Publications (15)54.69 Total impact
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Article: Development of bioinformatic tools to support EST-sequencing, in silico- and microarray-based transcriptome profiling in mycorrhizal symbioses.
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ABSTRACT: The great majority of terrestrial plants enters a beneficial arbuscular mycorrhiza (AM) or ectomycorrhiza (ECM) symbiosis with soil fungi. In the SPP 1084 "MolMyk: Molecular Basics of Mycorrhizal Symbioses", high-throughput EST-sequencing was performed to obtain snapshots of the plant and fungal transcriptome in mycorrhizal roots and in extraradical hyphae. To focus activities, the interactions between Medicago truncatula and Glomus intraradices as well as Populus tremula and Amanita muscaria were selected as models for AM and ECM symbioses, respectively. Together, almost, 20.000 expressed sequence tags (ESTs) were generated from different random and suppressive subtractive hybridization (SSH) cDNA libraries, providing a comprehensive overview of the mycorrhizal transcriptome. To automatically cluster and annotate EST-sequences, the BioMake and SAMS software tools were developed. In connection with the eNorthern software SteN, plant genes with a predicted mycorrhiza-induced expression were identified. To support experimental transcriptome profiling, macro- and microarray tools have been constructed for the two model mycorrhizae, based either on PCR-amplified cDNAs or 70mer oligonucleotides. These arrays were used to profile the transcriptome of AM and ECM roots under different conditions, and the data obtained were uploaded to the ArrayLIMS and EMMA databases that are designed to store and evaluate expression profiles from DNA arrays. Together, the EST- and transcriptome databases can be mined to identify candidate genes for targeted functional studies.Phytochemistry 02/2007; 68(1):19-32. · 3.35 Impact Factor -
Article: Identification and expression regulation of symbiotically activated legume genes.
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ABSTRACT: Legume plants are able to enter two different endosymbioses with soil prokaryotes and soil fungi, leading to nitrogen-fixing root nodules and to arbuscular mycorrhiza (AM), respectively. We applied in silico and microarray-based transcriptome profiling approaches to uncover the transcriptome of developing root nodules and AM roots of the model legume Medicago truncatula. Several hundred genes were found to be activated in different stages of either symbiosis, with almost 100 genes being co-induced during nodulation and in arbuscular mycorrhiza. These co-induced genes can be associated with different cellular functions required for symbiotic efficiency, such as the facilitation of transport processes across the perisymbiotic membranes that surround the endosymbiotic bacteroids in root nodules and the arbuscules in AM roots. To specify promoter elements required for gene expression in arbuscule-containing cells, reporter gene fusions of the promoter of the Vicia faba leghemoglobin gene VfLb29 were studied by loss-of-function and gain-of-function approaches in transgenic hairy roots. These analyses specified a 85-bp fragment that was necessary for gene expression in arbuscule-containing cells but was dispensible for gene activation in root nodules. In contrast to promoters mediating gene expression in the infected cells of root nodules, the activation of genes in AM appears to be governed by more complex regulatory systems requiring different promoter modules.Phytochemistry 02/2007; 68(1):8-18. · 3.35 Impact Factor -
Article: Combined transcriptome profiling reveals a novel family of arbuscular mycorrhizal-specific Medicago truncatula lectin genes.
