Symbiotic Nitrogen Fixation
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Domestication: Sweet! A naturally transgenic crop

Domestication: Sweet! A naturally transgenic crop | Symbiotic Nitrogen Fixation | Scoop.it
Nature Plants, Published online: 2 June 2015; | doi:10.1038/nplants.2015.77
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ARP2/3-Mediated Actin Nucleation Associated With Symbiosome Membrane Is Essential for the Development of Symbiosomes in Infected Cells of Medicago truncatula Root Nodules

The nitrogen-fixing rhizobia in the symbiotic infected cells of root nodules are kept in membrane compartments derived from the host cell plasma membrane, forming what are known as symbiosomes. These are maintained as individual units, with mature symbiosomes having a specific radial position in the host cell cytoplasm. The mechanisms that adapt the host cell architecture to accommodate intracellular bacteria are not clear. The intracellular organization of any cell depends heavily on the actin cytoskeleton. Dynamic rearrangement of the actin cytoskeleton is crucial for cytoplasm organization and intracellular trafficking of vesicles and organelles. A key component of the actin cytoskeleton rearrangement is the ARP2/3 complex, which nucleates new actin filaments and forms branched actin networks. To clarify the role of the ARP2/3 complex in the development of infected cells and symbiosomes, we analyzed the pattern of actin microfilaments and the functional role of ARP3 in Medicago truncatula root nodules. In infected cells, ARP3 protein and actin were spatially associated with maturing symbiosomes. Partial ARP3 silencing causes defects in symbiosome development; in particular, ARP3 silencing disrupts the final differentiation steps in functional maturation into nitrogen-fixing units.

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Jean-Michel Ané's curator insight, May 30, 2015 3:29 PM

Nice piece of work!

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Bacterial killing via a type IV secretion system : Nature Communications : Nature Publishing Group

Bacterial killing via a type IV secretion system : Nature Communications : Nature Publishing Group | Symbiotic Nitrogen Fixation | Scoop.it
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Soybean miR172c Targets the Repressive AP2 Transcription Factor NNC1 to Activate ENOD40 Expression and Regulate Nodule Initiation

Soybean miR172c Targets the Repressive AP2 Transcription Factor NNC1 to Activate ENOD40 Expression and Regulate Nodule Initiation | Symbiotic Nitrogen Fixation | Scoop.it
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Host plant peptides elicit a transcriptional response to control the S. meliloti cell cycle during symbiosis

Host plant peptides elicit a transcriptional response to control the S. meliloti cell cycle during symbiosis | Symbiotic Nitrogen Fixation | Scoop.it

The α-proteobacterium Sinorhizobium meliloti establishes a chronic intracellular infection during the symbiosis with its legume hosts. Within specialized host cells, S. meliloti differentiates into highly polyploid, enlarged nitrogen-fixing bacteroids. This differentiation is driven by host cells through the production of defensin-like peptides called "nodule-specific cysteine-rich" (NCR) peptides. Recent research has shown that synthesized NCR peptides exhibit antimicrobial activity at high concentrations but cause bacterial endoreduplication at sublethal concentrations. We leveraged synchronized S. meliloti populations to determine how treatment with a sublethal NCR peptide affects the cell cycle and physiology of bacteria at the molecular level. We found that at sublethal levels a representative NCR peptide specifically blocks cell division and antagonizes Z-ring function. Gene-expression profiling revealed that the cell division block was produced, in part, through the substantial transcriptional response elicited by sublethal NCR treatment that affected ∼15% of the genome. Expression of critical cell-cycle regulators, including ctrA, and cell division genes, including genes required for Z-ring function, were greatly attenuated in NCR-treated cells. In addition, our experiments identified important symbiosis functions and stress responses that are induced by sublethal levels of NCR peptides and other antimicrobial peptides. Several of these stress-response pathways also are found in related α-proteobacterial pathogens and might be used by S. meliloti to sense host cues during infection. Our data suggest a model in which, in addition to provoking stress responses, NCR peptides target intracellular regulatory pathways to drive S. meliloti endoreduplication and differentiation during symbiosis.

