Medicago Truncatula Roots Research Articles

Size matters: three methods for estimating nuclear size in mycorrhizal roots of Medicago truncatula by image analysis

BackgroundThe intracellular accommodation of arbuscular mycorrhizal (AM) fungi involves a profound molecular reprogramming of the host cell architecture and metabolism, based on the activation of a symbiotic signaling pathway. In analogy with other plant biotrophs, AM fungi are reported to trigger cell cycle reactivation in their host tissues, possibly in support of the enhanced metabolic demand required for the symbiosis.ResultsWe here compare the efficiency of three Fiji/ImageJ image analysis plugins in localizing and quantifying the increase in nuclear size - a hallmark of recursive events of endoreduplication - in M. truncatula roots colonized by the AM fungus Gigaspora margarita.All three approaches proved to be versatile and upgradeable, allowing the investigation of nuclear changes in a complex tissue; 3D Object Counter provided more detailed information than both TrackMate and Round Surface Detector plugins.On this base we challenged 3D Object Counter with two case studies: verifying the lack of endoreduplication-triggering responses in Medicago truncatula mutants with a known non-symbiotic phenotype; and analysing the correlation in space and time between the induction of cortical cell division and endoreduplication upon AM colonization.Both case studies revealed important biological aspects. Mutant phenotype analyses have demonstrated that the knock-out mutation of different key genes in the symbiotic signaling pathway block AM-associated endoreduplication. Furthermore, our data show that cell divisions occur during initial stages of root colonization and are followed by recursive activation of the endocycle in preparation for arbuscule accommodation.ConclusionsIn conclusion, our results indicate 3D Object Counter as the best performing Fiji/ImageJ image analysis script in plant root thick sections and its application highlighted endoreduplication as a major feature of the AM pre-penetration response in root cortical cells.

Mycorrhizal and non-mycorrhizal Medicago truncatula roots exhibit differentially regulated NADPH oxidase and antioxidant response under Pb stress

The regulation of reactive oxygen species (ROS) generation and ROS scavenging is essential to understand the redox balance state under heavy metal stress. In order to figure out the effect of arbuscular mycorrhizal (AM) fungus on ROS generation and ROS scavenging ability under Pb stress, the ROS content, NADPH oxidase activity, the expression levels of MtRbohs, and the antioxidant response were evaluated in AM roots under Pb stress. AM inoculation decreased the malondialdehyde content by 18%–38% and decreased the O2− production rate by 20%–38% compared with non-mycorrhizal (NM) roots. The water-soluble Pb complex content was decreased by AM inoculation. Without Pb stress, the H2O2 content and NADPH oxidase activity in roots were increased by AM inoculation by 4-fold and 1-fold, respectively. Pb stress decreased H2O2 content by 12%–39% and decreased NADPH oxidase activity by 14%–43% in AM roots. When exposed to Pb stress, MtRbohC-G were down-regulated in AM roots, but MtRbohA-G were up-regulated in NM roots. AM inoculation increased the glutathione content in roots by 2-fold, 2-fold, and 3-fold under 0, 500, and 1000 mg kg−1 Pb treatment, respectively. The antioxidant enzyme activity was increased 43%–322% by AM inoculation under 1000 mg kg−1 Pb treatment. These results reveal that AM inoculation might maintain ROS homeostasis by decreasing Pb induced ROS, by decreasing the NADPH oxidase-mediated H2O2 generation, and by increasing the antioxidant response under Pb stress.

Local endoreduplication as a feature of intracellular fungal accommodation in arbuscular mycorrhizas.

