Natalija Hohnjec

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.

Expression Profiling in Medicago truncatula Identifies More Than 750 Genes Differentially Expressed during Nodulation, Including Many Potential Regulators of the Symbiotic Program

In this study, we describe a large-scale expression-profiling approach to identify genes differentially regulated during the symbiotic interaction between the model legume Medicago truncatula and the nitrogen-fixing bacterium Sinorhizobium meliloti. Macro- and microarrays containing about 6,000 probes were generated on the basis of three cDNA libraries dedicated to the study of root symbiotic interactions. The experiments performed on wild-type and symbiotic mutant material led us to identify a set of 756 genes either up- or down-regulated at different stages of the nodulation process. Among these, 41 known nodulation marker genes were up-regulated as expected, suggesting that we have identified hundreds of new nodulation marker genes. We discuss the possible involvement of this wide range of genes in various aspects of the symbiotic interaction, such as bacterial infection, nodule formation and functioning, and defense responses. Importantly, we found at least 13 genes that are good candidates to play a role in the regulation of the symbiotic program. This represents substantial progress toward a better understanding of this complex developmental program.

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.

Laser Microdissection Unravels Cell-Type-Specific Transcription in Arbuscular Mycorrhizal Roots, Including CAAT-Box Transcription Factor Gene Expression Correlating with Fungal Contact and Spread

Arbuscular mycorrhizae (AM) are the most widespread symbioses on Earth, promoting nutrient supply of most terrestrial plant species. To unravel gene expression in defined stages of Medicago truncatula root colonization by AM fungi, we here combined genome-wide transcriptome profiling based on whole mycorrhizal roots with real-time reverse transcription-PCR experiments that relied on characteristic cell types obtained via laser microdissection. Our genome-wide approach delivered a core set of 512 genes significantly activated by the two mycorrhizal fungi Glomus intraradices and Glomus mossae. Focusing on 62 of these genes being related to membrane transport, signaling, and transcriptional regulation, we distinguished whether they are activated in arbuscule-containing or the neighboring cortical cells harboring fungal hyphae. In addition, cortical cells from nonmycorrhizal roots served as a reference for gene expression under noncolonized conditions. Our analysis identified 25 novel arbuscule-specific genes and 37 genes expressed both in the arbuscule-containing and the adjacent cortical cells colonized by fungal hyphae. Among the AM-induced genes specifying transcriptional regulators were two members encoding CAAT-box binding transcription factors (CBFs), designated MtCbf1 and MtCbf2. Promoter analyses demonstrated that both genes were already activated by the first physical contact between the symbionts. Subsequently, and corresponding to our cell-type expression patterns, they were progressively up-regulated in those cortical areas colonized by fungal hyphae, including the arbuscule-containing cells. The encoded CBFs thus represent excellent candidates for regulators that mediate a sequential reprogramming of root tissues during the establishment of an AM symbiosis.

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.

Transcriptional Responses toward Diffusible Signals from Symbiotic Microbes Reveal MtNFP- and MtDMI3-Dependent Reprogramming of Host Gene Expression by Arbuscular Mycorrhizal Fungal Lipochitooligosaccharides

The formation of root nodules and arbuscular mycorrhizal (AM) roots is controlled by a common signaling pathway including the calcium/calmodulin-dependent kinase Doesn’t Make Infection3 (DMI3). While nodule initiation by lipochitooligosaccharide (LCO) Nod factors is well characterized, diffusible AM fungal signals were only recently identified as sulfated and nonsulfated LCOs. Irrespective of different outcomes, the perception of symbiotic LCOs in Medicago truncatula is mediated by the LysM receptor kinase M. truncatula Nod factor perception (MtNFP). To shed light on transcriptional responses toward symbiotic LCOs and their dependence on MtNFP and Ca2+ signaling, we performed genome-wide expression studies of wild-type, Nod-factor-perception mutant1, and dmi3 mutant roots challenged with Myc- and Nod-LCOs. We show that Myc-LCOs lead to transient, quick responses in the wild type, whereas Nod-LCOs require prolonged incubation for maximal expression activation. While Nod-LCOs are most efficient for an induction of persistent transcriptional changes, sulfated Myc-LCOs are less active, and nonsulfated Myc-LCOs display the lowest capacity to activate and sustain expression. Although all symbiotic LCOs up-regulated a common set of genes, discrete subsets were induced by individual LCOs, suggesting common and specific functions for these in presymbiotic signaling. Surprisingly, even sulfated fungal Myc-LCOs and Sinorhizobium meliloti Nod-LCOs, having very similar structures, each elicited discrete subsets of genes, while a mixture of both Myc-LCOs activated responses deviating from those induced by single treatments. Focusing on the precontact phase, we identified signaling-related and transcription factor genes specifically up-regulated by Myc-LCOs. Comparative gene expression studies in symbiotic mutants demonstrated that transcriptional reprogramming by AM fungal LCOs strictly depends on MtNFP and largely requires MtDMI3.

