ABSTRACT
Legumes acquire fixed nitrogen (N) from the soil and through endosymbiotic association with diazotrophic bacteria. However, establishing and maintaining N2-fixing nodules are expensive for the host plant, relative to taking up N from the soil. Therefore, plants suppress symbiosis when N is plentiful and enhance symbiosis when N is sparse. Here, we show that the nitrate transporter MtNRT2.1 is required for optimal nodule establishment in Medicago truncatula under low-nitrate conditions and the repression of nodulation under high-nitrate conditions. The NIN-like protein (NLP) MtNLP1 is required for MtNRT2.1 expression and regulation of nitrate uptake/transport under low- and high-nitrate conditions. Under low nitrate, the gene encoding the C-terminally encoded peptide (CEP) MtCEP1 was more highly expressed, and the exogenous application of MtCEP1 systemically promoted MtNRT2.1 expression in a compact root architecture 2 (MtCRA2)-dependent manner. The enhancement of nodulation by MtCEP1 and nitrate uptake were both impaired in the Mtnrt2.1 mutant under low nitrate. Our study demonstrates that nitrate uptake by MtNRT2.1 differentially affects nodulation at low- and high-nitrate conditions through the actions of MtCEP1 and MtNLP1.
Subject(s)
Medicago truncatula , Nitrates , Gene Expression Regulation, Plant , Medicago truncatula/metabolism , Nitrates/pharmacology , Nitrates/metabolism , Peptides/genetics , Peptides/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism , Plant Root Nodulation/genetics , Root Nodules, Plant/genetics , Root Nodules, Plant/metabolism , Symbiosis/physiologyABSTRACT
The conservation of GOLVEN (GLV)/ROOT MERISTEM GROWTH FACTOR (RGF) peptide encoding genes across plant genomes capable of forming roots or root-like structures underscores their potential significance in the terrestrial adaptation of plants. This study investigates the function and role of GOLVEN peptide-coding genes in Medicago truncatula. Five out of fifteen GLV/RGF genes were notably upregulated during nodule organogenesis and were differentially responsive to nitrogen deficiency and auxin treatment. Specifically, the expression of MtGLV9 and MtGLV10 at nodule initiation sites was contingent upon the NODULE INCEPTION transcription factor. Overexpression of these five nodule-induced GLV genes in hairy roots of M. truncatula and application of their synthetic peptide analogues led to a decrease in nodule count by 25-50%. Uniquely, the GOLVEN10 peptide altered the positioning of the first formed lateral root and nodule on the primary root axis, an observation we term 'noduletaxis'; this decreased the length of the lateral organ formation zone on roots. Histological section of roots treated with synthetic GOLVEN10 peptide revealed an increased cell number within the root cortical cell layers without a corresponding increase in cell length, leading to an elongation of the root likely introducing a spatiotemporal delay in organ formation. At the transcription level, the GOLVEN10 peptide suppressed expression of microtubule-related genes and exerted its effects by changing expression of a large subset of Auxin responsive genes. These findings advance our understanding of the molecular mechanisms by which GOLVEN peptides modulate root morphology, nodule ontogeny, and interactions with key transcriptional pathways.
Subject(s)
Gene Expression Regulation, Plant , Medicago truncatula , Plant Proteins , Plant Roots , Root Nodules, Plant , Medicago truncatula/genetics , Medicago truncatula/growth & development , Medicago truncatula/metabolism , Medicago truncatula/drug effects , Medicago truncatula/physiology , Plant Proteins/genetics , Plant Proteins/metabolism , Plant Roots/growth & development , Plant Roots/genetics , Plant Roots/drug effects , Plant Roots/metabolism , Root Nodules, Plant/genetics , Root Nodules, Plant/growth & development , Root Nodules, Plant/metabolism , Root Nodules, Plant/drug effects , Indoleacetic Acids/metabolism , Indoleacetic Acids/pharmacology , Plant Root Nodulation/genetics , Meristem/genetics , Meristem/growth & development , Meristem/drug effects , Peptides/metabolism , Peptides/geneticsABSTRACT
The preference for nitrate over chloride through regulation of transporters is a fundamental feature of plant ion homeostasis. We show that Medicago truncatula MtNPF6.5, an ortholog of Arabidopsis thaliana AtNPF6.3/NRT1.1, can mediate nitrate and chloride uptake in Xenopus oocytes but is chloride selective and that its close homologue, MtNPF6.7, can transport nitrate and chloride but is nitrate selective. The MtNPF6.5 mutant showed greatly reduced chloride content relative to wild type, and MtNPF6.5 expression was repressed by high chloride, indicating a primary role for MtNPF6.5 in root chloride uptake. MtNPF6.5 and MtNPF6.7 were repressed and induced by nitrate, respectively, and these responses required the transcription factor MtNLP1. Moreover, loss of MtNLP1 prevented the rapid switch from chloride to nitrate as the main anion in nitrate-starved plants after nitrate provision, providing insight into the underlying mechanism for nitrate preference. Sequence analysis revealed three sub-types of AtNPF6.3 orthologs based on their predicted substrate-binding residues: A (chloride selective), B (nitrate selective), and C (legume specific). The absence of B-type AtNPF6.3 homologues in early diverged plant lineages suggests that they evolved from a chloride-selective MtNPF6.5-like protein.