 

Penterman J1, Abo RP, De Nisco NJ, Arnold MF, Longhi R, Zanda M, Walker GC. (2014). Proc Natl Acad Sci U S A. Mar 4;111(9):3561-6. 

 


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From Peter Young's Blog-Some 2013 papers

From Peter Young's Blog-Some 2013 papers | Symbiotic Nitrogen Fixation | Scoop.it

Happy New Year!

2013 has gone, and this blog seems to have missed most of it.  To make amends, here are a few rather randomly chosen papers from the past year.  You may have missed some of them.  For all I know, you may have written some of them.  They are not the most important papers of the year, but they are in the general area of rhizobial diversity and evolution.  There are dozens of others I could equally well have chosen – maybe I will add some more over the next few days.  If you have any suggestions, feel free to add them as comments on this post.


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The Sinorhizobium fredii HH103 Lipopolysaccharide Is Not Only Relevant at Early Soybean Nodulation Stages but Also for Symbiosome Stability in Mature Nodules

The Sinorhizobium fredii HH103 Lipopolysaccharide Is Not Only Relevant at Early Soybean Nodulation Stages but Also for Symbiosome Stability in Mature Nodules | Symbiotic Nitrogen Fixation | Scoop.it

In this work we have characterised the Sinorhizobium fredii HH103 greA lpsB lpsCDE genetic region and analysed for the first time the symbiotic performance of Sinorhizobium fredii lps mutants on soybean. The organization of the S. fredii HH103 greA, lpsB, and lpsCDE genes was equal to that of Sinorhizobium meliloti 1021. S. fredii HH103 greA, lpsB, and lpsE mutant derivatives produced altered LPS profiles that were characteristic of the gene mutated. In addition, S. fredii HH103 greA mutants showed a reduction in bacterial mobility and an increase of auto-agglutination in liquid cultures. RT-PCR and qPCR experiments demonstrated that the HH103 greA gene has a positive effect on the transcription of lpsB. Soybean plants inoculated with HH103 greA, lpsB or lpsE mutants formed numerous ineffective pseudonodules and showed severe symptoms of nitrogen starvation. However, HH103 greA and lps mutants were also able to induce the formation of a reduced number of soybean nodules of normal external morphology, allowing the possibility of studying the importance of bacterial LPS in later stages of the S. fredii HH103-soybean symbiosis. The infected cells of these nodules showed signs of early termination of symbiosis and lytical clearance of bacteroids. These cells also had very thick walls and accumulation of phenolic-like compounds, pointing to induced defense reactions. Our results show the importance of bacterial LPS in later stages of the S. fredii HH103-soybean symbiosis and their role in preventing host cell defense reactions. S. fredii HH103 lpsB mutants also showed reduced nodulation with Vigna unguiculata, although the symbiotic impairment was less pronounced than in soybean.

 

Margaret I, Lucas MM, Acosta-Jurado S, Buendía-Clavería AM, Fedorova E, Hidalgo A, Rodríguez-Carvajal MA, Rodriguez-Navarro DN, Ruiz-Sainz JE,Vinardell JM. (2013). PLoS One. Oct 1;8(10):e74717.


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The Nod factor hydrolase of Medicago truncatula: Characterization of an enzyme specifically cleaving rhizobial nodulation signals