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ABSTRACT: The large majority of plants are capable of undergoing a tight symbiosis with arbuscular mycorrhizal (AM) fungi. During this symbiosis, highly specialized new structures called arbuscules are formed within the host cells, indicating that, during interaction with AM fungi, plants express AM-specific genetic programs. Despite increasing efforts, the number of genes known to be induced in the AM symbiosis is still low. In order to identify novel AM-induced genes which have not been listed before, 5,646 expressed sequence tags (ESTs) were generated from two Medicago truncatula cDNA libraries: a random cDNA library (MtAmp) and a suppression subtractive hybridization (SSH) library (MtGim), the latter being designed to enhance the cloning of mycorrhiza-upregulated genes. In silico expression analysis was applied to identify those tentative consensus sequences (TCs) of The Institute for Genomic Research M. truncatula gene index (MtGI) that are composed exclusively of ESTs deriving from the MtGim or MtAmp library, but not from any other cDNA library of the MtGI. This search revealed 115 MtAmp- or MTGim-specific TCs. For the majority of these TCs with sequence similarities to plant genes, the AM-specific expression was verified by quantitative reverse-transcription polymerase chain reaction. Annotation of the novel genes induced in mycorrhizal roots suggested their involvement in different transport as well as signaling processes and revealed a novel family of AM-specific lectin genes. The expression of reporter gene fusions in transgenic roots revealed an arbuscule-related expression of two members of the lectin gene family, indicating a role for AM-specific lectins during arbuscule formation or functioning.Molecular Plant-Microbe Interactions 09/2005; 18(8):771-82. · 4.43 Impact Factor -
Article: The promoter of the leghaemoglobin gene VfLb29: functional analysis and identification of modules necessary for its activation in the infected cells of root nodules and in the arbuscule-containing cells of mycorrhizal roots.
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ABSTRACT: In this study the further characterization of the Vicia faba leghaemoglobin promoter pVfLb29 is presented that was previously shown to be specifically active in the infected cells of root nodules and in arbuscule-containing cells of mycorrhizal roots. Using promoter studies in transgenic hairy roots of the Pisum sativum mutant RisNod24, disabled in the formation of functional arbuscules, VfLb29 promoter activity is assigned to later stages of arbuscule development. In order to narrow down the regions containing cis-acting elements of pVfLb29, the activity of five VfLb29 promoter deletions (-797/-31 to -175/-31 in relation to the start codon) fused to the gusAint coding region were tested in transgenic V. hirsuta hairy roots. The results specify a promoter region ranging from position -410 to -326 (85 bp) as necessary for gus expression in arbuscule-containing cells, whereas this segment is not involved in the nodule-specific activity. Sequence analysis of the pVfLb29 fragment -410/-326 (85 bp) revealed sequence motifs previously shown to be cis-acting elements of diverse promoters. To investigate the autonomous function of pVfLb29 regions for activation in arbuscule-containing cells, different regions of pVfLb29 from positions -410 to -198 were used to prepare chimeric promoter constructs for trans-activation studies. These fragments alone did not activate the mycorrhiza inactive promoter of the Vicia faba leghaemoglobin gene VfLb3, showing that the activation of pVfLb29 in arbuscule-containing cells is governed by a complex regulatory system that requires at least two modules located between position -410 and -31 of the VfLb29 gene.Journal of Experimental Botany 04/2005; 56(413):799-806. · 5.36 Impact Factor -
Article: Transcriptome profiling in root nodules and arbuscular mycorrhiza identifies a collection of novel genes induced during Medicago truncatula root endosymbioses.
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ABSTRACT: Transcriptome profiling based on cDNA array hybridizations and in silico screening was used to identify Medicago truncatula genes induced in both root nodules and arbuscular mycorrhiza (AM). By array hybridizations, we detected several hundred genes that were upregulated in the root nodule and the AM symbiosis, respectively, with a total of 75 genes being induced during both interactions. The second approach based on in silico data mining yielded several hundred additional candidate genes with a predicted symbiosis-enhanced expression. A subset of the genes identified by either expression profiling tool was subjected to quantitative real-time reverse-transcription polymerase chain reaction for a verification of their symbiosis-induced expression. That way, induction in root nodules and AM was confirmed for 26 genes, most of them being reported as symbiosis-induced for the first time. In addition to delivering a number of novel symbiosis-induced genes, our approach identified several genes that were induced in only one of the two root endosymbioses. The spatial expression patterns of two symbiosis-induced genes encoding an annexin and a beta-tubulin were characterized in transgenic roots using promoter-reporter gene fusions.Molecular Plant-Microbe Interactions 11/2004; 17(10):1063-77. · 4.43 Impact Factor -
Article: The Medicago truncatula sucrose synthase gene MtSucS1 is activated both in the infected region of root nodules and in the cortex of roots colonized by arbuscular mycorrhizal fungi.