Subject(s)
Anion Transport Proteins/genetics , Chlorides/metabolism , Gene Expression Regulation, Plant , Medicago truncatula/metabolism , Nitrates/metabolism , Plant Proteins/genetics , Plant Roots/metabolism , Transcription Factors/genetics , Animals , Anion Transport Proteins/metabolism , Arabidopsis/genetics , Arabidopsis/growth & development , Arabidopsis/metabolism , Biological Evolution , Biological Transport , Conserved Sequence , Homeostasis , Medicago truncatula/genetics , Medicago truncatula/growth & development , Oocytes , Phylogeny , Plant Proteins/metabolism , Plant Roots/genetics , Plant Roots/growth & development , Protein Binding , Protein Isoforms/genetics , Protein Isoforms/metabolism , Seedlings/genetics , Seedlings/growth & development , Seedlings/metabolism , Signal Transduction , Transcription Factors/metabolism , Xenopus laevisABSTRACT
Most land plants benefit from endosymbiotic interactions with mycorrhizal fungi, including legumes and some nonlegumes that also interact with endosymbiotic nitrogen (N)-fixing bacteria to form nodules. In addition to these helpful interactions, plants are continuously exposed to would-be pathogenic microbes: discriminating between friends and foes is a major determinant of plant survival. Recent breakthroughs have revealed how some key signals from pathogens and symbionts are distinguished. Once this checkpoint has been passed and a compatible symbiont is recognized, the plant coordinates the sequential development of two types of specialized structures in the host. The first serves to mediate infection, and the second, which appears later, serves as sophisticated intracellular nutrient exchange interfaces. The overlap in both the signaling pathways and downstream infection components of these symbioses reflects their evolutionary relatedness and the common requirements of these two interactions. However, the different outputs of the symbioses, phosphate uptake versus N fixation, require fundamentally different components and physical environments and necessitated the recruitment of different master regulators, NODULE INCEPTION-LIKE PROTEINS, and PHOSPHATE STARVATION RESPONSES, for nodulation and mycorrhization, respectively.
Subject(s)
Fabaceae , Mycorrhizae , Rhizobium , Mycorrhizae/physiology , Nitrogen Fixation , Phosphates , Plants/microbiology , Rhizobium/physiology , Symbiosis/physiologyABSTRACT
The subcellular events occurring in cells of legume plants as they form transcellular symbiotic-infection structures have been compared with those occurring in premitotic cells. Here, we demonstrate that Aurora kinase 1 (AUR1), a highly conserved mitotic regulator, is required for intracellular infection by rhizobia in Medicago truncatula. AUR1 interacts with microtubule-associated proteins of the TPXL and MAP65 families, which, respectively, activate and are phosphorylated by AUR1, and localizes with them within preinfection structures. MYB3R1, a rhizobia-induced mitotic transcription factor, directly regulates AUR1 through two closely spaced, mitosis-specific activator cis elements. Our data are consistent with a model in which the MYB3R1-AUR1 regulatory module serves to properly orient preinfection structures to direct the transcellular deposition of cell wall material for the growing infection thread, analogous to its role in cell plate formation. Our findings indicate that the eukaryotically conserved MYB3R1-TPXL-AUR1-MAP65 mitotic module was conscripted to support endosymbiotic infection in legumes.