Nodule formation induced by nitrogen-fixing rhizobia depends on bacterial nodulation factors (NFs), modified chitin oligosaccharides with a fatty acid moiety. Certain NFs can be cleaved and inactivated by plant chitinases. However, the most abundant NF of Sinorhizobium meliloti, an O-acetylated and sulphated tetramer, is resistant to hydrolysis by all plant chitinases tested so far. Nevertheless, this NF is rapidly degraded in the host rhizosphere. Here, we identify and characterize MtNFH1 (Medicago truncatula Nod factor hydrolase 1), a legume enzyme structurally related to defense-related class V chitinases (glycoside hydrolase family 18). MtNFH1 lacks chitinase activity but efficiently hydrolyzes all tested NFs of S. meliloti. The enzyme shows a high cleavage preference, releasing exclusively lipo-disaccharides from NFs. Substrate specificity and kinetic properties of MtNFH1 were compared to those of class V chitinases from Arabidopsis thaliana and Nicotiana tabacum, which cannot hydrolyze tetrameric NFs of S. meliloti. The Michaelis-Menten constants of MtNFH1 for NFs are in the micromolar concentration range, whereas non-modified chitin oligosaccharides represent neither substrates nor inhibitors for MtNFH1. The three-dimensional structure of MtNFH1 was modeled on the basis of the known structure of class V chitinases. Docking simulation of NFs to MtNFH1 predicted a distinct binding cleft for the fatty acid moiety, which is absent in the class V chitinases. Point mutation analysis confirmed the modeled NF-MtNFH1 interaction. Silencing of MtNFH1 by RNA interference resulted in reduced NF degradation in the rhizosphere of M. truncatula. In conclusion, we have found a novel legume hydrolase that specifically inactivates NFs.


Via Jean-Michel Ané, IvanOresnik
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Possible role of glutamine synthetase in the NO signaling response in root nodules by contributing to the antioxidant defenses

Possible role of glutamine synthetase in the NO signaling response in root nodules by contributing to the antioxidant defenses | Symbiotic Nitrogen Fixation | Scoop.it

Nitric oxide (NO) is emerging as an important regulatory player in the Rhizobium-legume symbiosis. The occurrence of NO during several steps of the symbiotic interaction suggests an important, but yet unknown, signaling role of this molecule for root nodule formation and functioning. The identification of the molecular targets of NO is key for the assembly of the signal transduction cascade that will ultimately help to unravel NO function. We have recently shown that the key nitrogen assimilatory enzyme glutamine synthetase (GS) is a molecular target of NO in root nodules of Medicago truncatula, being post-translationally regulated by tyrosine nitration in relation to nitrogen fixation. In functional nodules of M. truncatula NO formation has been located in the bacteroid containing cells of the fixation zone, where the ammonium generated by bacterial nitrogenase is released to the plant cytosol and assimilated into the organic pools by plant GS. We propose that the NO-mediated GS post-translational inactivation is connected to nitrogenase inhibition induced by NO and is related to metabolite channeling to boost the nodule antioxidant defenses. Glutamate, a substrate for GS activity is also the precursor for the synthesis of glutathione (GSH), which is highly abundant in root nodules of several plant species and known to play a major role in the antioxidant defense participating in the ascorbate/GSH cycle. Existing evidence suggests that upon NO-mediated GS inhibition, glutamate could be channeled for the synthesis of GSH. According to this hypothesis, GS would be involved in the NO-signaling responses in root nodules and the NO-signaling events would meet the nodule metabolic pathways to provide an adaptive response to the inhibition of symbiotic nitrogen fixation by reactive nitrogen species.

 

Silva L, Carvalho H. (2013). Front Plant Sci. Sep 19;4:372


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Going back to the roots: the microbial ecology of the rhizosphere

Going back to the roots: the microbial ecology of the rhizosphere | Symbiotic Nitrogen Fixation | Scoop.it

The rhizosphere is the interface between plant roots and soil where interactions among a myriad of microorganisms and invertebrates affect biogeochemical cycling, plant growth and tolerance to biotic and abiotic stress. The rhizosphere is intriguingly complex and dynamic, and understanding its ecology and evolution is key to enhancing plant productivity and ecosystem functioning. Novel insights into key factors and evolutionary processes shaping the rhizosphere microbiome will greatly benefit from integrating reductionist and systems-based approaches in both agricultural and natural ecosystems. Here, we discuss recent developments in rhizosphere research in relation to assessing the contribution of the micro- and macroflora to sustainable agriculture, nature conservation, the development of bio-energy crops and the mitigation of climate change.


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The calcium-stimulated lipid A 3-O deacylase from Rhizobium etli is not essential for plant nodulation.

