NERNST: a genetically-encoded ratiometric non-destructive sensing tool to estimate NADP(H) redox status in bacterial, plant and animal systems.

NADP(H) is a central metabolic hub providing reducing equivalents to multiple biosynthetic, regulatory and antioxidative pathways in all living organisms. While biosensors are available to determine NADP+ or NADPH levels in vivo, no probe exists to estimate the NADP(H) redox status, a determinant of the cell energy availability. We describe herein the design and characterization of a genetically-encoded ratiometric biosensor, termed NERNST, able to interact with NADP(H) and estimate ENADP(H). NERNST consists of a redox-sensitive green fluorescent protein (roGFP2) fused to an NADPH-thioredoxin reductase C module which selectively monitors NADP(H) redox states via oxido-reduction of the roGFP2 moiety. NERNST is functional in bacterial, plant and animal cells, and organelles such as chloroplasts and mitochondria. Using NERNST, we monitor NADP(H) dynamics during bacterial growth, environmental stresses in plants, metabolic challenges to mammalian cells, and wounding in zebrafish. NERNST estimates the NADP(H) redox poise in living organisms, with various potential applications in biochemical, biotechnological and biomedical research.

Regulation of Heterogenous LexA Expression in Staphylococcus aureus by an Antisense RNA Originating from Transcriptional Read-Through upon Natural Mispairings in the sbrB Intrinsic Terminator.

Bacterial genomes are pervasively transcribed, generating a wide variety of antisense RNAs (asRNAs). Many of them originate from transcriptional read-through events (TREs) during the transcription termination process. Previous transcriptome analyses revealed that the lexA gene from Staphylococcus aureus, which encodes the main SOS response regulator, is affected by the presence of an asRNA. Here, we show that the lexA antisense RNA (lexA-asRNA) is generated by a TRE on the intrinsic terminator (TTsbrB) of the sbrB gene, which is located downstream of lexA, in the opposite strand. Transcriptional read-through occurs by a natural mutation that destabilizes the TTsbrB structure and modifies the efficiency of the intrinsic terminator. Restoring the mispairing mutation in the hairpin of TTsbrB prevented lexA-asRNA transcription. The level of lexA-asRNA directly correlated with cellular stress since the expressions of sbrB and lexA-asRNA depend on the stress transcription factor SigB. Comparative analyses revealed strain-specific nucleotide polymorphisms within TTsbrB, suggesting that this TT could be prone to accumulating natural mutations. A genome-wide analysis of TREs suggested that mispairings in TT hairpins might provide wider transcriptional connections with downstream genes and, ultimately, transcriptomic variability among S. aureus strains.

The MicroRNA390/TAS3 Pathway Mediates Symbiotic Nodulation and Lateral Root Growth.

Legume roots form two types of postembryonic organs, lateral roots and symbiotic nodules. Nodule formation is the result of the interaction of legumes with rhizobia and requires the mitotic activation and differentiation of root cells as well as an independent, but coordinated, program that allows infection by rhizobia. MicroRNA390 (miR390) is an evolutionarily conserved microRNA that targets the Trans-Acting Short Interference RNA3 (TAS3) transcript. Cleavage of TAS3 by ARGONAUTE7 results in the production of trans-acting small interference RNAs, which target mRNAs encoding AUXIN RESPONSE FACTOR2 (ARF2), ARF3, and ARF4. Here, we show that activation of the miR390/TAS3 regulatory module by overexpression of miR390 in Medicago truncatula promotes lateral root growth but prevents nodule organogenesis, rhizobial infection, and the induction of two key nodulation genes, Nodulation Signaling Pathway1 (NSP1) and NSP2 Accordingly, inactivation of the miR390/TAS3 module, either by expression of a miR390 target mimicry construct or mutations in ARGONAUTE7, enhances nodulation and rhizobial infection, alters the spatial distribution of the nodules, and increases the percentage of nodules with multiple meristems. Our results revealed a key role of the miR390/TAS3 pathway in legumes as a modulator of lateral root organs, playing opposite roles in lateral root and nodule development.

Function of glutathione peroxidases in legume root nodules.

