The signal peptide of the Medicago truncatula modular nodulin MtNOD25 operates as an address label for the specific targeting of proteins to nitrogen-fixing symbiosomes.

The nodule-specific MtNOD25 gene of the model legume Medicago truncatula encodes a modular nodulin composed of different repetitive modules flanked by distinct N- and C-termini. Although similarities are low with respect to all repetitive modules, both the N-terminal signal peptide (SP) and the C-terminus are highly conserved in modular nodulins from different legumes. On the cellular level, MtNOD25 is only transcribed in the infected cells of root nodules, and this activation is mediated by a 299-bp minimal promoter containing an organ-specific element. By expressing mGFP6 translational fusions in transgenic nodules, we show that MtNOD25 proteins are exclusively translocated to the symbiosomes of infected cells. This specific targeting only requires an N-terminal MtNOD25 SP that is highly conserved across a family of legume-specific symbiosome proteins. Our finding sheds light on one possible mechanism for the delivery of host proteins to the symbiosomes of infected root nodule cells and, in addition, defines a short molecular address label of only 24 amino acids whose N-terminal presence is sufficient to translocate proteins across the peribacteroid membrane.

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.

Overlaps in the transcriptional profiles of Medicago truncatula roots inoculated with two different Glomus fungi provide insights into the genetic program activated during arbuscular mycorrhiza.

Arbuscular mycorrhiza (AM) is a widespread symbiotic association between plants and fungal microsymbionts that supports plant development under nutrient-limiting and various stress conditions. In this study, we focused on the overlapping genetic program activated by two commonly studied microsymbionts in addition to identifying AM-related genes. We thus applied 16,086 probe microarrays to profile the transcriptome of the model legume Medicago truncatula during interactions with Glomus mosseae and Glomus intraradices and specified a total of 201 plant genes as significantly coinduced at least 2-fold, with more than 160 being reported as AM induced for the first time. Several hundred genes were additionally up-regulated during a sole interaction, indicating that the plant genetic program activated in AM to some extent depends on the colonizing microsymbiont. Genes induced during both interactions specified AM-related nitrate, ion, and sugar transporters, enzymes involved in secondary metabolism, proteases, and Kunitz-type protease inhibitors. Furthermore, coinduced genes encoded receptor kinases and other components of signal transduction pathways as well as AM-induced transcriptional regulators, thus reflecting changes in signaling. By the use of reporter gene expression, we demonstrated that one member of the AM-induced gene family encoding blue copper binding proteins (MtBcp1) was both specifically and strongly up-regulated in arbuscule-containing regions of mycorrhizal roots. A comparison of the AM expression profiles to those of nitrogen-fixing root nodules suggested only a limited overlap between the genetic programs orchestrating root endosymbioses.

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.

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.

Two genes encoding different truncated hemoglobins are regulated during root nodule and arbuscular mycorrhiza symbioses of Medicago truncatula.

The MtTrHb1 and MtTrHb2 genes of the model legume Medicago truncatula Gaertn. encode proteins homologous to truncated hemoglobins (TrHb) from plants and a range of different microorganisms. Induction of MtTrHb1 in root nodules and expression of MtTrHb2 in root nodules, as well as in mycorrhizal roots, were shown by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR). The promoters of both genes were PCR-amplified and fused to the gusAint coding region. By analysing these gusAint-fusions in transgenic root tissues, we were able to localize their activity in root nodules and in roots colonized by arbuscular mycorrhizal (AM) fungi. Whereas the promoter of MtTrHb1 was activated in the infected cells of the nitrogen-fixing zone of root nodules, the MtTrHb2 promoter was predominantly active in the nodule vascular tissue. This expression pattern correlates with the presence of an 'organ-specific element' (OSE)-like sequence in the MtTrHb1 promoter, which is not present in the MtTrHb2 regulatory unit. Concerning the AM symbiosis, only the MtTrHb2 promoter mediated an expression in arbuscule-containing cells and in the root vascular tissue of mycorrhizal root segments colonized by the fungus Glomus intraradices.

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.