The lipid A component of lipopolysaccharide from the nitrogen-fixing plant endosymbiont, Rhizobium etli, is structurally very different from that found in most enteric bacteria. The lipid A from free-living R. etli is structurally heterogeneous and exists as a mixture of species which are either pentaacylated or tetraacylated. In contrast, the lipid A from R. etli bacteroids is reported to consist exclusively of tetraacylated lipid A species. The tetraacylated lipid A species in both cases lack a beta-hydroxymyristoyl chain at the 3-position of lipid A. Here, we show that the lipid A modification enzyme responsible for 3-O deacylation in R. etli is a homolog of the PagL protein originally described in Salmonella enterica sv. typhimurium. In contrast to the PagL proteins described from other species, R. etli PagL displays a calcium dependency. To determine the importance of the lipid A modification catalyzed by PagL, we isolated and characterized a R. etli mutant deficient in the pagL gene. Mass spectrometric analysis confirmed that the mutant strain was exclusively tetraacylated and radiochemical analysis revealed that 3-O deacylase activity was absent in membranes prepared from the mutant. The R. etli mutant was not impaired in its ability to form nitrogen-fixing nodules on Phaseolus vulgaris but it displayed slower nodulation kinetics relative to the wild-type strain. The lipid A modification catalyzed by R. etli PagL, therefore, is not required for nodulation but may play other roles such as protecting bacterial endosymbionts from plant immune responses during infection.

  Sohlenkamp C, Raetz CR, Ingram BO. (2013). Biochim Biophys Acta. Jul;1831(7):1250-9.


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A Paradigm for Endosymbiotic Life: Cell Differentiation of Rhizobium Bacteria Provoked by Host Plant Factors

A Paradigm for Endosymbiotic Life: Cell Differentiation of Rhizobium Bacteria Provoked by Host Plant Factors | Symbiotic Nitrogen Fixation | Scoop.it

Symbiosis between Rhizobium bacteria and legumes leads to the formation of the root nodule. The endosymbiotic bacteria reside in polyploid host cells as membrane-surrounded vesicles where they reduce atmospheric nitrogen to support plant growth by supplying ammonia in exchange for carbon sources and energy. The morphology and physiology of endosymbionts, despite their common function, are highly divergent in different hosts. In galegoid plants, the endosymbionts are terminally differentiated, uncultivable polyploid cells, with remarkably elongated and even branched Y-shaped cells. Bacteroid differentiation is controlled by host peptides, many of which have antibacterial activity and require the bacterial function of BacA. Although the precise and combined action of several hundred host peptides and BacA has yet to be discovered, similarities, especially to certain insect-bacterium symbioses involving likewise host peptides for manipulation of endosymbionts, suggest convergent evolution. Rhizobium-legume symbiosis provides a rich source of information for understanding host-controlled endosymbiotic life in eukaryotic cells.

 

Eva Kondorosi, Peter Mergaert,and Attila Kereszt (2013) Annual Review Microniology 67: 611-628 (Volume publication date September 2013)DOI: 10.1146/annurev-micro-092412-155630

 


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Peribacteroid space acidification: a marker of mature bacteroid functioning in Medicago truncatula nodules - PIERRE - 2013 - Plant, Cell & Environment - Wiley Online Library

Peribacteroid space acidification: a marker of mature bacteroid functioning in Medicago truncatula nodules - PIERRE - 2013 - Plant, Cell & Environment - Wiley Online Library | Symbiotic Nitrogen Fixation | Scoop.it
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Recent developments in arbuscular mycorrhizal signaling

Plants can establish root endosymbioses with both arbuscular mycorrhizal fungi and rhizobial bacteria to improve their nutrition. Our understanding of the molecular events underlying the establishment of these symbioses has significantly advanced in the last few years. Here I highlight major recent findings in the field of endosymbiosis signaling. Despite the identification of new signaling components and the definition, or in some cases better re-definition of the molecular functions of previously known players, major questions still remain that need to be addressed. Most notably the mechanisms defining signaling specificities within either symbiosis remain unclear.