Glutathione peroxidases (Gpxs) are antioxidant enzymes not studied so far in legume nodules, despite the fact that reactive oxygen species are produced at different steps of the symbiosis. The function of two Gpxs that are highly expressed in nodules of the model legume Lotus japonicus was examined. Gene expression analysis, enzymatic and nitrosylation assays, yeast cell complementation, in situ mRNA hybridization, immunoelectron microscopy, and LjGpx-green fluorescent protein (GFP) fusions were used to characterize the enzymes and to localize each transcript and isoform in nodules. The LjGpx1 and LjGpx3 genes encode thioredoxin-dependent phospholipid hydroperoxidases and are differentially regulated in response to nitric oxide (NO) and hormones. LjGpx1 and LjGpx3 are nitrosylated in vitro or in plants treated with S-nitrosoglutathione (GSNO). Consistent with the modification of the peroxidatic cysteine of LjGpx3, in vitro assays demonstrated that this modification results in enzyme inhibition. The enzymes are highly expressed in the infected zone, but the LjGpx3 mRNA is also detected in the cortex and vascular bundles. LjGpx1 is localized to the plastids and nuclei, and LjGpx3 to the cytosol and endoplasmic reticulum. Based on yeast complementation experiments, both enzymes protect against oxidative stress, salt stress, and membrane damage. It is concluded that both LjGpxs perform major antioxidative functions in nodules, preventing lipid peroxidation and other oxidative processes at different subcellular sites of vascular and infected cells. The enzymes are probably involved in hormone and NO signalling, and may be regulated through nitrosylation of the peroxidatic cysteine essential for catalytic function.

The small RNA diversity from Medicago truncatula roots under biotic interactions evidences the environmental plasticity of the miRNAome.

BACKGROUND: Legume roots show a remarkable plasticity to adapt their architecture to biotic and abiotic constraints, including symbiotic interactions. However, global analysis of miRNA regulation in roots is limited, and a global view of the evolution of miRNA-mediated diversification in different ecotypes is lacking. RESULTS: In the model legume Medicago truncatula, we analyze the small RNA transcriptome of roots submitted to symbiotic and pathogenic interactions. Genome mapping and a computational pipeline identify 416 miRNA candidates, including known and novel variants of 78 miRNA families present in miRBase. Stringent criteria of pre-miRNA prediction yield 52 new mtr-miRNAs, including 27 miRtrons. Analyzing miRNA precursor polymorphisms in 26 M. truncatula ecotypes identifies higher sequence polymorphism in conserved rather than Medicago-specific miRNA precursors. An average of 19 targets, mainly involved in environmental responses and signalling, is predicted per novel miRNA. We identify miRNAs responsive to bacterial and fungal pathogens or symbionts as well as their related Nod and Myc-LCO symbiotic signals. Network analyses reveal modules of new and conserved co-expressed miRNAs that regulate distinct sets of targets, highlighting potential miRNA-regulated biological pathways relevant to pathogenic and symbiotic interactions. CONCLUSIONS: We identify 52 novel genuine miRNAs and large plasticity of the root miRNAome in response to the environment, and also in response to purified Myc/Nod signaling molecules. The new miRNAs identified and their sequence variation across M. truncatula ecotypes may be crucial to understand the adaptation of root growth to the soil environment, notably in the agriculturally important legume crops.

A Medicago truncatula rdr6 allele impairs transgene silencing and endogenous phased siRNA production but not development.

RNA-dependent RNA polymerase 6 (RDR6) and suppressor of gene silencing 3 (SGS3) act together in post-transcriptional transgene silencing mediated by small interfering RNAs (siRNAs) and in biogenesis of various endogenous siRNAs including the tasiARFs, known regulators of auxin responses and plant development. Legumes, the third major crop family worldwide, has been widely improved through transgenic approaches. Here, we isolated rdr6 and sgs3 mutants in the model legume Medicago truncatula. Two sgs3 and one rdr6 alleles led to strong developmental defects and impaired biogenesis of tasiARFs. In contrast, the rdr6.1 homozygous plants produced sufficient amounts of tasiARFs to ensure proper development. High throughput sequencing of small RNAs from this specific mutant identified 354 potential MtRDR6 substrates, for which siRNA production was significantly reduced in the mutant. Among them, we found a large variety of novel phased loci corresponding to protein-encoding genes or transposable elements. Interestingly, measurement of GFP expression revealed that post-transcriptional transgene silencing was reduced in rdr6.1 roots. Hence, this novel mis-sense mutation, affecting a highly conserved amino acid residue in plant RDR6s, may be an interesting tool both to analyse endogenous pha-siRNA functions and to improve transgene expression, at least in legume species.