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.

Antisense Repression of the Medicago truncatula Nodule-Enhanced Sucrose Synthase Leads to a Handicapped Nitrogen Fixation Mirrored by Specific Alterations in the Symbiotic Transcriptome and Metabolome

We analyzed the role of the sucrose (Suc) synthase MtSucS1 during nodulation of the model legume Medicago truncatula, integrating data for the developmental, transcriptional, and metabolic processes affected downstream of an impaired Suc cleavage in root nodules. To reduce carbohydrate supply to nodule tissues, transgenic plants expressing a p35S-driven MtSucS1-antisense fusion were constructed. These plants displayed an up to 90% reduction of MtSucS1 proteins in roots and nodules. Phenotypic studies of two independent MtSucS1-reduced lines demonstrated that only under conditions depending on nodulation, these plants appeared to be impaired in above-ground growth. Specifically plant height, shoot weight, leaf development, flowering, as well as seed maturation were reduced, and the efficiency of photosynthesis was affected. Concomitantly, a significantly enhanced root to shoot ratio with a marked increase in root tip numbers was observed. Root nodule formation was found retarded and the impaired nodulation was accompanied by a less efficient nitrogen (N) acquisition. The decreased total N content of MtSucS1-antisense lines and an enhanced carbon to N ratio in roots, nodules, and shoots correlated with the extent of MtSucS1 knockdown. On the level of transcription, effects of an MtSucS1 reduction were evident for genes representing important nodes of the nodule carbon and N metabolism, while metabolite profiling revealed significantly lower levels of amino acids and their derivatives particularly in strongly MtSucS1-reduced nodules. Our results support the model that nodule-enhanced Suc synthase 1 of the model legume M. truncatula is required for the establishment and maintenance of an efficient N-fixing symbiosis.

Evidence for the involvement in nodulation of the two small putative regulatory peptide-encoding genes MtRALFL1 and MtDVL1.

Nod factors are key bacterial signaling molecules regulating the symbiotic interaction between bacteria known as rhizobia and leguminous plants. Studying plant host genes whose expression is affected by Nod factors has given insights into early symbiotic signaling and development. Here, we used a double supernodulating mutant line that shows increased sensitivity to Nod factors to study the Nod factor-regulated transcriptome. Using microarrays containing more than 16,000 70-mer oligonucleotide probes, we identified 643 Nod-factor-regulated genes, including 225 new Nod-factor-upregulated genes encoding many potential regulators. Among the genes found to be Nod factor upregulated, we identified and characterized MtRALFL1 and MtDVL1, which code for two small putative peptide regulators of 135 and 53 amino acids, respectively. Expression analysis confirmed that these genes are upregulated during initial phases of nodulation. Overexpression of MtRALFL1 and MtDVL1 in Medicago truncatula roots resulted in a marked reduction in the number of nodules formed and in a strong increase in the number of aborted infection threads. In addition, abnormal nodule development was observed when MtRALFL1 was overexpressed. This work provides evidence for the involvement of new putative small-peptide regulators during nodulation.

Knockdown of the Symbiotic Sucrose Synthase MtSucS1 Affects Arbuscule Maturation and Maintenance in Mycorrhizal Roots of Medicago truncatula

The relevance of the symbiosis-induced Medicago truncatula sucrose synthase gene MtSucS1 for an efficient arbuscular mycorrhiza (AM) was studied using two independent antisense lines that displayed up to 10-fold reduced SucS1 levels in roots. Mycorrhizal MtSucS1-reduced lines exhibited an overall stunted aboveground growth under inorganic phosphorus limitation. Apart from a reduced plant height, shoot weight, and leaf development, a delayed flowering, resulting in a lower seed yield, was observed. In addition, the root-to-shoot and root weight ratios increased significantly. Gene expression studies demonstrated a major reversion of AM-associated transcription, exhibiting a significant repression of well-known plant AM marker and mycosymbiont genes, together indicating a diminished AM fungus colonization of MtSucS1-antisense lines. Concomitantly, gas chromatography-mass spectrometry-based metabolite profiling revealed that mycorrhizal MtSucS1-reduced lines were affected in important nodes of the carbon, nitrogen, and phosphorus metabolism, accentuating a physiological significance of MtSucS1 for AM. In fact, antisensing MtSucS1 provoked an impaired fungal colonization within the less abundant infected regions, evident from strongly reduced frequencies of internal hyphae, vesicles, and arbuscules. Moreover, arbuscules were early senescing, accompanied with a reduced development of mature arbuscules. This defective mycorrhiza status correlated with reduced phosphorus and nitrogen levels and was proportional to the extent of MtSucS1 knockdown. Together, our results point to an important role for MtSucS1 in the establishment and maintenance of arbuscules in the AM symbiosis.