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Frontiers | Perception of pathogenic or beneficial bacteria and their evasion of host immunity: pattern recognition receptors in the frontline | Plant-Microbe Interaction

Plants are continuously monitoring the presence of microorganisms to establish an adapted response. Plants commonly use pattern recognition receptors (PRRs) to perceive microbe- or pathogen-associated molecular patterns (MAMPs/PAMPs) which are microorganism molecular signatures. Located at the plant plasma membrane, the PRRs are generally receptor-like kinases (RLKs) or receptor-like proteins (RLPs). MAMP detection will lead to the establishment of a plant defense program called MAMP-triggered immunity (MTI). In this review, we overview the RLKs and RLPs that assure early recognition and control of pathogenic or beneficial bacteria. We also highlight the crucial function of PRRs during plant-microbe interactions, with a special emphasis on the receptors of the bacterial flagellin and peptidoglycan. In addition, we discuss the multiple strategies used by bacteria to evade PRR-mediated recognition.
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Medicago truncatula symbiotic peptide NCR247 contributes to bacteroid differentiation through multiple mechanisms

Medicago truncatula symbiotic peptide NCR247 contributes to bacteroid differentiation through multiple mechanisms | Symbiotic Nitrogen Fixation | Scoop.it

Symbiosis between rhizobia soil bacteria and legume plants results in the formation of root nodules where plant cells are fully packed with nitrogen fixing bacteria. In the host cells, the bacteria adapt to the intracellular environment and gain the ability for nitrogen fixation. Depending on the host plants, the symbiotic fate of bacteria can be either reversible or irreversible. In Medicago and related legume species, the bacteria undergo a host-directed multistep differentiation process culminating in the formation of elongated and branched polyploid bacteria with definitive loss of cell division ability. The plant factors are nodule-specific symbiotic peptides. Approximately 600 of them are nodule-specific cysteine-rich (NCR) peptides produced in the rhizobium-infected plant cells. NCRs are targeted to the endosymbionts, and concerted action of different sets of peptides governs different stages of endosymbiont maturation, whereas the symbiotic function of individual NCRs is unknown. This study focused on NCR247, a cationic peptide exhibiting in vitro antimicrobial activities. We show that NCR247 acts in those nodule cells where bacterial cell division is arrested and cell elongation begins. NCR247 penetrates the bacteria and forms complexes with many bacterial proteins. Interaction with FtsZ required for septum formation is one of the host interventions for inhibiting bacterial cell division. Complex formation with the ribosomal proteins affects translation and contributes to altered proteome and physiology of the endosymbiont. Binding to the chaperone GroEL amplifies the NCR247-modulated biological processes. We show that GroEL1 ofSinorhizobium meliloti is required for efficient infection, terminal differentiation, and nitrogen fixation.


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Cell Host & Microbe: CYCLOPS, A DNA-Binding Transcriptional Activator, Orchestrates Symbiotic Root Nodule Development (2014)

Cell Host & Microbe: CYCLOPS, A DNA-Binding Transcriptional Activator, Orchestrates Symbiotic Root Nodule Development (2014) | Symbiotic Nitrogen Fixation | Scoop.it

Nuclear calcium oscillations are a hallmark of symbiotically stimulated plant root cells. Activation of the central nuclear decoder, calcium- and calmodulin-dependent kinase (CCaMK), triggers the entire symbiotic program including root nodule organogenesis, but the mechanism of signal transduction by CCaMK was unknown. We show that CYCLOPS, a direct phosphorylation substrate of CCaMK, is a DNA-binding transcriptional activator. Two phosphorylated serine residues within the N-terminal negative regulatory domain of CYCLOPS are necessary for its activity. CYCLOPS binds DNA in a sequence-specific and phosphorylation-dependent manner and transactivates the NODULE INCEPTION (NIN) gene. A phosphomimetic version of CYCLOPS was sufficient to trigger root nodule organogenesis in the absence of rhizobia and CCaMK. CYCLOPS thus induces a transcriptional activation cascade, in which NIN and a heterotrimeric NF-Y complex act in hierarchical succession to initiate symbiotic root nodule development


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Which role for nitric oxide in symbiotic N2-fixing nodules: toxic by-product or useful signaling/metabolic intermediate?

