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Background and aims: Fatigue is a frequent and disabling symptom reported by patients with chronic hepatitis C (CHC). Its mechanism is poorly understood. Recent attention has focused on the role [...]

1. The loss of metabolic capacities during culture time constitutes a major limitation for the use of hepatocyte primary cultures in in vitro metabolism measurements. A new strategy is presented that permits one to calculate the Michaelis-Menten parameters V[sub max] and K[sub m] from extended experiments, by modelling V[sub max] as a variable dependent on time using exponential or sigmoidal equations. 2. This method was tested with cortisol depletion in cultured rat hepatocytes. V[sub max] and K[sub m] were used to calculate intrinsic clearance, and comparisons were made with methods already described in the literature. Intrinsic clearances given by our method were scaled to in vivo hepatic clearances that were close to those reported in the literature. 3. Our method could quantify the V[sub max] decrease with culture time from estimates of time parameters, t[sub 1/2] or t[sub 50]. In our system, this V[sub max] decrease was in agreement with P450 cytochrome inactivation rates published for the rat liver. 4. In conclusion, we propose a convenient, simple and useful general method for both Michaelis-Menten parameter estimation and modelling of variations in the metabolic capacities observed in in vitro systems. Such an approach should improve the usefulness of hepatocytes in primary cultures for long-term metabolism experiments. [ABSTRACT FROM AUTHOR]

Background: Certain chronic hepatitis C carriers have persistently normal transaminase activity. The aims of this study were to determine the virologic and histologic effects of 1 year of interferon-alpha treatment in such patients. Methods: Thirty-one patients were followed up in our Liver Unit. Eleven accepted interferon-alpha therapy; the 20 others were not treated and served as controls. Interferon-alpha, 3 MU, was given thrice weekly for 1 year. Serum was examined for hepatitis C virus (HCV)-RNA before, at the end of, and 6 months after treatment. Liver biopsy was performed 6 months after the cessation of treatment in 10 of 11 treated patients (one refused biopsy) and after a mean of 30.6 ± 22.7 months in the 20 untreated patients. Results: At the end of follow-up two of the treated patients had undetectable serum HCV-RNA and five had increased alanine aminotransferase (ALAT) values. In contrast, only one of the untreated patients had abnormal ALAT activity. All 20 untreated patients were constantly viremic. No significant histologic improvement was observed in the treated patients evaluated by means of post-treatment liver biopsy. The mean annual progression rate of fibrosis was very slow and similar in the treated and untreated patients (0.09 (range, 0-0.62) versus 0.07 (range, 0-0.60) fibrosis units). Conclusions: One year of interferon-alpha treatment can suppress HCV-RNA in patients with chronic hepatitis C and persistently normal ALAT values followed up over long periods. The rate of fibrosis progression in such patients is very slow, and therapeutic strategies should take this fact into account. Antiviral treatment is debated for patients without fibrosis in initial biopsy specimens. [ABSTRACT FROM AUTHOR]

The aim of this work was to specify the time course of response to interferon (IFN) of hepatitis G virus (HGV) and hepatitis C virus (HCV) in coinfected individuals. A group of 33 patients, undergoing 12 months of IFN therapy for chronic hepatitis C, was screened for the presence of both HGV and HCV RNAs to select seven coinfected patients. Spontaneous recovery from HGV infection was excluded through the detection of antibodies to the envelope-2 protein of HGV and HCV isolates were genotyped. Within three months of treatment, we found that HGV RNA was transiently cleared in 6/7 patients, but the rate of long-term favorable response was very low (1/7). In addition, considering the same individuals separately, it was shown that HGV and HCV responded to IFN with different kinetics in 5/7 patients. Taken together, these results underscore the importance of the virological basis of the resistance to IFN treatment. [ABSTRACT FROM AUTHOR]

The host immune responses have been suggested to play a role in liver injury occurring in patients with chronic hepatitis C. In order to explore the relationship between the relative proportions of intrahepatic and peripheral blood lymphocytes (IHL, PBL), the levels of viremia, and the histological hepatitis activity score, three-color fluorescence-activated cytometric analysis was performed for 36 patients with chronic hepatitis C and six control subjects without chronic hepatitis. The liver biopsy was performed before any antiviral therapy. Each liver specimen was divided into two parts: one for histological examination and one for immunological analysis. Tricolor CD45 was used to improve "lymphogating." Fluorescein isothiocyanate- or phycoerythrin-conjugated monoclonal antibodies with specificity for CD3, CD4, CD8, and CD20 (lymphocyte subpopulations), for CD69 (activated lymphocytes), and for CD16/56 (natural killer cells) were used. The livers of patients with chronic hepatitis C contained a greater proportion of CD4+ lymphocytes that exhibited marked expression of CD69 than in control subjects (20.7 +/- 7.3% vs 10.2 +/- 4.6%, P = 0.027). Moreover, in patients with chronic hepatitis C, the proportion of CD4+ IHL correlated with the histological hepatitis activity evaluated by the Knodell score (r = 0.48, P = 0.004). No correlation was found between the percentage of CD4+ IHL and the level of viremia or transaminase activities. Our findings clearly indicate that a cellular immune response does take place in HCV-infected livers and could thus contribute to the outcome of hepatitis C virus infection. [ABSTRACT FROM AUTHOR]

