Identification and expression regulation of symbiotically activated legume genes.

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.

Development of bioinformatic tools to support EST-sequencing, in silico- and microarray-based transcriptome profiling in mycorrhizal symbioses.

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.

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.

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.

Construction and validation of cDNA-based Mt6k-RIT macro- and microarrays to explore root endosymbioses in the model legume Medicago truncatula.

To construct macro- and microarray tools suitable for expression profiling in root endosymbioses of the model legume Medicago truncatula, we PCR-amplified a total of 6048 cDNA probes representing genes expressed in uninfected roots, mycorrhizal roots and young root nodules [Nucleic Acids Res. 30 (2002) 5579]. Including additional probes for either tissue-specific or constitutively expressed control genes, 5651 successfully amplified gene-specific probes were used to grid macro- and to spot microarrays designated Mt6k-RIT (M. truncatula 6k root interaction transcriptome). Subsequent to a technical validation of microarray printing, we performed two pilot expression profiling experiments using Cy-labeled targets from Sinorhizobium meliloti-induced root nodules and Glomus intraradices-colonized arbuscular mycorrhizal roots. These targets detected marker genes for nodule and arbuscular mycorrhiza development, amongst them different nodule-specific leghemoglobin and nodulin genes as well as a mycorrhiza-specific phosphate transporter gene. In addition, we identified several dozens of genes that have so far not been reported to be differentially expressed in nodules or arbuscular mycorrhiza thus demonstrating that Mt6k-RIT arrays serve as useful tools for an identification of genes relevant for legume root endosymbioses. A comprehensive profiling of such candidate genes will be very helpful to the development of breeding strategies and for the improvement of cultivation management targeted at increasing legume use in sustainable agricultural systems.