Small RNA pathways and diversity in model legumes: lessons from genomics.

Small non-coding RNAs (smRNA) participate in the regulation of development, cell differentiation, adaptation to environmental constraints and defense responses in plants. They negatively regulate gene expression by degrading specific mRNA targets, repressing their translation or modifying chromatin conformation through homologous interaction with target loci. MicroRNAs (miRNA) and short-interfering RNAs (siRNA) are generated from long double stranded RNA (dsRNA) that are cleaved into 20-24-nucleotide dsRNAs by RNase III proteins called DICERs (DCL). One strand of the duplex is then loaded onto effective complexes containing different ARGONAUTE (AGO) proteins. In this review, we explored smRNA diversity in model legumes and compiled available data from miRBAse, the miRNA database, and from 22 reports of smRNA deep sequencing or miRNA identification genome-wide in three legumes: Medicago truncatula, soybean (Glycine max) and Lotus japonicus. In addition to conserved miRNAs present in other plant species, 229, 179, and 35 novel miRNA families were identified respectively in these 3 legumes, among which several seems legume-specific. New potential functions of several miRNAs in the legume-specific nodulation process are discussed. Furthermore, a new category of siRNA, the phased siRNAs, which seems to mainly regulate disease-resistance genes, was recently discovered in legumes. Despite that the genome sequence of model legumes are not yet fully completed, further analysis was performed by database mining of gene families and protein characteristics of DCLs and AGOs in these genomes. Although most components of the smRNA pathways are conserved, identifiable homologs of key smRNA players from non-legumes, like AGO10 or DCL4, could not yet be detected in M. truncatula available genomic and expressed sequence (EST) databases. In contrast to Arabidopsis, an important gene diversification was observed in the three legume models (for DCL2, AGO4, AGO2, and AGO10) or specifically in soybean for DCL1 and DCL4. Functional significance of these variant isoforms may reflect peculiarities of smRNA biogenesis and functions in legumes.

miR396 affects mycorrhization and root meristem activity in the legume Medicago truncatula.

The root system is crucial for acquisition of resources from the soil. In legumes, the efficiency of mineral and water uptake by the roots may be reinforced due to establishment of symbiotic relationships with mycorrhizal fungi and interactions with soil rhizobia. Here, we investigated the role of miR396 in regulating the architecture of the root system and in symbiotic interactions in the model legume Medicago truncatula. Analyses with promoter-GUS fusions suggested that the mtr-miR396a and miR396b genes are highly expressed in root tips, preferentially in the transition zone, and display distinct expression profiles during lateral root and nodule development. Transgenic roots of composite plants that over-express the miR396b precursor showed lower expression of six growth-regulating factor genes (MtGRF) and two bHLH79-like target genes, as well as reduced growth and mycorrhizal associations. miR396 inactivation by mimicry caused contrasting tendencies, with increased target expression, higher root biomass and more efficient colonization by arbuscular mycorrhizal fungi. In contrast to MtbHLH79, repression of three GRF targets by RNA interference severely impaired root growth. Early activation of mtr-miR396b, concomitant with post-transcriptional repression of MtGRF5 expression, was also observed in response to exogenous brassinosteroids. Growth limitation in miR396 over-expressing roots correlated with a reduction in cell-cycle gene expression and the number of dividing cells in the root apical meristem. These results link the miR396 network to the regulation of root growth and mycorrhizal associations in plants.

Analyzing small and long RNAs in plant development using non-radioactive in situ hybridization.