The Animal Health Modeling & Simulation Society (AHM&S) is a newly founded association (2012) that aims to promote the development, application, and dissemination of modeling and simulation techniques in the field of Veterinary Pharmacology and Toxicology. The association is co-chaired by Pr. Johan Gabrielsson (Europe), Pr. Jim Riviere (USA), and secretary Dr. Jonathan Mochel (Switzerland). This short communication aims at presenting the membership, rationale and objectives of this group. [ABSTRACT FROM AUTHOR]

Bacterial infections often involve more than one pathogen. While it is well established that polymicrobial infections can impact disease outcomes, we know little about how pathogens interact and affect each other's behaviour and fitness. Here, we used a microscopy approach to explore interactions between Pseudomonas aeruginosa and six human opportunistic pathogens that often co-occur in polymicrobial infections: Acinetobacter baumannii, Burkholderia cenocepacia, Escherichia coli, Enterococcus faecium, Klebsiella pneumoniae, and Staphylococcus aureus. When following growing microcolonies on agarose pads over time, we observed a broad spectrum of species-specific ecological interactions, ranging from mutualism to antagonism. For example, P. aeruginosa engaged in a mutually beneficial interaction with E. faecium but suffered from antagonism by E. coli. While we found little evidence for active directional growth towards or away from cohabitants, we observed that some pathogens increased growth in double layers in response to competition and that physical forces due to fast colony expansion had a major impact on fitness. Overall, our work provides an atlas of pathogen interactions, highlighting the diversity of potential species dynamics that may occur in polymicrobial infections. We discuss possible mechanisms driving pathogen interactions and offer predictions of how the different ecological interactions could affect virulence., (© 2024 The Author(s). Environmental Microbiology Reports published by John Wiley & Sons Ltd.)

Legume plants can acquire mineral nitrogen (N) either through their roots or via a symbiotic interaction with N-fixing rhizobia bacteria housed in root nodules. To identify shoot-to-root systemic signals acting in Medicago truncatula plants at N deficit or N satiety, plants were grown in a split-root experimental design in which either high or low N was provided to half of the root system, allowing the analysis of systemic pathways independently of any local N response. Among the plant hormone families analyzed, the cytokinin trans-zeatin accumulated in plants at N satiety. Cytokinin application by petiole feeding led to inhibition of both root growth and nodulation. In addition, an exhaustive analysis of miRNAs revealed that miR2111 accumulates systemically under N deficit in both shoots and non-treated distant roots, whereas a miRNA related to inorganic phosphate (Pi) acquisition, miR399, accumulates in plants grown under N satiety. These two accumulation patterns are dependent on Compact Root Architecture 2 (CRA2), a receptor required for C-terminally Encoded Peptide (CEP) signaling. Constitutive ectopic expression of miR399 reduced nodule numbers and root biomass depending on Pi availability, suggesting that the miR399-dependent Pi-acquisition regulatory module controlled by N availability affects the development of the whole legume plant root system., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Most habitats host diverse bacterial communities, offering opportunities for inter-species interactions. While competition might often dominate such interactions, little is known about whether bacteria can sense competitors and mount adequate responses. The competition sensing hypothesis proposes that bacteria can use cues such as nutrient stress and cell damage to prepare for battle. Here, we tested this hypothesis by measuring transcriptome changes in Pseudomonas aeruginosa exposed to the supernatant of its competitor Burkholderia cenocepacia. We found that P. aeruginosa exhibited significant growth-medium-dependent transcriptome changes in response to competition. In an iron-rich medium, P. aeruginosa upregulated genes encoding the type-VI secretion system and the siderophore pyoverdine, whereas genes encoding phenazine toxins and hydrogen cyanide were upregulated under iron-limited conditions. Moreover, general stress response and quorum sensing regulators were upregulated upon supernatant exposure. Altogether, our results reveal nuanced competitive responses of P. aeruginosa when confronted with B. cenocepacia supernatant, integrating both environmental and social cues., (© 2024. The Author(s).)