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ABSTRACT: The MtSucS1 gene encodes a sucrose synthase (EC 2.4.1.13) in the model legume Medicago truncatula. To determine the expression pattern of this gene in different organs and in particular during root endosymbioses, we transformed M. truncatula with specific regions of MtSucS1 fused to the gusAint reporter gene. These fusions directed an induction to the vasculature of leaves, stems, and roots as well as to flowers, developing seeds, young pods, and germinating seedlings. In root nodules, strong promoter activity occurred in the infected cells of the nitrogen-fixing zone but was additionally observed in the meristematic region, the prefixing zone, and the inner cortex, including the vasculature. Concerning endomycorrhizal roots, the MtSucS1 promoter mediated strongest expression in cortical cells harboring arbuscules. Specifically in highly colonized root sections, GUS-staining was furthermore detected in the surrounding cortical cells, irrespective of a direct contact with fungal structures. In accordance with the presence of an orthologous PsSus1 gene, we observed a comparable regulation of MtSucS1 expression in the grain legume Pisum sativum in response to microbial symbionts. Unlike other members of the MtSucS gene family, the presence of rhizobial or Glomus microsymbionts significantly altered and enhanced MtSucS1 gene expression, leading us to propose that MtSucS1 is involved in generating sink-strength, not only in root nodules but also in mycorrhizal roots.Molecular Plant-Microbe Interactions 11/2003; 16(10):903-15. · 4.43 Impact Factor -
Article: Transcriptional changes in response to arbuscular mycorrhiza development in the model plant Medicago truncatula.
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ABSTRACT: Significant changes in root morphology and physiology during arbuscular mycorrhiza (AM) development are likely to be controlled by specific gene expression pattern in the host plant. Until now, little was known about transcriptional changes which occur AM-exclusively; that is, they do not occur during other root-microbe associations, nor are they induced by improved phosphate nutrition. In order to identify such AM-exclusive gene inductions of Medicago truncatula, we used a pool of different RNA samples as subtractor population in a suppressive subtractive hybridization (SSH) experiment. This approach resulted in the identification of a number of new AM-regulated genes. None of these genes were expressed in nonmycorrhiza roots or leaves. Electronic data obtained by comparison of the cDNA sequences to expressed sequence tag (EST) sequences from a wide range of cDNA libraries in the M. truncatula EST database (Gene Index, MtGI) support the mycorrhiza specificity of the corresponding genes, because sequences in the MtGI that were found to match the identified SSH-cDNA sequences originated exclusively from AM cDNA libraries. The promoter of one of those genes, MtGst1, showing similarities to plant glutathione-S-transferase (GST) encoding genes, was cloned and used in reporter gene studies. In contrast to studies with the potato GST gene PRP, MtGst 1 promoter activity was detected in all zones of the root cortex colonized by Glomus intraradices, but nowhere else.Molecular Plant-Microbe Interactions 05/2003; 16(4):306-14. · 4.43 Impact Factor -
Article: The nodulin VfENOD18 is an ATP-binding protein in infected cells of Vicia faba L. nodules