Which role for nitric oxide in symbiotic N2-fixing nodules: toxic by-product or useful signaling/metabolic intermediate? | Symbiotic Nitrogen Fixation | Scoop.it

The interaction between legumes and rhizobia leads to the establishment of a symbiotic relationship characterized by the formation of new organs called nodules, in which bacteria have the ability to fix atmospheric nitrogen (N2) via the nitrogenase activity. Significant nitric oxide (NO) production was evidenced in the N2-fixing nodules suggesting that it may impact the symbiotic process. Indeed, NO was shown to be a potent inhibitor of nitrogenase activity and symbiotic N2 fixation. It has also been shown that NO production is increased in hypoxic nodules and this production was supposed to be linked - via a nitrate/NO respiration process - with improved capacity of the nodules to maintain their energy status under hypoxic conditions. Other data suggest that NO might be a developmental signal involved in the induction of nodule senescence. Hence, the questions were raised of the toxic effects versus signaling/metabolic functions of NO, and of the regulation of NO levels compatible with nitrogenase activity. The present review analyses the different roles of NO in functioning nodules, and discusses the role of plant and bacterial (flavo)hemoglobins in the control of NO level in nodules.

 

Boscari A, Meilhoc E, Castella C, Bruand C, Puppo A, Brouquisse R. (2013).  Front Plant Sci. 4:384.


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Hijacking of leguminous nodulation signaling by the rhizobial type III secretion system

Hijacking of leguminous nodulation signaling by the rhizobial type III secretion system | Symbiotic Nitrogen Fixation | Scoop.it

Root–nodule symbiosis between leguminous plants and nitrogen-fixing bacteria (rhizobia) involves molecular communication between the two partners. Key components for the establishment of symbiosis are rhizobium-derived lipochitooligosaccharides (Nod factors; NFs) and their leguminous receptors (NFRs) that initiate nodule development and bacterial entry. Here we demonstrate that the soybean microsymbiontBradyrhizobium elkanii uses the type III secretion system (T3SS), which is known for its delivery of virulence factors by pathogenic bacteria, to promote symbiosis. Intriguingly, wild-type B. elkanii, but not the T3SS-deficient mutant, was able to form nitrogen-fixing nodules on soybean nfr mutant En1282. Furthermore, even the NF-deficient B. elkanii mutant induced nodules unless T3SS genes were mutated. Transcriptional analysis revealed that expression of the soybean nodulation-specific genes ENOD40 and NIN was increased in the roots of En1282 inoculated with B. elkanii but not with its T3SS mutant, suggesting that T3SS activates host nodulation signaling by bypassing NF recognition. Root-hair curling and infection threads were not observed in the roots of En1282 inoculated with B. elkanii, indicating that T3SS is involved in crack entry or intercellular infection. These findings suggest that B. elkanii has adopted a pathogenic system for activating host symbiosis signaling to promote its infection.


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Steve Marek's curator insight, October 2, 2013 11:44 AM

Changing the paradigm..."nod factors? we don't need no stinking nod factors'

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Iron: an essential micronutrient for the legume-rhizobium symbiosis

Iron: an essential micronutrient for the legume-rhizobium symbiosis | Symbiotic Nitrogen Fixation | Scoop.it

Legumes, which develop a symbiosis with nitrogen-fixing bacteria, have an increased demand for iron. Iron is required for the synthesis of iron-containing proteins in the host, including the highly abundant leghemoglobin, and in bacteroids for nitrogenase and cytochromes of the electron transport chain. Deficiencies in iron can affect initiation and development of the nodule. Within root cells, iron is chelated with organic acids such as citrate and nicotianamine and distributed to other parts of the plant. Transport to the nitrogen-fixing bacteroids in infected cells of nodules is more complicated. Formation of the symbiosis results in bacteroids internalized within root cortical cells of the legume where they are surrounded by a plant-derived membrane termed the symbiosome membrane (SM). This membrane forms an interface that regulates nutrient supply to the bacteroid. Consequently, iron must cross this membrane before being supplied to the bacteroid. Iron is transported across the SM as both ferric and ferrous iron. However, uptake of Fe(II) by both the symbiosome and bacteroid is faster than Fe(III) uptake. Members of more than one protein family may be responsible for Fe(II) transport across the SM. The only Fe(II) transporter in nodules characterized to date is GmDMT1 (Glycine max divalent metal transporter 1), which is located on the SM in soybean. Like the root plasma membrane, the SM has ferric iron reductase activity. The protein responsible has not been identified but is predicted to reduce ferric iron accumulated in the symbiosome space prior to uptake by the bacteroid. With the recent publication of a number of legume genomes including Medicago truncatula and G. max, a large number of additional candidate transport proteins have been identified. Members of the NRAMP (natural resistance-associated macrophage protein), YSL (yellow stripe-like), VIT (vacuolar iron transporter), and ZIP (Zrt-, Irt-like protein) transport families show enhanced expression in nodules and are expected to play a role in the transport of iron and other metals across symbiotic membranes.