C-terminally encoded peptides (CEP) signaling peptides are drivers of systemic pathways regulating nitrogen (N) acquisition in different plants, from Arabidopsis to legumes, depending on mineral N availability (e.g. nitrate) and on the whole plant N demand. Recent studies in the Medicago truncatula model legume revealed how root-produced CEP peptides control the root competence for endosymbiosis with N fixing rhizobia soil bacteria through the activity of the Compact Root Architecture 2 (CRA2) CEP receptor in shoots. Among CEP genes, MtCEP7 was shown to be tightly linked to nodulation, and the dynamic temporal regulation of its expression reflects the plant ability to maintain a different symbiotic root competence window depending on the symbiotic efficiency of the rhizobium strain, as well as to reinitiate a new window of root competence for nodulation., (© 2023 The Authors New Phytologist © 2023 New Phytologist Foundation.)

Legumes acquire soil nutrients through nitrogen-fixing root nodules and lateral roots. To balance the costs and benefits of nodulation, legumes negatively control root nodule number by autoregulatory and hormonal pathways. How legumes simultaneously coordinate root nodule and lateral root development to procure nutrients remains poorly understood. In Medicago (Medicago truncatula), a subset of mature C-TERMINALLY ENCODED PEPTIDE (CEP) hormones can systemically promote nodule number, but all CEP hormones tested to date negatively regulate lateral root number. Here we showed that Medicago CEP7 produces a mature peptide, SymCEP7, that promotes nodulation from the shoot without compromising lateral root number. Rhizobial inoculation induced CEP7 in the susceptible root nodulation zone in a Nod factor-dependent manner, and, in contrast to other CEP genes, its transcription level was elevated in the ethylene signaling mutant sickle. Using mass spectrometry, fluorescence microscopy and expression analysis, we demonstrated that SymCEP7 activity requires the COMPACT ROOT ARCHITECTURE 2 receptor and activates the shoot-to-root systemic effector, miR2111. Shoot-applied SymCEP7 rapidly promoted nodule number in the pM to nM range at concentrations up to five orders of magnitude lower than effects mediated by root-applied SymCEP7. Shoot-applied SymCEP7 also promoted nodule number in White Clover (Trifolium repens) and Lotus (Lotus japonicus), which suggests that this biological function may be evolutionarily conserved. We propose that SymCEP7 acts in the Medicago shoot to counter balance the autoregulation pathways induced rapidly by rhizobia to enable nodulation without compromising lateral root growth, thus promoting the acquisition of nutrients other than nitrogen to support their growth., Competing Interests: Conflict of interest statement. None declared., (© American Society of Plant Biologists 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Medicago truncatula is a model legume species that has been studied for decades to understand the symbiotic relationship between legumes and soil bacteria collectively named rhizobia. This symbiosis called nodulation is initiated in roots with the infection of root hair cells by the bacteria, as well as the initiation of nodule primordia from root cortical, endodermal, and pericycle cells, leading to the development of a new root organ, the nodule, where bacteria fix and assimilate the atmospheric dinitrogen for the benefit of the plant. Here, we report the isolation and use of the nuclei from mock and rhizobia-inoculated roots for the single nuclei RNA-seq (sNucRNA-seq) profiling to gain a deeper understanding of early responses to rhizobial infection in Medicago roots. A gene expression map of the Medicago root was generated, comprising 25 clusters, which were annotated as specific cell types using 119 Medicago marker genes and orthologs to Arabidopsis cell-type marker genes. A focus on root hair, cortex, endodermis, and pericycle cell types, showing the strongest differential regulation in response to a short-term (48 h) rhizobium inoculation, revealed not only known genes and functional pathways, validating the sNucRNA-seq approach, but also numerous novel genes and pathways, allowing a comprehensive analysis of early root symbiotic responses at a cell type-specific level., (Copyright © 2022 The Author. Published by Elsevier Inc. All rights reserved.)

Plant nutrient acquisition is tightly regulated by resource availability and metabolic needs, implying the existence of communication between roots and shoots to ensure their integration at the whole-plant level. Here, we focus on systemic signaling pathways controlling nitrogen (N) nutrition, achieved both by the root import of mineral N and, in legume plants, through atmospheric N fixation by symbiotic bacteria inside dedicated root nodules. We explore features conserved between systemic pathways repressing or enhancing symbiotic N fixation and the regulation of mineral N acquisition by roots, as well as their integration with other environmental factors, such as phosphate, light, and CO 2 availability., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

To overcome the metal restriction imposed by the host's nutritional immunity, pathogenic bacteria use high metal affinity molecules called metallophores. Metallophore-mediated metal uptake pathways necessitate complex cycles of synthesis, secretion, and recovery of the metallophore across the bacterial envelope. We recently discovered staphylopine and pseudopaline, two members of a new family of broad-spectrum metallophores important for bacterial survival during infections. Here, we are expending the molecular understanding of the pseudopaline transport cycle across the diderm envelope of the Gram-negative bacterium Pseudomonas aeruginosa. We first explored pseudopaline secretion by performing in vivo quantifications in various genetic backgrounds and revealed the specific involvement of the MexAB-OprM efflux pump in pseudopaline transport across the outer membrane. We then addressed the recovery part of the cycle by investigating the fate of the recaptured metal-loaded pseudopaline. To do so, we combined in vitro reconstitution experiments and in vivo phenotyping in absence of pseudopaline transporters to reveal the existence of a pseudopaline modification mechanism, possibly involved in the metal release following pseudopaline recovery. Overall, our data allowed us to provide an improved molecular model of secretion, recovery, and fate of this important metallophore by P. aeruginosa., (© 2020 John Wiley & Sons Ltd.)