The intracellular accommodation of arbuscular mycorrhizal (AM) fungi is a paradigmatic feature of this plant symbiosis that depends on the activation of a dedicated signaling pathway and the extensive reprogramming of host cells, including striking changes in nuclear size and transcriptional activity. By combining targeted sampling of early root colonization sites, detailed confocal imaging, flow cytometry and gene expression analyses, we demonstrate that local, recursive events of endoreduplication are triggered in the Medicago truncatula root cortex during AM colonization. AM colonization induces an increase in ploidy levels and the activation of endocycle specific markers. This response anticipates the progression of fungal colonization and is limited to arbusculated and neighboring cells in the cortical tissue. Furthermore, endoreduplication is not induced in M.truncatula mutants for symbiotic signaling pathway genes. On this basis, we propose endoreduplication as part of the host cell prepenetration responses that anticipate AM fungal accommodation in the root cortex.

An arbuscular mycorrhizal fungus and a root pathogen induce different volatiles emitted by Medicago truncatula roots

Plants are in permanent contact with various microorganisms and are always impacted by them. To better understand the first steps of a plant’s recognition of soil-borne microorganisms, the early release of volatile organic compounds (VOCs) emitted from roots of Medicago truncatula in response to the symbiont Rhizophagus irregularis or the pathogenic oomycete Aphanomyces euteiches was analysed. More than 90 compounds were released from roots as detected by an untargeted gas chromatography-mass spectrometry approach. Principal component analyses clearly distinguished untreated roots from roots treated with either R. irregularis or A. euteiches. Several VOCs were found to be emitted specifically in response to each of the microorganisms. Limonene was specifically emitted from wild-type roots after contact with R. irregularis spores but not from roots of the mycorrhiza-deficient mutant does not make infections3. The application of limonene to mycorrhizal roots, however, did not affect the mycorrhization rate. Inoculation of roots with A. euteiches zoospores resulted in the specific emission of several sesquiterpenes, such as nerolidol, viridiflorol and nerolidol-epoxyacetate but application of nerolidol to zoospores of A. euteiches did not affect their vitality. Therefore, plants discriminate between different microorganisms at early stages of their interaction and respond differently to the level of root-emitted volatiles.

Transcriptomic Changes in Medicago truncatula and Lotus japonicus Root Nodules during Drought Stress.

Drought is one of the major environmental factors limiting biomass and seed yield production in agriculture. In this research, we focused on plants from the Fabaceae family, which has a unique ability for the establishment of symbiosis with nitrogen-fixing bacteria, and are relatively susceptible to water limitation. We have presented the changes in nitrogenase activity and global gene expression occurring in Medicago truncatula and Lotus japonicus root nodules during water deficit. Our results proved a decrease in the efficiency of nitrogen fixation, as well as extensive changes in plant and bacterial transcriptomes, shortly after watering cessation. We showed for the first time that not only symbiotic plant components but also Sinorhizobium meliloti and Mesorhizobium loti bacteria residing in the root nodules of M. truncatula and L. japonicus, respectively, adjust their gene expression in response to water shortage. Although our results demonstrated that both M. truncatula and L. japonicus root nodules were susceptible to water deprivation, they indicated significant differences in plant and bacterial response to drought between the tested species, which might be related to the various types of root nodules formed by these species.

Arbuscular mycorrhizal fungi in soil, roots and rhizosphere of Medicago truncatula: diversity and heterogeneity under semi-arid conditions.

