Andreas M. Perlick

Transcriptome profiling in root nodules and arbuscular mycorrhiza identifies a collection of novel genes induced during Medicago truncatula root endosymbioses

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.

Transcriptional changes in response to arbuscular mycorrhiza development in the model plant Medicago truncatula

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.

The promoter of the Vicia faba L. leghemoglobin gene VfLb29 is specifically activated in the infected cells of root nodules and in the arbuscule-containing cells of mycorrhizal roots from different legume and nonlegume plants

The VfLb29 leghemoglobin gene promoter was polymerase chain reaction-amplified from a Vicia faba genomic library and was fused to the gusAint coding region. Expression of the chimeric gene was analyzed in transgenic hairy roots of the legumes V. faba, V. hirsuta, and Medicago truncatula as well as in transgenic Nicotiana tabacum plants. The VfLb29 promoter was found to be specifically active not only in the infected cells of the nitrogen-fixing zone of root nodules but also in arbuscule-containing cells of transgenic V. faba and M. truncatula roots colonized by the endomycorrhizal fungus Glomus intraradices. In addition to these two legumes, specific expression in arbuscule-containing cells was also observed in the nonlegume N. tabacum. All studies were done in comparison to the V. faba leghemoglobin gene promoter VfLb3 that as VfLb29 was expressed in the infected cells of root nodules but showed no activity in endomycorrhiza. An activation of the VfLb29 promoter due to hypoxia in metabolically active tissues was excluded. The conserved activation in arbuscule-containing cells of legumes and the nonlegume N. tabacum suggests a conserved trigger for this promoter in legume and nonlegume endomycorrhiza symbioses.

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

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.

The Vicia faba Leghemoglobin Gene VfLb29 Is Induced in Root Nodules and in Roots Colonized by the Arbuscular Mycorrhizal Fungus Glomus fasciculatum

To investigate similarities between symbiotic interactions of broad bean (Vicia faba) with rhizobia and mycorrhizal fungi, plant gene expression induced by both microsymbionts was compared. We demonstrated the exclusive expression of 19 broad bean genes, including VfENOD2, VfENOD5, VfENOD12 and three different leghemoglobin genes, in root nodules. In contrast, the leghemoglobin gene VfLb29 was found to be induced not only in root nodules, but also in broad bean roots colonized by the mycorrhizal fungus Glomus fasciculatum. In uninfected roots, none of the 20 nodulin transcripts investigated was detectable. VfLb29 has an unusually low sequence homology with all other broad bean leghemoglobins as well as with leghemoglobins from other legumes. It can be regarded as a novel kind of leghemoglobin gene not described until now and the induction of which is common to symbiotic interactions of broad bean with both Rhizobium and a mycorrhizal fungus.

Combined Transcriptome Profiling Reveals a Novel Family of Arbuscular Mycorrhizal-Specific Medicago truncatula Lectin Genes

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.

The Broad Bean Gene VfNOD32 Encodes a Nodulin with Sequence Similarities to Chitinases That Is Homologous to ([alpha]/[beta])8-Barrel-Type Seed Proteins

Nodulin gene transcripts isolated from a broad bean (Vicia faba L.) root nodule cDNA library and designated VfNOD32 are detectable in the nitrogen-fixing zone III of nodules and in much smaller amounts in flowers. In nodules, these transcripts are detectable for the first time 7 d after inoculation, at least 1 d before leghemoglobin gene transcription starts. Two putative full-length cDNAs representing different transcript sequences of 92.5% identity were sequenced. The corresponding broad bean genes were termed VfNOD32-A1 and VfNOD32-A2, and the encoded proteins were termed Nvf32-A1 and Nvf32-A2. The derived amino acid sequences of the Nvf32 proteins are highly homologous to the Vicia narbonensis ([alpha]/[beta])8-barrel seed protein narbonin. Considering this homology, Nvf32 is assumed to have a similar structure consisting of [beta]-sheets forming a central barrel surrounded by [alpha]-helices. The two Nvf32 sequences also contain two conserved amino acid motifs that are characteristic of class-III chitinases. Several amino acids demonstrated to be essential for chitinase activity are conserved in both regions, whereas one essential glutamic acid was changed to glycine in the Nvf32-A1 isoform but not in the Nvf32-A2 isoform.

Genomic organization and expression properties of the MtSucS1 gene, which encodes a nodule-enhanced sucrose synthase in the model legume Medicago truncatula.

Abstract We have isolated and sequenced a sucrose synthase (SucS) cDNA from the model legume Medicago truncatula. This cDNA (MtSucS1) contains an ORF of 2418 bp, coding for a protein of 805 amino acids with a molecular mass of 92.29 kDa. The deduced amino acid sequence shows significant homology to other plant sucrose synthases, in particular to the nodule-enhanced sucrose synthases from pea and broad bean. Northern analysis revealed that the corresponding gene shows a ten-fold higher expression level in root nodules than in uninfected root, stem and leaf tissues. SucS protein was detected in root nodules from a variety of legumes, including M. truncatula. Whereas only one SucS isoform was detectable in root nodules, an additional sucrose synthase of slightly larger molecular weight was present in uninfected root, stem and flower tissues of M. truncatula. From our expression and sequence data we infer that the MtSucS1 gene encodes a nodule-enhanced sucrose synthase in M. truncatula. Southern hybridization data indicate that MtSucS1 is a single-copy gene. An analysis of a genomic MtSucS1 sequence revealed that the gene consists of 14 exons with the start codon being located on exon II. As is common for SucS genes, the MtSucS1 gene contains a large intron of 747 bp in the 5′ untranslated region. The transcriptional start of MtSucS1 was mapped and putative regulatory elements in the MtSucS1 promoter were identified.

A survey of transcripts expressed specifically in root nodules of broadbean (Vicia faba L.)

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 α, which is expressed in all broadbean tissues tested.

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

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.