In the past decade, hundreds of non-coding RNAs (small and long RNAs) have been identified as crucial elements in developmental processes and stress response in plants. Among small RNAs, the microRNAs or miRNAs control levels of specific mRNA by inhibiting translation or reducing the stability of their mRNA targets through integration into different ribonucleoproteins (RNP). Spatio-temporal expression of small and long RNAs, using reporter genes or in situ hybridization, is essential to understand their functions. We are interested in understanding the role of various non-coding RNAs (including miRNAs) in the regulation of root and nodule development in legumes, which are agriculturally important crops. Here, we present the protocol we are currently using for detection of small and long RNA in model legume plants and tissues, like nodules and roots. The probe selection, as well as the fixation and permeabilization steps allowing to preserve tissues and cell integrity and to increase accessibility to RNA targets, will be specifically discussed.

Analyzing protein distribution in plant tissues using "whole-mount" immunolocalization.

Proteins are distributed in different cellular compartments. Our group studies the role of non-coding RNAs and associated RNPs in the development and stress response in legumes. Ribonucleoproteins (RNPs) are RNA-protein complexes that play different roles in many cellular processes. Long and small non-coding RNAs determine the specificity of action of several RNPs as the RNA Induced Silencing Complex (RISC), or affect mRNA translation, splicing and stability by interacting with other RNPs such as P-bodies, spliceosome or polysomes. Together with small and long RNAs (Chapter 20), the precise localization of the associated RNPs or the translational products regulated by small RNAs (ie target proteins regulated by miRNAs, or translationally-regulated products) by immunocytochemistry could bring novel insights into these regulatory processes. The protocol described is currently used for detection of RNP associated proteins in nodules and roots of Medicago truncatula but could be extended to any other protein. The critical points, as the choice of the antibody and the fixation and permeabilization steps, that allow preservation of tissue and cell integrity and increase the accessibility to epitopes, will be discussed.

Overexpression of miR160 affects root growth and nitrogen-fixing nodule number in Medicago truncatula.

Auxin action is mediated by a complex signalling pathway involving transcription factors of the auxin response factor (ARF) family. In Arabidopsis, microRNA160 (miR160) negatively regulates three ARF genes (ARF10/ARF16/ARF17) and therefore controls several developmental processes, including primary and lateral root growth. Here, we analysed the role of miR160 in root development and nodulation in Medicago truncatula Gaertn. Bioinformatic analyses identified two main mtr-miR160 variants (mtr-miR160abde and mtr-miR160c) and 17 predicted ARF targets. The miR160-dependent cleavage of four predicted targets in roots was confirmed by analysis of parallel analysis of RNA ends (PARE) data and RACE-PCR experiments. Promoter-GUS analyses for mtr-miR160d and mtr-miR160c genes revealed overlapping but distinct expression profiles during root and nodule development. In addition, the early miR160 activation in roots during symbiotic interaction was not observed in mutants of the nodulation signalling or autoregulation pathways. Composite plants that overexpressed mtr-miR160a under two different promoters exhibited distinct defects in root growth and nodulation: the p35S:miR160a construct led to reduced root length associated to a severe disorganisation of the RAM, whereas pCsVMV:miR160a roots showed gravitropism defects and lower nodule numbers. Our results suggest that a regulatory loop involving miR160/ARFs governs root and nodule organogenesis in M. truncatula.

Complexity of miRNA-dependent regulation in root symbiosis.

The development of root systems may be strongly affected by the symbiotic interactions that plants establish with soil organisms. Legumes are able to develop symbiotic relationships with both rhizobial bacteria and arbuscular mycorrhizal fungi leading to the formation of nitrogen-fixing nodules and mycorrhizal arbuscules, respectively. Both of these symbiotic interactions involve complex cellular reprogramming and profound morphological and physiological changes in specific root cells. In addition, the repression of pathogenic defence responses seems to be required for successful symbiotic interactions. Apart from typical regulatory genes, such as transcription factors, microRNAs (miRNAs) are emerging as riboregulators that control gene networks in eukaryotic cells through interactions with specific target mRNAs. In recent years, the availability of deep-sequencing technologies and the development of in silico approaches have allowed for the identification of large sets of miRNAs and their targets in legumes. A number of conserved and legume-specific miRNAs were found to be associated with symbiotic interactions as shown by their expression patterns or actions on symbiosis-related targets. In this review, we combine data from recent literature and genomic and deep-sequencing data on miRNAs controlling nodule development or restricting defence reactions to address the diversity and specificity of miRNA-dependent regulation in legume root symbiosis. Phylogenetic analysis of miRNA isoforms and their potential targets suggests a role for miRNAs in the repression of plant defence during symbiosis and revealed the evolution of miRNA-dependent regulation in legumes to allow for the modification of root cell specification, such as the formation of mycorrhized roots and nitrogen-fixing nodules.