Mycorrhizal symbioses are considered indicators of ecosystem biodiversity. However, their diversity and relevance in arid and semi-arid ecosystems are poorly understood. This study addressed this subject, the main objective being to evaluate arbuscular mycorrhizal fungi (AMF) diversity and heterogeneity in a semi-arid region. Samples of bulk and rhizosphere soil and fine roots of Medicago truncatula were collected at four different sites with the same aridity index (6.1), in Bou-Hedma National Park, Tunisia, a semi-arid ecosystem. AMF taxa were assessed by 454- pyrosequencing and identified by BLAST matching of operational taxonomic units (OTUs) against the MaarjAM database, targeting AMF SSU rRNA gene diversity. Roots were the hotspots of AMF diversity (107 OTUs out of a total of 138). Of the 138 OTUs, 113 found correspondence in the MaarjAM database, with 32 AMF virtual taxa (VTX),19 Site-exclusive (SE) and 13 common to at least two sites (Non-site exclusive, NSE); the remaining 25 OTUs grouped in 16 putative new AMF taxa (pNTX), each one consisting of OTUs sharing pairwise distances not higher than 3%. We found a high diversity and heterogeneity of AMF across the four sites, which showed, in a regression analysis, significant relation to six out of the eight environmental parameters evaluated: grazing activity and soil texture, electrical conductivity, organic matter, total phosphorus and total nitrogen. AMF colonization of plants also presented significant differences among the four sites, as well as spore density, microbial biomass and several enzymatic activities (dehydrogenase, β-glucosidase and phosphatase) evaluated in rhizosphere soils. The four sites clustered in two groups in a hierarchical clustering evaluation based on their AMF diversity (total numbers of OTU, VTX and pNTX) and the parameters referred above. The crucial role of abiotic factors, other than aridity index, on AMF community composition, was evidenced by the high heterogeneity found between AMF communities across sites under identical aridity conditions.

Physiological and genetic changes during natural senescence of Medicago truncatula root nodules

AbstractUnder nitrogen‐limiting conditions, legumes are able to form a symbiotic interaction with bacteria of the Rhizobiaceae family to produce root nodules. These new root organs satisfy plant nitrogen needs by reducing atmospheric nitrogen to ammonium. However, the senescence of these organs disturbs the assimilation of nitrogen. In this study, we present different histological, biochemical, and genetic markers of the natural nodule senescence in Medicago truncatula over a 10‐week period following bacterial inoculation. During aging the length and the weight of nodules increased, whereas the nitrogen‐fixing capacity of the nodules decreased. The development of the nodule senescence zone correlated with a reduction in leghemoglobin levels without significant reduction in the total protein concentration of the nodule. In contrast, no difference in glutathione and homoglutathione concentration was detected at the onset of senescence at 6 and 8 weeks after bacterial infection. Furthermore, we observed a significant decrease in the relative transcription levels of Nodule Cysteine Rich 001, MtLb1, sucrose synthase 1, thioredoxin S1 and thioredoxin S2 genes, which are involved in nodule development and functioning, thus demonstrating that natural senescence impacts the transcription of genes involved in the expansion and the metabolism of the nitrogen‐fixing zone. Finally, the induction of amine oxidase and cysteine protease CP6 transcription was unstable, suggesting that these two genes are related to senescence but are not robust gene markers of the natural senescence process. Considered together, our results define novel biochemical and genetic markers for natural nodule senescence and show that leghemoglobin gene transcription and protein concentration are robust markers that closely correlate with nitrogen fixation efficiency.

Gibberellins negatively regulate the development of Medicago truncatula root system

The root system displays a remarkable plasticity that enables plants to adapt to changing environmental conditions. This plasticity is tightly linked to the activity of root apical meristems (RAMs) and to the formation of lateral roots, both controlled by related hormonal crosstalks. In Arabidopsis thaliana, gibberellins (GAs) were shown to positively control RAM growth and the formation of lateral roots. However, we showed in Medicago truncatula that GAs negatively regulate root growth and RAM size as well as the number of lateral roots depending at least on the MtDELLA1 protein. By using confocal microscopy and molecular analyses, we showed that GAs primarily regulate RAM size by affecting cortical cell expansion and additionally negatively regulate a subset of cytokinin-induced root expansin encoding genes. Moreover, GAs reduce the number of cortical cell layers, resulting in the formation of both shorter and thinner roots. These results suggest contrasting effects of GA regulations on the root system architecture depending on plant species.

Lipo-chitooligosaccharide signalling blocks a rapid pathogen-induced ROS burst without impeding immunity.