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ABSTRACT: Recently we described the novel nodulin gene VfENOD18, whose corresponding transcripts were restricted to the nitrogen-fixing zone III of broad bean root nodules. To characterize VfENOD18 on the protein level, polyclonal antibodies were generated using the purified recombinant VfENOD18 protein produced in Escherichia coli by employing the pMAL-c expression system. These antibodies recognized immunoreactive proteins isolated from indeterminate nodules of different leguminous plants, but also from non-symbiotic tissues of Glycine max and from tissues of Arabidopsis thaliana and Zea mays. Using immunogold labelling the nodulin VfENOD18 was localized to the cytoplasm of infected cells in the nitrogen-fixing zone of broad bean nodules. Due to the homology of the VfENOD18 sequence to that of the ATP-binding protein MJ0577 from the hyperthermophile Methanococcus jannaschii the recombinant VfENOD18 protein was tested for ATP-binding. Using the biotin photoaffinity ATP analogue 8N3ATP[]biotin it could be demonstrated that VfENOD18 is an ATP-binding protein. PCR experiments revealed that the amino acid sequences of the putative C-terminal ATP-binding sites of the VfENOD18 homologues from Lens culinaris, Vicia hirsuta, Vicia sativa and Vicia villosa were conserved. We propose that VfENOD18 is a member of a novel family of ATP-binding proteins in plants.Plant Molecular Biology 11/2001; 47(6):749-759. · 4.15 Impact Factor -
Article: The temporal and spatial transcription pattern in root nodules of Vicia faba nodulin genes encoding glycine-rich proteins
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ABSTRACT: Four different transcript sequences encoding gene products with an unusually high glycine content were identified in Vicia faba root nodules. Northern blot analysis revealed a strong nodule specific expression of the corresponding genes. Time course experiments showed that two of these genes were transcribed before the onset of leghemoglobin expression and hence were designated VfENOD-GRP2 and VfENOD-GRP5, whereas the first detection of VfNOD-GRP1 and VfNOD-GRP4 transcripts coincided with the appearance of leghemoglobin transcripts in V. faba root nodules. A characteristic feature of all encoded nodulins was a hydrophobic N-terminus, which in the case of the nodulins ENOD-GRP2 and ENOD-GRP5 has the characteristics of a signal peptide. Such a structure is comparable to other plant glycine-rich proteins described as components of the plant cell wall. Based on tissue print hybridizations, we found that VfNOD-GRP1, VfENOD-GRP2 and VfNOD-GRP4 were expressed in the interzone II-III and in the whole nitrogen-fixing zone III. In contrast to VfENOD-GRP2 and VfNOD-GRP4, the signal intensity of hybridizing VfNOD-GRP1 transcripts was slightly reduced in the more proximal part of broad bean root nodules. Apart from the interzone II-III and the nitrogen fixing zone III, VfENOD-GRP5 RNA was also detected in large areas of the prefixing zone II.Plant Molecular Biology 12/1996; 33(1):113-123. · 4.15 Impact Factor -
Article: The promoter of the Vicia faba L. VfENOD-GRP3 gene encoding a glycine-rich early nodulin mediates a predominant gene expression in the interzone II-III region of transgenic Vicia hirsuta root nodules
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ABSTRACT: We recently reported on the broad bean gene VfENOD-GRP3 encoding a glycine-rich early nodulin. This gene was predominantly expressed in the interzone II–III region of Vicia faba root nodules. The VfENOD-GRP3 promoter contained several sequence motifs potentially involved in the regulation of gene expression. To investigate the molecular basis for the specific VfENOD-GRP3 expression, defined VfENOD-GRP3 promoter fragments were fused to an intron-containing gusAint gene. Agrobacterium rhizogenes ARqual strains carrying these fusions integrated into the TL DNA were used to generate hairy roots on Vicia hirsuta, which subsequently were nodulated. Histochemical analysis of transgenic nodules indicated that a strong gusAint expression in the interzone II–III region was mediated by the –1252/ + 10 VfENOD-GRP3 promoter region. This reporter gene expression in V. hirsuta was comparable to the location of VfENOD-GRP3 transcripts in V. faba nodules. An analysis of defined promoter fragments revealed that a strong gusAint expression in the interzone II–III region was also mediated by the –737/ + 10 promoter, whereas the –239/ + 10 promoter only mediated a weak gusAint expression in the interzone II–III region. Since the –239/ + 10 promoter fragment did not resemble published nodulin gene promoters, we propose that it contains new sequence motifs involved in mediating gene expression in the interzone II–III region of vicia nodules.Plant Molecular Biology 10/1995; 29(4):759-772. · 4.15 Impact Factor -