Nicotianamine (NA) is a metabolite synthesized by all plants, in which it is involved in the homeostasis of different micronutrients such as iron, nickel or zinc. In some plants it also serves as a precursor of phytosiderophores, which are used for extracellular iron scavenging. Previous studies have also established the presence of NA in filamentous fungi and some mosses, whereas an analogue of NA was inferred in an archaeon. More recently, opine-type metallophores with homology to NA were uncovered in bacteria, especially in human pathogens such as Staphylococcus aureus, Pseudomonas aeruginosa or Yersinia pestis, synthesizing respectively staphylopine, pseudopaline and yersinopine. Here, we review the current state of knowledge regarding the discovery, biosynthesis, function and regulation of these metallophores. We also discuss the genomic environment of the cntL gene, which is homologous to the plant NA synthase (NAS) gene, and plays a central role in the synthesis of NA-like metallophores. This reveals a large diversity of biosynthetic, export and import pathways. Using sequence similarity networks, we uncovered that these metallophores are widespread in numerous bacteria thriving in very different environments, such as those living at the host-pathogen interface, but also in the soil. We additionally established a phylogeny of the NAS/cntL gene and, as a result, we propose that this gene is an ancient gene and NA, or its derivatives, is an ancient metallophore that played a prominent role in metal acquisition or metal resistance. Indeed, our phylogenetic analysis suggests an evolutionary model where the possibility to synthesize this metallophore was present early in the appearance of life, although it was later lost by most living microorganisms, unless facing metal starvation such as at the host-pathogen interface or in some soils. According to our model, NA then re-emerged as a central metabolite for metal homeostasis in fungi, mosses and all known higher plants.

Nitrogen-fixing root nodulation in legumes challenged with nitrogen-limiting conditions requires infection of the root hairs by soil symbiotic bacteria, collectively referred to as rhizobia, and the initiation of cell divisions in the root cortex. Cytokinin hormones are critical for early nodulation to coordinate root nodule organogenesis and the progression of bacterial infections. Cytokinin signaling involves regulation of the expression of cytokinin primary response genes by type-B response regulator (RRB) transcription factors. RNA interference or mutation of MtRRB3 , the RRB-encoding gene most strongly expressed in Medicago truncatula roots and nodules, significantly decreased the number of nodules formed, indicating a function of this RRB in nodulation initiation. Fewer infection events were also observed in rrb3 mutant roots associated with a reduced Nod factor induction of the Early Nodulin 11 ( MtENOD11 ) infection marker, and of the cytokinin-regulated Nodulation Signaling Pathway 2 ( Mt NSP2 ) gene. Rhizobial infections correlate with an expansion of the nuclear area, suggesting the activation of endoreduplication cycles linked to the cytokinin-regulated Cell Cycle Switch 52A ( Mt CCS52A ) gene. Although no significant difference in nucleus size and endoreduplication were detected in rhizobia-infected rrb3 mutant roots, expression of the MtCCS52A endoreduplication marker was reduced. As the MtRRB3 expression pattern overlaps with those of MtNSP2 and MtCCS52A in roots and nodule primordia, chromatin immunoprecipitation-quantitative PCR and protoplast trans-activation assays were used to show that MtRRB3 can interact with and trans-activate MtNSP2 and MtCCS52A promoters. Overall, we highlight that the MtRRB3 cytokinin signaling transcription factor coordinates the expression of key early nodulation genes., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

Legumes tightly regulate nodule number to balance the cost of supporting symbiotic rhizobia with the benefits of nitrogen fixation. C-terminally Encoded Peptides (CEPs) and CLAVATA3-like (CLE) peptides positively and negatively regulate nodulation, respectively, through independent systemic pathways, but how these regulations are coordinated remains unknown. Here, we show that rhizobia, Nod Factors, and cytokinins induce a symbiosis-specific CEP gene, MtCEP7, which positively regulates rhizobial infection. Via grafting and split root studies, we reveal that MtCEP7 increases nodule number systemically through the MtCRA2 receptor. MtCEP7 and MtCLE13 expression in rhizobia-inoculated roots rely on the MtCRE1 cytokinin receptor and on the MtNIN transcription factor. MtNIN binds and transactivates MtCEP7 and MtCLE13, and a NIN Binding Site (NBS) identified within the proximal MtCEP7 promoter is required for its symbiotic activation. Overall, these results demonstrate that a cytokinin-MtCRE1-MtNIN regulatory module coordinates the expression of two antagonistic, symbiosis-related, peptide hormones from different families to fine-tune nodule number.