Molecular signals released by microbes at the surface of plant roots and leaves largely determine host responses, notably by triggering either immunity or symbiosis. How these signalling pathways cross-talk upon coincident perception of pathogens and symbionts is poorly described in plants forming symbiosis. Nitrogen fixing symbiotic Rhizobia spp. and arbuscular mycorrhizal fungi produce lipo-chitooligosaccharides (LCOs) to initiate host symbiotic programmes. In Medicago truncatula roots, the perception of LCOs leads to reduced efflux of reactive oxygen species (ROS). By contrast, pathogen perception generally triggers a strong ROS burst and activates defence gene expression. Here we show that incubation of M.truncatula seedlings with culture filtrate (CF) of the legume pathogen Aphanomyces euteiches alone or simultaneously with Sinorhizobium meliloti LCOs, resulted in a strong ROS release. However, this response was completely inhibited if CF was added after pre-incubation of seedlings with LCOs. By contrast, expression of immunity-associated genes in response to CF and disease resistance to A.euteiches remained unaffected by LCO treatment of M.truncatula roots. Our findings suggest that symbiotic plants evolved ROS inhibition response to LCOs to facilitate early steps of symbiosis whilst maintaining a parallel defence mechanisms toward pathogens.

Unified Transcriptomic Signature of Arbuscular Mycorrhiza Colonization in Roots of Medicago truncatula by Integration of Machine Learning, Promoter Analysis, and Direct Merging Meta-Analysis.

Plant root symbiosis with Arbuscular mycorrhizal (AM) fungi improves uptake of water and mineral nutrients, improving plant development under stressful conditions. Unraveling the unified transcriptomic signature of a successful colonization provides a better understanding of symbiosis. We developed a framework for finding the transcriptomic signature of Arbuscular mycorrhiza colonization and its regulating transcription factors in roots of Medicago truncatula. Expression profiles of roots in response to AM species were collected from four separate studies and were combined by direct merging meta-analysis. Batch effect, the major concern in expression meta-analysis, was reduced by three normalization steps: Robust Multi-array Average algorithm, Z-standardization, and quartiling normalization. Then, expression profile of 33685 genes in 18 root samples of Medicago as numerical features, as well as study ID and Arbuscular mycorrhiza type as categorical features, were mined by seven models: RELIEF, UNCERTAINTY, GINI INDEX, Chi Squared, RULE, INFO GAIN, and INFO GAIN RATIO. In total, 73 genes selected by machine learning models were up-regulated in response to AM (Z-value difference > 0.5). Feature weighting models also documented that this signature is independent from study (batch) effect. The AM inoculation signature obtained was able to differentiate efficiently between AM inoculated and non-inoculated samples. The AP2 domain class transcription factor, GRAS family transcription factors, and cyclin-dependent kinase were among the highly expressed meta-genes identified in the signature. We found high correspondence between the AM colonization signature obtained in this study and independent RNA-seq experiments on AM colonization, validating the repeatability of the colonization signature. Promoter analysis of upregulated genes in the transcriptomic signature led to the key regulators of AM colonization, including the essential transcription factors for endosymbiosis establishment and development such as NF-YA factors. The approach developed in this study offers three distinct novel features: (I) it improves direct merging meta-analysis by integrating supervised machine learning models and normalization steps to reduce study-specific batch effects; (II) seven attribute weighting models assessed the suitability of each gene for the transcriptomic signature which contributes to robustness of the signature (III) the approach is justifiable, easy to apply, and useful in practice. Our integrative framework of meta-analysis, promoter analysis, and machine learning provides a foundation to reveal the transcriptomic signature and regulatory circuits governing Arbuscular mycorrhizal symbiosis and is transferable to the other biological settings.

RiCRN1, a Crinkler Effector From the Arbuscular Mycorrhizal Fungus Rhizophagus irregularis, Functions in Arbuscule Development.