Article: The nodule-specific VfENOD-GRP3 gene encoding a glycine-rich early nodulin is located on chromosome I of Vicia faba L. and is predominantly expressed in the interzone II-III of root nodules
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ABSTRACT: A nodule-specific cDNA was isolated from a Vicia faba L. nodule cDNA library. Since time course experiments revealed an early expression of this transcript in the nodule, this cDNA coded for an early nodulin and was designated VfENOD-GRP3. Based on tissue print hybridizations, we found a predominant expression of VfENOD-GRP3 transcripts in the interzone II-III region of broad bean root nodules. The encoded early nodulin ENOD-GRP3 was characterized by an N-terminal signal peptide and a C-terminal domain displaying a glycine content of 31%. Sequence analysis of a genomic VfENOD-GRP3 clone revealed that the signal peptide and the glycine-rich domain were specified by two separate exons. Primer extension experiments identified two adjacent transcription start sites for VfENOD-GRP3 transcripts. The common nodulin sequences AAAGAT and CTCTT were present five and three times on both DNA strands of the putative VfENOD-GRP3 promoter, respectively. Additionally, three sequence motifs resembling organ-specific elements of the soybean lbc3 gene promoter and a sequence similar to the binding site 1 for the nodule trans-acting factor Nat2 were identified. From Southern blot data and from sequence analysis of genomic PCR fragments, the presence of a VfENOD-GRP3 gene family was inferred. By PCR experiments using sequence-specific primers and DNA of microisolated chromosomes as a template, this family was located on the long arm of chromosome I.Plant Molecular Biology 05/1995; 28(3):405-421. · 4.15 Impact Factor -
Article: Members of a broadbean nodulin family with partial homologies to the alfalfa nodulin 25 are composed of two types of amino acid repeats flanked by unique amino acid sequence termini
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ABSTRACT: Five cross-hybridizing cDNAs from clone group VfNDS-L of a broadbean nodule-specific cDNA library differed by five specific in-frame deletions within the coding region. Northern blot analysis revealed that the transcripts represented by these clones were expressed in a nodule-specific way and therefore encoded a new family of broadbean nodulins. These nodulins have been designated Nvf-28/32. The Nvf-28/32 proteins were between 269 and 299 amino acids long with a high proportion of charged amino acids and contained a putative signal peptide of 20 amino acids. Sequence analysis indicated that the central part of the Nvf-28/32 proteins was composed of two different types of repeating amino acid stretches designated repeat 1 and repeat 2, whereas the N- and C-termini were unique. In contrast to repeat 1 stretches, which contained both positively and negatively charged amino acid residues, repeat 2 sequences did not contain any positively, but a total of 13 negatively charged amino acid residues. We could demonstrate that the five deletions identified exactly corresponded to complete repeats. The unique sequence termini of the Nvf-28/32 proteins displayed strong homologies to the late nodulin 25 from alfalfa. In addition, the repeating units identified were significantly homologous to several, but not all exon parts of this protein. We speculate that the VfNDS-L transcripts were derived from a differential splicing mechanism and that the Nvf-28/32 proteins fulfil a structural rather than an enzymatic function within the broadbean root nodule.Plant Molecular Biology 12/1993; 24(1):143-157. · 4.15 Impact Factor -
Article: A survey of transcripts expressed specifically in root nodules of broadbean (Vicia faba L.)