Thiol synthetases of legumes: immunogold localization and differential gene regulation by phytohormones.

In plants and other organisms, glutathione (GSH) biosynthesis is catalysed sequentially by γ-glutamylcysteine synthetase (γECS) and glutathione synthetase (GSHS). In legumes, homoglutathione (hGSH) can replace GSH and is synthesized by γECS and a specific homoglutathione synthetase (hGSHS). The subcellular localization of the enzymes was examined by electron microscopy in several legumes and gene expression was analysed in Lotus japonicus plants treated for 1-48 h with 50 µM of hormones. Immunogold localization studies revealed that γECS is confined to chloroplasts and plastids, whereas hGSHS is also in the cytosol. Addition of hormones caused differential expression of thiol synthetases in roots. After 24-48 h, abscisic and salicylic acids downregulated GSHS whereas jasmonic acid upregulated it. Cytokinins and polyamines activated GSHS but not γECS or hGSHS. Jasmonic acid elicited a coordinated response of the three genes and auxin induced both hGSHS expression and activity. Results show that the thiol biosynthetic pathway is compartmentalized in legumes. Moreover, the similar response profiles of the GSH and hGSH contents in roots of non-nodulated and nodulated plants to the various hormonal treatments indicate that thiol homeostasis is independent of the nitrogen source of the plants. The differential regulation of the three mRNA levels, hGSHS activity, and thiol contents by hormones indicates a fine control of thiol biosynthesis at multiple levels and strongly suggests that GSH and hGSH play distinct roles in plant development and stress responses.

Peroxiredoxins and NADPH-dependent thioredoxin systems in the model legume Lotus japonicus.

Peroxiredoxins (Prxs), thioredoxins (Trxs), and NADPH-thioredoxin reductases (NTRs) constitute central elements of the thiol-disulfide redox regulatory network of plant cells. This study provides a comprehensive survey of this network in the model legume Lotus japonicus. The aims were to identify and characterize these gene families and to assess whether the NTR-Trx systems are operative in nodules. Quantitative reverse transcription-polymerase chain reaction and immunological and proteomic approaches were used for expression profiling. We identified seven Prx, 14 Trx, and three NTR functional genes. The PrxQ1 gene was found to be transcribed in two alternative spliced variants and to be expressed at high levels in leaves, stems, petals, pods, and seeds and at low levels in roots and nodules. The 1CPrx gene showed very high expression in the seed embryos and low expression in vegetative tissues and was induced by nitric oxide and cytokinins. In sharp contrast, cytokinins down-regulated all other Prx genes, except PrxQ1, in roots and nodules, but only 2CPrxA and PrxQ1 in leaves. Gene-specific changes in Prx expression were also observed in response to ethylene, abscisic acid, and auxins. Nodules contain significant mRNA and protein amounts of cytosolic PrxIIB, Trxh1, and NTRA and of plastidic NTRC. Likewise, they express cytosolic Trxh3, Trxh4, Trxh8, and Trxh9, mitochondrial PrxIIF and Trxo, and plastidic Trxm2, Trxm4, and ferredoxin-Trx reductase. These findings reveal a complex regulation of Prxs that is dependent on the isoform, tissue, and signaling molecule and support that redox NTR-Trx systems are functional in the cytosol, mitochondria, and plastids of nodules.

Regulation of nonsymbiotic and truncated hemoglobin genes of Lotus japonicus in plant organs and in response to nitric oxide and hormones.