Arbuscular mycorrhizal (AM) symbiosis is one of the most prominent and beneficial plant–microbe interactions that facilitates mineral nutrition and confers tolerance to biotic and abiotic stresses. AM fungi colonize the root cortex and develop specialized structures called arbuscules where the nutrient exchange takes place. Arbuscule development is a highly controlled and coordinated process requiring the involvement of many plant proteins recruited at that interface. In contrast, much less is known about the fungal proteins involved in this process. Here, we have identified an AM fungal effector that participates in this developmental step of the symbiosis. RiCRN1 is a crinkler (CRN) effector that belongs to a subfamily of secreted CRN proteins from R. irregularis. CRNs have been so far only functionally characterized in pathogenic microbes and shown to participate in processes controlling plant cell death and immunity. RiCRN1 accumulates during symbiosis establishment parallel to MtPT4, the gene coding for an arbuscule-specific phosphate transporter. Expression in Nicotiana benthamiana leaves and in Medicago truncatula roots suggest that RiCRN1 is not involved in cell death processes. RiCRN1 dimerizes and localizes to nuclear bodies, suggesting that, similar to other CRNs, it functions in the plant nucleus. Downregulation of RiCRN1 using host-induced gene silencing led to an impairment of the symbiosis in M. truncatula and to a reduction of MtPT4, while ectopic expression of RiCRN1, surprisingly, led to a drastic reduction in arbuscule size that correlated with a decrease not only in MtPT4 but also in MtBCP1, a marker for initial stages of arbuscule development. Altogether, our results suggest that a tightly regulated expression in time and space of RiCRN1 is critical for symbiosis progression and for the proper initiation of arbuscule development.

Ectopic activation of cortical cell division during the accommodation of arbuscular mycorrhizal fungi

Arbuscular mycorrhizas (AMs) between plants and soil fungi are widespread symbioses with a major role in soil nutrient uptake. In this study we investigated the induction of root cortical cell division during AM colonization by combining morphometric and gene expression analyses with promoter activation and protein localization studies of the cell-plate-associated exocytic marker TPLATE. Our results show that TPLATE promoter is activated in colonized cells of the root cortex where we also observed the appearance of cells that are half the size of the surrounding cells. Furthermore, TPLATE-green fluorescent protein recruitment to developing cell plates highlighted ectopic cell division events in the inner root cortex during early AM colonization. Lastly, transcripts of TPLATE, KNOLLE and Cyclinlike 1 (CYC1) are all upregulated in the same context, alongside endocytic markers Adaptor-Related Protein complex 2 alpha 1 subunit (AP2A1) and Clathrin Heavy Chain 2 (CHC2), known to be active during cell plate formation. This pattern of gene expression was recorded in wild-type Medicago truncatula roots, but not in a common symbiotic signalling pathway mutant where fungal colonization is blocked at the epidermal level. Altogether, these results suggest the activation of cell-division-related mechanisms by AM hosts during the accommodation of the symbiotic fungus.

Comparison of Vacuum MALDI and AP-MALDI Platforms for the Mass Spectrometry Imaging of Metabolites Involved in Salt Stress in Medicago truncatula.

Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) is routinely used to determine the spatial distributions of various biomolecules in tissues. Recently, there has been an increased interest in creating higher resolution images using sources with more focused beams. One such source, an atmospheric pressure (AP) MALDI source from MassTech, has a laser capable of reaching spatial resolutions of 10 μm. Here, the AP-MALDI source coupled with a Q Exactive HF Orbitrap platform is compared to the commercial MALDI LTQ Orbitrap XL system using Medicago truncatula root nodules. AP-MALDI parameters, such as the S-lens value, capillary temperature, and spray voltage, were optimized on the Q Exactive-HF platform for optimal detection of plant metabolites. The performance of the two systems was evaluated for sensitivity, spatial resolution, and overall ability to detect plant metabolites. The commercial MALDI LTQ Orbitrap XL was superior regarding the number of compounds detected, as at least two times more m/z were detected compared to the AP-MALDI system. However, although the AP-MALDI source requires a spatial resolution higher than 10 μm to get the best signal, the spatial resolution at 30 μm is still superior compared to the 75 μm spatial resolution achieved on the MALDI platform. The AP-MALDI system was also used to investigate the metabolites present in M. truncatula roots and root nodules under high salt and low salt conditions. A discriminative analysis with SCiLS software revealed m/z ions specific to the control and salt conditions. This analysis revealed 44 m/z ions present at relatively higher abundances in the control samples, and 77 m/z enriched in the salt samples. Liquid chromatography-tandem MS was performed to determine the putative molecular identities of some of the mass ions enriched in each sample, including, asparagine, adenosine, and nicotianamine in the control samples, and arginine and soyasaponin I in the salt treated samples.