Nitrogen-deprived legume plants form new root organs, the nodules, following a symbiosis with nitrogen-fixing rhizobial bacteria [1]. Because this interaction is beneficial for the plant but has a high energetic cost, nodulation is tightly controlled by host plants through systemic pathways (acting at long distance) to promote or limit rhizobial infections and nodulation depending on earlier infections and on nitrogen availability [2]. In the Medicago truncatula model legume, CLE12 (Clavata3/Embryo surrounding region 12) and CLE13 signaling peptides produced in nodulated roots act in shoots through the SUNN (Super Numeric Nodule) receptor to negatively regulate nodulation and therefore autoregulate nodule number [3-5]. Conversely, CEP (C-terminally Encoded Peptide) signaling peptides produced in nitrogen-starved roots act in shoots through the CRA2 (Compact Root Architecture 2) receptor to promote nodulation already in the absence of rhizobia [6-9]. We show in this study that a downstream shoot-to-root signaling effector of these systemic pathways is the shoot-produced miR2111 microRNA [10] that negatively regulates TML1 (Too Much Love 1) and TML2 [11] transcripts accumulation in roots, ultimately promoting nodulation. Low nitrogen conditions and CEP1 signaling peptides induce in the absence of rhizobia the production of miR2111 depending on CRA2 activity in shoots, thus favoring root competence for nodulation. Together with the SUNN pathway negatively regulating the same miR2111 systemic effector when roots are nodulated, this allows a dynamic fine-tuning of the nodulation capacity of legume roots by nitrogen availability and rhizobial cues., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Varroa destructor, an acarian parasite of the Western honey bee Apis mellifera L., is a serious threat to colonies and beekeeping worldwide. The parasite lifecycle occurs in close synchrony with its host development. The females have to discriminate between different developmental stages of the host and trigger an appropriate behavioral response. Many studies have focused on these behavioral aspects, whether it is the choice of a precise host stage or the reproduction of female mites. Behavioral tests often require laboratory settings that are very different from the mite's environment. Our first experiment was designed to study the impact of the surface of test arena on the mite behavior. We found that plastic from Petri dishes commonly used as test arenas disturbs the female mites and can cause death. We searched for a substrate that does not harm mites and found that gelatin-coated plastic Petri dishes responded to these expectations. We then investigated the host choice behavior of phoretic mites confronted with larval stages of the bee on gelatin-coated arenas to watch if the well-documented orientation towards 5th instar larva was observable in our conditions. Pupal stages were included in the host choice experiments, initially to act as neutral stimuli. As white-eyed pupae were revealed attractive to the mite, several pupal stages were then included in a series of host choice bioassays. These additional experiments tend to show that the positive response to the white-eyed pupa stage depends on cues only delivered by living pupae. Further investigation on the nature and impact of these cues are needed as they could shed light on key signals involved in the parasite lifecycle.

In metal-scarce environments, some pathogenic bacteria produce opine-type metallophores mainly to face the host's nutritional immunity. This is the case of staphylopine, pseudopaline and yersinopine, identified in Staphylococcus aureus , Pseudomonas aeruginosa and Yersinia pestis , respectively. Depending on the species, these metallophores are synthesized by two (CntLM) or three enzymes (CntKLM), CntM catalyzing the last step of biosynthesis using diverse substrates (pyruvate or α-ketoglutarate), pathway intermediates (xNA or yNA) and cofactors (NADH or NADPH). Here, we explored the substrate specificity of CntM by combining bioinformatic and structural analysis with chemical synthesis and enzymatic studies. We found that NAD(P)H selectivity is mainly due to the amino acid at position 33 ( S. aureus numbering) which ensures a preferential binding to NADPH when it is an arginine. Moreover, whereas CntM from P. aeruginosa preferentially uses yNA over xNA, the staphylococcal enzyme is not stereospecific. Most importantly, selectivity toward α-ketoacids is largely governed by a single residue at position 150 of CntM ( S. aureus numbering): an aspartate at this position ensures selectivity toward pyruvate, whereas an alanine leads to the consumption of both pyruvate and α-ketoglutarate. Modifying this residue in P. aeruginosa led to a complete reversal of selectivity. Thus, the diversity of opine-type metallophore is governed by the absence/presence of a cntK gene encoding a histidine racemase, and the amino acid residue at position 150 of CntM. These two simple rules predict the production of a fourth metallophore by Paenibacillus mucilaginosus , which was confirmed in vitro and called bacillopaline., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Plant systemic signaling pathways allow the integration and coordination of shoot and root organ metabolism and development at the whole-plant level depending on nutrient availability. In legumes, two systemic pathways have been reported in the Medicago truncatula model to regulate root nitrogen-fixing symbiotic nodulation. Both pathways involve leucine-rich repeat receptor-like kinases acting in shoots and proposed to perceive signaling peptides produced in roots depending on soil nutrient availability. In this study, we characterized in the M. truncatula Jemalong A17 genotype a mutant allelic series affecting the Compact Root Architecture2 (CRA2) receptor. These analyses revealed that this pathway acts systemically from shoots to positively regulate nodulation and is required for the activity of carboxyl-terminally encoded peptides (CEPs). In addition, we generated a double mutant to test genetic interactions of the CRA2 systemic pathway with the CLAVATA3/EMBRYO SURROUNDING REGION peptide (CLE)/Super Numeric Nodule (SUNN) receptor systemic pathway negatively regulating nodule number from shoots, which revealed an intermediate nodule number phenotype close to the wild type. Finally, we showed that the nitrate inhibition of nodule numbers was observed in cra2 mutants but not in sunn and cra2 sunn mutants. Overall, these results suggest that CEP/CRA2 and CLE/SUNN systemic pathways act independently from shoots to regulate nodule numbers., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