 

Brear EM, Day DA, Smith PM. (2013). Front Plant Sci. Sep 13;4:359.


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Electron transfer precedes ATP hydrolysis during nitrogenase catalysis

 

Abstract

The biological reduction of N2 to NH3 catalyzed by Mo-dependent nitrogenase requires at least eight rounds of a complex cycle of events associated with ATP-driven electron transfer (ET) from the Fe protein to the catalytic MoFe protein, with each ET coupled to the hydrolysis of two ATP molecules. Although steps within this cycle have been studied for decades, the nature of the coupling between ATP hydrolysis and ET, in particular the order of ET and ATP hydrolysis, has been elusive. Here, we have measured first-order rate constants for each key step in the reaction sequence, including direct measurement of the ATP hydrolysis rate constant: kATP = 70 s−1, 25 °C. Comparison of the rate constants establishes that the reaction sequence involves four sequential steps: (i) conformationally gated ET (kET = 140 s−1, 25 °C), (ii) ATP hydrolysis (kATP = 70 s−1, 25 °C), (iii) Phosphate release (kPi = 16 s−1, 25 °C), and (iv) Fe protein dissociation from the MoFe protein (kdiss = 6 s−1, 25 °C). These findings allow completion of the thermodynamic cycle undergone by the Fe protein, showing that the energy of ATP binding and protein–protein association drive ET, with subsequent ATP hydrolysis and Pi release causing dissociation of the complex between the Feox(ADP)2 protein and the reduced MoFe protein.

 

Simon Duval, Karamatullah Danyal, Sudipta Shaw, Anna K. Lytle, Dennis R. Dean, Brian M. Hoffman, Edwin Antony and Lance C. Seefeldt. (2013). Proc Natl Acad Sci USA published ahead of print September 23,, doi:10.1073/pnas.1311218110


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IvanOresnik's curator insight, September 25, 2013 1:35 PM
Significance (from article)

Biological reduction of dinitrogen (N2) to ammonia (NH3) occurs in a select group of bacteria that contain the metalloenzyme nitrogenase. To catalyze this difficult reaction, nitrogenase requires electrons, protons, and ATP. From the earliest studies of nitrogenase, it was realized that ATP hydrolysis is coupled to delivery of electrons and reduction of N2, yet the order of ATP hydrolysis and electron transfer, which determines the nature of the coupling, was never established. In this work, we establish the order of all the key events during one catalytic cycle of electron delivery in nitrogenase, showing that ATP hydrolysis follows electron transfer. These findings guide future studies aimed at understanding what roles ATP binding and hydrolysis play in the nitrogenase mechanism.

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Predicting gene regulatory networks of soybean nodulation from RNA-Seq transcriptome data.

Predicting gene regulatory networks of soybean nodulation from RNA-Seq transcriptome data. | Symbiotic Nitrogen Fixation | Scoop.it

BACKGROUND: High-throughput RNA sequencing (RNA-Seq) is a revolutionary technique to study the transcriptome of a cell under various conditions at a systems level. Despite the wide application of RNA-Seq techniques to generate experimental data in the last few years, few computational methods are available to analyze this huge amount of transcription data. The computational methods for constructing gene regulatory networks from RNA-Seq expression data of hundreds or even thousands of genes are particularly lacking and urgently needed.