Functional analysis of the taproot and fibrous roots of Medicago truncatula: Sucrose and proline catabolism primary response to water deficit

Root performance represents a target factor conditioning plant development under drought conditions. Moreover, recent root phenotyping studies remark relevant differences on functionality of the different root types. However, despite its relevance, the performance of different types of roots such as primary/taproot (tapR) and lateral/fibrous roots (fibR) under water stress conditions is largely unknown. In the current study, the impact of water stress on target C and N metabolism (namely sucrose and proline) processes were characterized in tapR and fibR of Medicago truncatula plants exposed to different water stress severity regimes (moderate versus severe). While both root types exhibit some common responses to face water stress, the study highlighted important physiological and metabolic differences between them. The tapR proved to have an essential role on carbon and nitrogen partitioning rather than just on storage. Moreover, this root type showed a higher resilience towards water deficit stress. Sucrose metabolization at sucrose synthase level was early blocked in this tissue together with a selective accumulation of some amino acids such as proline and branched chain amino acids, which may act as alternative carbon sources under water deficit stress conditions. The decline in respiration, despite the over-accumulation of carbon compounds, suggests a modulation at sucrose cleavage level by sucrose synthase and invertase. These data not only provide new information on the carbon and nitrogen metabolism modulation upon water deficit stress but also on the different role, physiology, and metabolism of the taproot and fibrous roots. In addition, obtained results highlight the fact that both root types show distinct performance under water deficit stress; this factor can be of great relevance to improve breeding programs for increasing root efficiency under adverse conditions.

Localization of calreticulin and calcium ions in mycorrhizal roots of Medicago truncatula in response to aluminum stress

Aluminum (Al) toxicity limits growth and symbiotic interactions of plants. Calcium plays essential roles in abiotic stresses and legume-Rhizobium symbiosis, but the sites and mechanism of Ca2+ mobilization during mycorrhizae have not been analyzed. In this study, the changes of cytoplasmic Ca2+ and calreticulin (CRT) in Medicago truncatula mycorrhizal (MR) and non-mycorrizal (NM) roots under short Al stress [50 μM AlCl3 pH 4.3 for 3 h] were analyzed.Free Ca2+ ions were detected cytochemically by their reaction with potassium pyroantimonate and anti-CRT antibody was used to locate this protein in Medicago roots by immunocytochemical methods.In MR and NM roots, Al induced accumulation of CRT and free Ca2+. Similar calcium and CRT distribution in the MR were found at the surface of fungal structures (arbuscules and intercellular hyphae), cell wall and in plasmodesmata, and in plant and fungal intracellular compartments. Additionally, degenerated arbuscules were associated with intense Ca2+ and CRT accumulation. In NM roots, Ca2+ and CRT epitopes were observed in the stele, near wall of cortex and endodermis.The present study provides new insight into Ca2+ storage and mobilization in mycorrhizae symbiosis. The colocalization of CRT and Ca2+ suggests that CRT is essential for calcium mobilization for normal mycorrhiza development and response to Al stress.

Mycorrhizal lipochitinoligosaccharides (LCOs) depolarize root hairs of Medicago truncatula.