Enzymatic regulations are central processes for the adaptation to changing environments. In the particular case of metallophore-dependent metal uptake, there is a need to quickly adjust the production of these metallophores to the metal level outside the cell, to avoid metal shortage or overload, as well as waste of metallophores. In Staphylococcus aureus, CntM catalyzes the last biosynthetic step in the production of staphylopine, a broad-spectrum metallophore, through the reductive condensation of a pathway intermediate (xNA) with pyruvate. Here, we describe the chemical synthesis of this intermediate, which was instrumental in the structural and functional characterization of CntM and confirmed its opine synthase properties. The three-dimensional structure of CntM was obtained in an "open" form, in the apo state or as a complex with substrate or product. The xNA substrate appears mainly stabilized by its imidazole ring through a π-π interaction with the side chain of Tyr240. Intriguingly, we found that metals exerted various and sometime antagonistic effects on the reaction catalyzed by CntM: zinc and copper are moderate activators at low concentration and then total inhibitors at higher concentration, whereas manganese is only an activator and cobalt and nickel are only inhibitors. We propose a model in which the relative affinity of a metal toward xNA and an inhibitory binding site on the enzyme controls activation, inhibition, or both as a function of metal concentration. This metal-dependent regulation of a metallophore-producing enzyme might also take place in vivo, which could contribute to the adjustment of metallophore production to the internal metal level.

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.

The number of legume root nodules resulting from a symbiosis with rhizobia is tightly controlled by the plant. Certain members of the CLAVATA3/Embryo Surrounding Region (CLE) peptide family, specifically MtCLE12 and MtCLE13 in Medicago truncatula, act in the systemic autoregulation of nodulation (AON) pathway that negatively regulates the number of nodules. Little is known about the molecular pathways that operate downstream of the AON-related CLE peptides. Here, by means of a transcriptome analysis, we show that roots ectopically expressing MtCLE13 deregulate only a limited number of genes, including three down-regulated genes encoding lysin motif receptor-like kinases (LysM-RLKs), among which are the nodulation factor (NF) receptor NF Perception gene (NFP) and two up-regulated genes, MtTML1 and MtTML2, encoding Too Much Love (TML)-related Kelch-repeat containing F-box proteins. The observed deregulation was specific for the ectopic expression of nodulation-related MtCLE genes and depended on the Super Numeric Nodules (SUNN) AON RLK. Moreover, overexpression and silencing of these two MtTML genes demonstrated that they play a role in the negative regulation of nodule numbers. Hence, the identified MtTML genes are the functional counterpart of the Lotus japonicus TML gene shown to be central in the AON pathway. Additionally, we propose that the down-regulation of a subset of LysM-RLK-encoding genes, among which is NFP, might contribute to the restriction of further nodulation once the first nodules have been formed., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Peptides are signaling molecules regulating various aspects of plant development, including the balance between cell division and differentiation in different meristems. Among those, CLAVATA3/Embryo Surrounding Region-related (CLE-ESR) peptide activity depends on leucine-rich-repeat receptor-like-kinases (LRR-RLK) belonging to the subclass XI. In legume plants, such as the Medicago truncatula model, specific CLE peptides were shown to regulate root symbiotic nodulation depending on the LRR-RLK SUNN (Super Numeric Nodules). Amongst the ten M. truncatula LRR-RLK most closely related to SUNN, only one showed a nodule-induced expression, and was so-called MtNRLK1 (Nodule-induced Receptor-Like Kinase 1). MtNRLK1 expression is associated to root and nodule vasculature as well as to the proximal meristem and rhizobial infection zone in the nodule apex. Except for the root vasculature, the MtNRLK1 symbiotic expression pattern is different than the one of MtSUNN. Functional analyses either based on RNA interference, insertional mutagenesis, and overexpression of MtNRLK1 however failed to identify a significant nodulation phenotype, either regarding the number, size, organization or nitrogen fixation capacity of the symbiotic organs formed.

