamap.de/