Arbuscular Mycorrhiza and Root Nodule Symbiosis are symbiotic interactions with a high benefit for plant growth and crop production. Thus, it is of great interest to understand the developmental process of these symbioses in detail. We analysed very early symbiotic responses of Medicago truncatula root hair cells, by stimulation with lipochitinoligosaccharides specific for the induction of nodules (Nod-LCOs), or the interaction with mycorrhiza (Myc-LCOs). Intracellular micro electrodes were used, in combination with Ca2+ sensitive reporter dyes, to study the relations between cytosolic Ca2+ signals and membrane potential changes. We found that sulfated Myc- as well as Nod-LCOs initiate a membrane depolarization, which depends on the chemical composition of these signaling molecules, as well as the genotype of the plants that were studied. A successive application of sulfated Myc-LCOs and Nod-LCOs resulted only in a single transient depolarization, indicating that Myc-LCOs can repress plasma membrane responses to Nod-LCOs. In contrast to current models, the Nod-LCO-induced depolarization precedes changes in the cytosolic Ca2+ level of root hair cells. The Nod-LCO induced membrane depolarization thus is most likely independent of cytosolic Ca2+ signals and nuclear Ca2+ spiking.

Use of RNA Immunoprecipitation Method for Determining Sinorhizobium meliloti RNA-Hfq Protein Associations In Vivo

BackgroundSoil bacterium Sinorhizobium meliloti (S. meliloti) forms an endosymbiotic partnership with Medicago truncatula (M. truncatula) roots which results in root nodules. The bacteria live within root nodules where they function to fix atmospheric N2 and supply the host plant with reduced nitrogen. The bacterial RNA-binding protein Hfq (Hfq) is an important regulator for the effectiveness of the nitrogen fixation. RNA immunoprecipitation (RIP) method is a powerful method for detecting the association of Hfq protein with specific RNA in cultured bacteria, yet a RIP method for bacteria living in root nodules remains to be described.ResultsA modified S. meliloti gene encoding a His-tagged Hfq protein (HfqHis) was placed under the regulation of the native Hfq gene promoter (Phfqsm). The trans produced HfqHis protein was accumulated at its nature levels during all stages of the symbiosis, allowing RNAs that associated with the given protein to be immunoprecipitated with the anti-His antibody against the protein from root nodule lysates. RNAs that associated with the protein were selectively enriched in the immunoprecipitated sample. The RNAs were recovered by a simple method using heat and subsequently analyzed by RT-PCR. The nature of PCR products was determined by DNA sequencing. Hfq association with specific RNAs can be analyzed at different conditions (e. g. young or older root nodules) and/or in wild-type versus mutant strains.ConclusionsThis article describes the RIP method for determining Sinorhizobium meliloti RNA-Hfq associations in vivo. It is also applicable to other rhizobia living in planta, although some tissue-specific modification related to sample disruption and homogenization may be needed.

Dual Color Sensors for Simultaneous Analysis of Calcium Signal Dynamics in the Nuclear and Cytoplasmic Compartments of Plant Cells.

Spatiotemporal changes in cellular calcium (Ca2+) concentrations are essential for signal transduction in a wide range of plant cellular processes. In legumes, nuclear and perinuclear-localized Ca2+ oscillations have emerged as key signatures preceding downstream symbiotic signaling responses. Förster resonance energy transfer (FRET) yellow-based Ca2+ cameleon probes have been successfully exploited to measure the spatiotemporal dynamics of symbiotic Ca2+ signaling in legumes. Although providing cellular resolution, these sensors were restricted to measuring Ca2+ changes in single subcellular compartments. In this study, we have explored the potential of single fluorescent protein-based Ca2+ sensors, the GECOs, for multicolor and simultaneous imaging of the spatiotemporal dynamics of cytoplasmic and nuclear Ca2+ signaling in root cells. Single and dual fluorescence nuclear and cytoplasmic-localized GECOs expressed in transgenic Medicago truncatula roots and Arabidopsis thaliana were used to successfully monitor Ca2+ responses to microbial biotic and abiotic elicitors. In M. truncatula, we demonstrate that GECOs detect symbiosis-related Ca2+ spiking variations with higher sensitivity than the yellow FRET-based sensors previously used. Additionally, in both M. truncatula and A. thaliana, the dual sensor is now able to resolve in a single root cell the coordinated spatiotemporal dynamics of nuclear and cytoplasmic Ca2+ signaling in vivo. The GECO-based sensors presented here therefore represent powerful tools to monitor Ca2+ signaling dynamics in vivo in response to different stimuli in multi-subcellular compartments of plant cells.