A reversed-phase (RP) high-performance liquid chromatography (HPLC) method for the content determination of IR780-oleyl (IRO) dye in lipid nanoparticles was developed and validated. Chromatographic separation was performed on a RP C18 column with a gradient program of water and acetonitrile both with 0.1% (v/v) TFA, at a flow rate of 1.0mL/min and a total run of 21min. IRO dye detection was made by fluorescence at emission wavelength of 773nm (excitation wavelength: 744nm). According to ICH guidelines, the developed method was shown to be specific, linear in the range 3-8μg/mL (R 2 =0.9998), precise at the intra-day and inter-day levels as reflected by the coefficient of variation (CV≤1.98%) at three different concentrations (4, 6 and 8 μg/mL) and accurate, with recovery rates between 98.2-101.6% and 99.2-100.5%. The detection and quantitation limits were 0.41 and 1.24μg/mL, respectively. Stability studies of sample processing showed that IRO dye was stable after 24h in the autosampler or after three freeze/thaw cycles. Combined with fluorescence measurements, the developed method was successfully applied to optimize the loading capacity of IRO dye in the core of lipid nanoparticles., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Mitogen-activated protein kinase (MAPK) signaling cascades play critical roles in various cellular events in plants, including stress responses, innate immunity, hormone signaling, and cell specificity. MAPK-mediated stress signaling is also known to negatively regulate nitrogen-fixing symbiotic interactions, but the molecular mechanism of the MAPK signaling cascades underlying the symbiotic nodule development remains largely unknown. We show that the MtMKK5-MtMPK3/6 signaling module negatively regulates the early symbiotic nodule formation, probably upstream of ERN1 (ERF Required for Nodulation 1) and NSP1 (Nod factor Signaling Pathway 1) in Medicago truncatula . The overexpression of MtMKK5 stimulated stress and defense signaling pathways but also reduced nodule formation in M. truncatula roots. Conversely, a MAPK specific inhibitor, U0126, enhanced nodule formation and the expression of an early nodulation marker gene, MtNIN . We found that MtMKK5 directly activates MtMPK3/6 by phosphorylating the TEY motif within the activation loop and that the MtMPK3/6 proteins physically interact with the early nodulation-related transcription factors ERN1 and NSP1. These data suggest that the stress signaling-mediated MtMKK5/MtMPK3/6 module suppresses symbiotic nodule development via the action of early nodulation transcription factors.

We investigated the role of KNOX genes in legume root nodule organogenesis. Class 1 KNOX homeodomain transcription factors (TFs) are involved in plant shoot development and leaf shape diversity. Class 2 KNOX genes are less characterized, even though an antagonistic function relative to class 1 KNOXs was recently proposed. In silico expression data and further experimental validation identified in the Medicago truncatula model legume three class 2 KNOX genes, belonging to the KNAT3/4/5-like subclass (Mt KNAT3/4/5-like), as expressed during nodulation from early stages. RNA interference (RNAi)-mediated silencing and overexpression studies were used to unravel a function for KNOX TFs in nodule development. Mt KNAT3/4/5-like genes encoded four highly homologous proteins showing overlapping expression patterns during nodule organogenesis, suggesting functional redundancy. Simultaneous reduction of Mt KNAT3/4/5-like genes indeed led to an increased formation of fused nodule organs, and decreased the expression of the MtEFD (Ethylene response Factor required for nodule Differentiation) TF and its direct target MtRR4, a cytokinin response gene. Class 2 KNOX TFs therefore regulate legume nodule development, potentially through the MtEFD/MtRR4 cytokinin-related regulatory module, and may control nodule organ boundaries and shape like class 2 KNOX function in leaf development., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