• In legumes, symbiotic leghemoglobins facilitate oxygen diffusion to the bacteroids, but the roles of nonsymbiotic and truncated hemoglobins are largely unknown. Here the five hemoglobin genes of Lotus japonicus have been functionally characterized to gain insight into their regulatory mechanisms. • Plants were exposed to nitric oxide donors, stressful conditions, and hormones. Gene expression profiling was determined by quantitative PCR, and gene activities were localized using in situ hybridization and promoter-reporter gene fusions. • The LjGLB1-1, LjGLB2, and LjGLB3-1 mRNA expression levels were very high in nodules relative to other plant organs. The expression of these genes was localized in the vascular bundles, cortex, and infected tissue. LjGLB1-1 was the only gene induced by nitric oxide. Cytokinins caused nearly complete inactivation of LjGLB2 and LjGLB3-1 in nodules and induction of LjGLB1-1 in roots. Abscisic acid induced LjGLB1-1 in nodules and LjGLB1-2 and LjGLB2 in roots, whereas polyamines and jasmonic acid induced LjGLB1-1 only in roots. • The enhanced expression of the three types of hemoglobins in nodules, the colocalization of gene activities in nodule and root tissues with high metabolic rates, and their distinct regulatory mechanisms point out complementary roles of hemoglobins and strongly support the hypothesis that LjGLB1-1, LjGLB2, and LjGLB3-1 are required for symbiosis.

The FN3 and BRCT motifs in the exomer component Chs5p define a conserved module that is necessary and sufficient for its function.

Chs5p is a component of the exomer, a coat complex required to transport the chitin synthase Chs3p from the trans-Golgi network to the plasma membrane. The Chs5p N-terminal region exhibits fibronectin type III (FN3) and BRCT domains. FN3 domains are present in proteins that mediate adhesion processes, whereas BRCT domains are involved in DNA repair. Several fungi--including Schizosaccharomyces pombe, which has no detectable amounts of chitin--have proteins similar to Chs5p. Here we show that the FN3 and BRCT motifs in Chs5p behave as a module that is necessary and sufficient for Chs5p localization and for cargo delivery. The N-terminal regions of S. cerevisiae Chs5p and S. pombe Cfr1p are interchangeable in terms of Golgi localization, but not in terms of exomer assembly, showing that the conserved function of this module is protein retention in this organelle and that the interaction between the exomer components is organism-specific.

Effects of salt stress on the expression of antioxidant genes and proteins in the model legume Lotus japonicus.

Salt stress negatively affects many physiological processes in plants. Some of these effects may involve the oxidative damage of cellular components, which can be promoted by reactive oxygen species and prevented by antioxidants. The protective role of antioxidants was investigated in Lotus japonicus exposed to two salinization protocols: S1 (150 mM NaCl for 7 d) and S2 (50, 100 and 150 mM NaCl, each concentration for 6 d). Several markers of salt stress were measured and the expression of antioxidant genes was analyzed using quantitative reverse transcription­polymerase chain reaction and, in some cases, immunoblots and enzyme activity assays. Leaves of S1 plants suffered from mild osmotic stress, accumulated proline but noNa+, and showed induction of many superoxide dismutase and glutathione peroxidase genes. Leaves of S2 plants showed increases in Na+ and Ca2+, decreases in K+, and accumulation of proline and malondialdehyde. In leaves and roots of S1 and S2 plants, the mRNA, protein and activity levels of the ascorbate-glutathione enzymes remained constant, with a few exceptions. Notably, there was consistent up-regulation of the gene encoding cytosolic dehydroascorbate reductase, and this was possibly related to its role in ascorbate recycling in the apoplast. The overall results indicate that L. japonicus is more tolerant to salt stress than other legumes, which can be attributed to the capacity of the plant to prevent Na+reaching the shoot and to activate antioxidant defenses.

The fission yeast Map4 protein is a novel adhesin required for mating.

Cell adhesion is required for many cellular processes. In fungi, cell-cell contact during mating, flocculation or virulence is mediated by adhesins, which typically are glycosyl phosphatidyl inositol (GPI)-modified cell wall glycoproteins. Proteins with internal repeats (PIR) are surface proteins involved in the response to stress. In Schizosaccharomyces pombe no adhesins or PIR proteins have been described. Here we study the S. pombe Map4p, which defines a new class of surface protein that is not GPI-modified and has a serine/threonine rich domain and internal repeats that differ from those present in PIR proteins. Map4p is a mating type-specific adhesin required for mating in h(+) cells and enhances cell adhesion when overexpressed.