 

RESULTS: We developed an automated bioinformatics method to predict gene regulatory networks from the quantitative expression values of differentially expressed genes based on RNA-Seq transcriptome data of a cell in different stages and conditions, integrating transcriptional, genomic and gene function data. We applied the method to the RNA-Seq transcriptome data generated for soybean root hair cells in three different development stages of nodulation after rhizobium infection. The method predicted a soybean nodulation-related gene regulatory network consisting of 10 regulatory modules common for all three stages, and 24, 49 and 70 modules separately for the first, second and third stage, each containing both a group of co-expressed genes and several transcription factors collaboratively controlling their expression under different conditions. 8 of 10 common regulatory modules were validated by at least two kinds of validations, such as independent DNA binding motif analysis, gene function enrichment test, and previous experimental data in the literature.

 

CONCLUSIONS: We developed a computational method to reliably reconstruct gene regulatory networks from RNA-Seq transcriptome data. The method can generate valuable hypotheses for interpreting biological data and designing biological experiments such as ChIP-Seq, RNA interference, and yeast two hybrid experiments.

 

Zhu M, Dahmen JL, Stacey G, Cheng J. (2013).  BMC Bioinformatics. Sep 22;14(1):278. [Epub ahead of print]

 


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An endogenous artificial microRNA system for unraveling the function of root endosymbioses related genes in Medicago truncatula

An endogenous artificial microRNA system for unraveling the function of root endosymbioses related genes in Medicago truncatula | Symbiotic Nitrogen Fixation | Scoop.it

Background

Legumes have the unique capacity to undergo two important root endosymbioses: the root nodule symbiosis and the arbuscular mycorrhizal symbiosis. Medicago truncatula is widely used to unravel the functions of genes during these root symbioses. Here we describe the development of an artificial microRNA (amiR)-mediated gene silencing system for M. truncatula roots.

Results

The endogenous microRNA (miR) mtr-miR159b was selected as a backbone molecule for driving amiR expression. Heterologous expression of mtr-miR159b-amiR constructs in tobacco showed that the backbone is functional and mediates an efficient gene silencing. amiR-mediated silencing of a visible marker was also effective after root transformation of M. truncatula constitutively expressing the visible marker. Most importantly, we applied the novel amiR system to shed light on the function of a putative transcription factor, MtErf1, which was strongly induced in arbuscule-containing cells during mycorrhizal symbiosis. MtPt4 promoter driven amiR-silencing led to strongly decreased transcript levels and deformed, non-fully truncated arbuscules indicating that MtErf1 is required for arbuscule development.

Conclusions

The endogenous amiR system demonstrated here presents a novel and highly efficient tool to unravel gene functions during root endosymbioses.


Via Olivier ANDRE, Jean-Michel Ané, IvanOresnik
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Rescooped by Senlei Zhang from Plants and Microbes
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Nature Communications: Root-derived CLE glycopeptides control nodulation by direct binding to HAR1 receptor kinase (2013)

Nature Communications: Root-derived CLE glycopeptides control nodulation by direct binding to HAR1 receptor kinase (2013) | Symbiotic Nitrogen Fixation | Scoop.it

Leguminous plants establish a symbiosis with rhizobia to enable nitrogen fixation in root nodules under the control of the presumed root-to-shoot-to-root negative feedback called autoregulation of nodulation. In Lotus japonicus, autoregulation is mediated by CLE-RS genes that are specifically expressed in the root, and the receptor kinase HAR1 that functions in the shoot. However, the mature functional structures of CLE-RS gene products and the molecular nature of CLE-RS/HAR1signalling governed by these spatially distant components remain elusive. Here we show that CLE-RS2 is a post-translationally arabinosylated glycopeptide derived from the CLE domain. Chemically synthesized CLE-RS glycopeptides cause significant suppression of nodulation and directly bind to HAR1 in an arabinose-chain and sequence-dependent manner. In addition, CLE-RS2 glycopeptide specifically produced in the root is found in xylem sap collected from the shoot. We propose that CLE-RS glycopeptides are the long sought mobile signals responsible for the initial step of autoregulation of nodulation.


Via Kamoun Lab @ TSL
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