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ABSTRACT: More than 600 potentially nodule-specific clones have been detected by differential hybridization of a broadbean cDNA library constructed from root nodule poly(A)+ RNA. These isolated cDNAs belong to at least 28 different clone groups containing cross-hybridizing sequences. The number of clones within a clone group varies from about 200 to only one single clone.Northern hybridization experiments revealed nodule-specific transcripts for 14 clone groups and markedly nodule-enhanced transcripts for another 7 clone groups. Sequence homologies indicate that three transcript sequences code for different leghemoglobins. Two other transcripts encode a nodule-specific sucrose synthase and a nodule-enhanced asparagine synthetase, respectively. Four deduced gene products are proline-rich, two of them being the homologues of PsENOD2 and PsENOD12. The third proline-rich protein (PRP) is composed of similar amino acid repeats as the nodule-specific PsENOD12 but is expressed in nodules and roots in comparable amounts. The fourth PRP is a nodule-enhanced extensin-type protein built up by Ser-Pro4 repeats. Two further nodule-specific transcripts encode gene products showing some similarity to structural glycine-rich proteins. Additionally, transcripts could be identified for broadbean homologues of the nodulins MsNOD25, PsENOD3 and PsENOD5 and transcripts specifying a nodule-enhanced lipoxygenase and a translation elongation factor EF-1 , which is expressed in all broadbean tissues tested.Plant Molecular Biology 08/1993; 22(6):957-970. · 4.15 Impact Factor -
Article: The promoter of the Vicia faba L. gene VfEnod12 encoding an early nodulin is active in cortical cells and nodule primordia of transgenic hairy roots of Vicia hirsuta as well as in the prefixing zone II of mature transgenic V. hirsuta root nodules
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ABSTRACT: A full-length cDNA encoding the Viciafaba L. early nodulin VfEnod12 was isolated. The deduced protein sequence specified a 90 amino acid protein with a MW of 10 206 and contained a putative signal peptide sequence followed by PPX3 repeats characteristic of Enod12 proteins. The VfEnod12 gene was found to be expressed specifically in root nodules as early as 3 days post inoculation with Rhizobium leguminosarum bv. viciae. In mature nodules, VfEnod12 transcripts were confined to the prefixing zone II. A 3.3 kb genomic fragment carrying the complete VfEnod12 coding region was isolated. No intervening sequences were identified in the coding region. A promoter fragment carrying the -692/-41 region mediated reporter gene expression in root cortical cells, nodule primordia and the prefixing zone II of transgenic Vicia hirsuta root nodules. This fragment contained a putative binding site for the transcription factor ENBP1. In contrast to the highly conserved terminal AATAA motif of the ENBP1 binding site of known Enod12 promoters, the VfEnod12 promoter was characterized by an altered terminal AATAT sequence. This alteration did not interfere with VfEnod12 promoter activity in transgenic roots and nodules of V. hirsuta.Plant Science. -
Article: Genomic organization and expression properties of the VfENOD5 gene from broad bean (Vicia faba L.)
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ABSTRACT: A full-length cDNA encoding the broad bean (Vicia faba L.) early nodulin VfENOD5 was isolated from a nodule cDNA library. In addition to the ENOD5 homologues from other legumes the derived VfENOD5 amino acid sequence also displayed homologies to the phytocyanin-related nodulins GmENOD55-2, MtENOD16, and MtENOD20. A close inspection of the ENOD5 proteins from broad bean, pea and vetch indicated that all these nodulins possess a putative C-terminal GPI-anchor signal sequence. This novel finding supports the hypothesis that ENOD5 is an arabinogalactan protein. Tissue print hybridizations revealed that the broad bean ENOD5 gene was not only expressed in the central tissues of root nodules. In contrast to other legumes hybridizing transcripts were also be detected in a narrow zone within the peripheral nodule tissues. Sequence analysis of a genomic clone indicated the presence of a single intron interrupting the VfENOD5 coding region at a position precisely corresponding to the MtENOD16 and MtENOD20 introns.Plant Science.
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1993–2007
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Universität Bielefeld
- • CeBiTec - Center for Biotechnology
- • Faculty of Biology
Bielefeld, North Rhine-Westphalia, Germany
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