C-TERMINALLY ENCODED PEPTIDEs (CEPs) control root system architecture in a non-cell-autonomous manner. In Medicago truncatula, MtCEP1 affects root development by increasing nodule formation and inhibiting lateral root emergence by unknown pathways. Here, we show that the MtCEP1 peptide-dependent increase in nodulation requires the symbiotic signaling pathway and ETHYLENE INSENSITIVE2 (EIN2)/SICKLE (SKL), but acts independently of SUPER NUMERIC NODULES. MtCEP1-dependent inhibition of lateral root development acts through an EIN2-independent mechanism. MtCEP1 increases nodulation by promoting rhizobial infections, the developmental competency of roots for nodulation, the formation of fused nodules, and an increase in frequency of nodule development that initiates at proto-phloem poles. These phenotypes are similar to those of the ein2/skl mutant and support that MtCEP1 modulates EIN2-dependent symbiotic responses. Accordingly, MtCEP1 counteracts the reduction in nodulation induced by increasing ethylene precursor concentrations, and an ethylene synthesis inhibitor treatment antagonizes MtCEP1 root phenotypes. MtCEP1 also inhibits the development of EIN2-dependent pseudonodule formation. Finally, mutants affecting the COMPACT ROOT ARCHITECTURE2 (CRA2) receptor, which is closely related to the Arabidopsis CEP Receptor1, are unresponsive to MtCEP1 effects on lateral root and nodule formation, suggesting that CRA2 is a CEP peptide receptor mediating both organogenesis programs. In addition, an ethylene inhibitor treatment counteracts the cra2 nodulation phenotype. These results indicate that MtCEP1 and its likely receptor, CRA2, mediate nodulation and lateral root development through different pathways., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

Initiation of symbiotic nodules in legumes requires cytokinin signaling, but its mechanism of action is largely unknown. Here, we tested whether the failure to initiate nodules in the Medicago truncatula cytokinin perception mutant cre1 (cytokinin response1) is due to its altered ability to regulate auxin transport, auxin accumulation, and induction of flavonoids. We found that in the cre1 mutant, symbiotic rhizobia cannot locally alter acro- and basipetal auxin transport during nodule initiation and that these mutants show reduced auxin (indole-3-acetic acid) accumulation and auxin responses compared with the wild type. Quantification of flavonoids, which can act as endogenous auxin transport inhibitors, showed a deficiency in the induction of free naringenin, isoliquiritigenin, quercetin, and hesperetin in cre1 roots compared with wild-type roots 24 h after inoculation with rhizobia. Coinoculation of roots with rhizobia and the flavonoids naringenin, isoliquiritigenin, and kaempferol, or with the synthetic auxin transport inhibitor 2,3,5,-triiodobenzoic acid, rescued nodulation efficiency in cre1 mutants and allowed auxin transport control in response to rhizobia. Our results suggest that CRE1-dependent cytokinin signaling leads to nodule initiation through the regulation of flavonoid accumulation required for local alteration of polar auxin transport and subsequent auxin accumulation in cortical cells during the early stages of nodulation., (© 2015 American Society of Plant Biologists. All rights reserved.)

Cytokinins are phytohormones that regulate many developmental and environmental responses. The Medicago truncatula cytokinin receptor MtCRE1 (Cytokinin Response 1) is required for the nitrogen-fixing symbiosis with rhizobia. As several cytokinin signaling genes are modulated in roots depending on different biotic and abiotic conditions, we assessed potential involvement of this pathway in various root environmental responses. Phenotyping of cre1 mutant roots infected by the Gigaspora margarita arbuscular mycorrhizal (AM) symbiotic fungus, the Aphanomyces euteiches root oomycete, or subjected to an abiotic stress (salt), were carried out. Detailed histological analysis and quantification of cre1 mycorrhized roots did not reveal any detrimental phenotype, suggesting that MtCRE1 does not belong to the ancestral common symbiotic pathway shared by rhizobial and AM symbioses. cre1 mutants formed an increased number of emerged lateral roots compared to wild-type plants, a phenotype which was also observed under non-stressed conditions. In response to A. euteiches, cre1 mutants showed reduced disease symptoms and an increased plant survival rate, correlated to an enhanced formation of lateral roots, a feature previously linked to Aphanomyces resistance. Overall, we showed that the cytokinin CRE1 pathway is not only required for symbiotic nodule organogenesis but also affects both root development and resistance to abiotic and biotic environmental stresses.

In plants, root system architecture is determined by the activity of root apical meristems, which control the root growth rate, and by the formation of lateral roots. In legumes, an additional root lateral organ can develop: the symbiotic nitrogen-fixing nodule. We identified in Medicago truncatula ten allelic mutants showing a compact root architecture phenotype (cra2) independent of any major shoot phenotype, and that consisted of shorter roots, an increased number of lateral roots, and a reduced number of nodules. The CRA2 gene encodes a Leucine-Rich Repeat Receptor-Like Kinase (LRR-RLK) that primarily negatively regulates lateral root formation and positively regulates symbiotic nodulation. Grafting experiments revealed that CRA2 acts through different pathways to regulate these lateral organs originating from the roots, locally controlling the lateral root development and nodule formation systemically from the shoots. The CRA2 LRR-RLK therefore integrates short- and long-distance regulations to control root system architecture under non-symbiotic and symbiotic conditions.

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., (© 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.)