The Medicago truncatula GRAS protein RAD1 supports arbuscular mycorrhiza symbiosis and Phytophthora palmivora susceptibility.

The roots of most land plants are colonized by symbiotic arbuscular mycorrhiza (AM) fungi. To facilitate this symbiosis, plant genomes encode a set of genes required for microbial perception and accommodation. However, the extent to which infection by filamentous root pathogens also relies on some of these genes remains an open question. Here, we used genome-wide association mapping to identify genes contributing to colonization of Medicago truncatula roots by the pathogenic oomycete Phytophthora palmivora. Single-nucleotide polymorphism (SNP) markers most significantly associated with plant colonization response were identified upstream of RAD1, which encodes a GRAS transcription regulator first negatively implicated in root nodule symbiosis and recently identified as a positive regulator of AM symbiosis. RAD1 transcript levels are up-regulated both in response to AM fungus and, to a lower extent, in infected tissues by P. palmivora where its expression is restricted to root cortex cells proximal to pathogen hyphae. Reverse genetics showed that reduction of RAD1 transcript levels as well as a rad1 mutant are impaired in their full colonization by AM fungi as well as by P. palmivora. Thus, the importance of RAD1 extends beyond symbiotic interactions, suggesting a general involvement in M. truncatula microbe-induced root development and interactions with unrelated beneficial and detrimental filamentous microbes.

Effect of short-term aluminum stress and mycorrhizal inoculation on nitric oxide metabolism in Medicago truncatula roots

Aluminum (Al) toxicity can induce oxidative and nitrosative stress, which limits growth and yield of crop plants. Nevertheless, plant tolerance to stress may be improved by symbiotic associations including arbuscular mycorrhiza (AM). Nitric oxide (NO) is a signaling molecule involved in physiological processes and plant responses to abiotic and biotic stresses. However, almost no information about the NO metabolism has been gathered about AM. In the present work, Medicago truncatula seedlings were inoculated with Rhizophagus irregularis, and 7-week-old plants were treated with 50μM AlCl3 for 3h. Cytochemical and molecular techniques were used to measure the components of the NO metabolism, including NO content and localization, expression of genes encoding NO-synthesis (MtNR1, MtNR2 and MtNIR1) and NO-scavenging (MtGSNOR1, MtGSNOR2, MtHB1 and MtHB2) enzymes and the profile of protein tyrosine nitration (NO2-Tyr) in Medicago roots.For the first time, NO and NO2-Tyr accumulation was connected with fungal structures (arbuscules, vesicles and intercellular hyphae). Expression analysis of genes encoding NO-synthesis enzymes indicated that AM symbiosis results in lower production of NO in Al-treated roots in comparison to non-mycorrhizal roots. Elevated levels of transcription of genes encoding NO-scavenging enzymes indicated more active NO scavenging in AMF-inoculated Al-treated roots compared to non-inoculated roots. These results were confirmed by less NO accumulation and lower protein nitration in Al-stressed mycorrhizal roots in comparison to non-mycorrhizal roots.This study provides a new insight in NO metabolism in response to arbuscular mycorrhiza under normal and metal stress conditions. Our results suggest that mycorrhizal fungi decrease NO and tyrosine nitrated proteins content in Al-treated Medicago roots, probably via active NO scavenging system.