Subject(s)
Aurora Kinases , Medicago truncatula , Plant Proteins , Rhizobium , Symbiosis , Aurora Kinases/genetics , Aurora Kinases/metabolism , Gene Expression Regulation, Plant , Medicago truncatula/genetics , Medicago truncatula/microbiology , Microtubule-Associated Proteins/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism , Rhizobium/metabolism , Transcription Factors/metabolismABSTRACT
Legume nodulation requires the detection of flavonoids in the rhizosphere by rhizobia to activate their production of Nod factor countersignals. Here we investigated the flavonoids involved in nodulation of Medicago truncatula. We biochemically characterized five flavonoid-O-methyltransferases (OMTs) and a lux-based nod gene reporter was used to investigate the response of Sinorhizobium medicae NodD1 to various flavonoids. We found that chalcone-OMT 1 (ChOMT1) and ChOMT3, but not OMT2, 4, and 5, were able to produce 4,4'-dihydroxy-2'-methoxychalcone (DHMC). The bioreporter responded most strongly to DHMC, while isoflavones important for nodulation of soybean (Glycine max) showed no activity. Mutant analysis revealed that loss of ChOMT1 strongly reduced DHMC levels. Furthermore, chomt1 and omt2 showed strongly reduced bioreporter luminescence in their rhizospheres. In addition, loss of both ChOMT1 and ChOMT3 reduced nodulation, and this phenotype was strengthened by the further loss of OMT2. We conclude that: the loss of ChOMT1 greatly reduces root DHMC levels; ChOMT1 or OMT2 are important for nod gene activation in the rhizosphere; and ChOMT1/3 and OMT2 promote nodulation. Our findings suggest a degree of exclusivity in the flavonoids used for nodulation in M. truncatula compared to soybean, supporting a role for flavonoids in rhizobial host range.
Subject(s)
Chalcones , Medicago truncatula , Plant Root Nodulation , Rhizosphere , Medicago truncatula/genetics , Medicago truncatula/microbiology , Medicago truncatula/metabolism , Chalcones/metabolism , Plant Root Nodulation/genetics , Gene Expression Regulation, Plant , Mutation/genetics , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Flavonoids/metabolism , Plant Proteins/metabolism , Plant Proteins/genetics , Sinorhizobium/physiology , Sinorhizobium/genetics , Methyltransferases/metabolism , Methyltransferases/geneticsABSTRACT
Protecting haploid pollen and spores against UV-B light and high temperature, 2 major stresses inherent to the terrestrial environment, is critical for plant reproduction and dispersal. Here, we show flavonoids play an indispensable role in this process. First, we identified the flavanone naringenin, which serves to defend against UV-B damage, in the sporopollenin wall of all vascular plants tested. Second, we found that flavonols are present in the spore/pollen protoplasm of all euphyllophyte plants tested and that these flavonols scavenge reactive oxygen species to protect against environmental stresses, particularly heat. Genetic and biochemical analyses showed that these flavonoids are sequentially synthesized in both the tapetum and microspores during pollen ontogeny in Arabidopsis (Arabidopsis thaliana). We show that stepwise increases in the complexity of flavonoids in spores/pollen during plant evolution mirror their progressive adaptation to terrestrial environments. The close relationship between flavonoid complexity and phylogeny and its strong association with pollen survival phenotypes suggest that flavonoids played a central role in the progression of plants from aquatic environments into progressively dry land habitats.
Subject(s)
Arabidopsis , Flavonoids , Plants , Pollen/genetics , Arabidopsis/genetics , Flavonols , SporesABSTRACT
Most legumes can form an endosymbiotic association with soil bacteria called rhizobia, which colonize specialized root structures called nodules where they fix nitrogen. To colonize nodule cells, rhizobia must first traverse the epidermis and outer cortical cell layers of the root. In most legumes, this involves formation of the infection thread, an intracellular structure that becomes colonized by rhizobia, guiding their passage through the outer cell layers of the root and into the newly formed nodule cells. In this brief review, we recount the early research milestones relating to the rhizobial infection thread and highlight two relatively recent advances in the symbiotic infection mechanism, the eukaryotically conserved 'MYB-AUR1-MAP' mitotic module, which links cytokinesis mechanisms to intracellular infection, and the discovery of the 'infectosome' complex, which guides infection thread growth. We also discuss the potential intertwining of the two modules and the hypothesis that cytokinesis served as a foundation for intracellular infection of symbiotic microbes.
Subject(s)
Fabaceae , Rhizobium , Fabaceae/microbiology , Bacteria , Symbiosis , Root Nodules, PlantABSTRACT
Legume nodules express multiple leghemoglobins (Lbs) and non-symbiotic hemoglobins (Glbs), but how they are regulated is unclear. Here, we study the regulation of all Lbs and Glbs of Lotus japonicus in different physiologically relevant conditions and mutant backgrounds. We quantified hemoglobin expression, localized reactive oxygen species (ROS) and nitric oxide (NO) in nodules, and deployed mutants deficient in Lbs and in the transcription factors NLP4 (associated with nitrate sensitivity) and NAC094 (associated with senescence). Expression of Lbs and class 2 Glbs was suppressed by nitrate, whereas expression of class 1 and 3 Glbs was positively correlated with external nitrate concentrations. Nitrate-responsive elements were found in the promoters of several hemoglobin genes. Mutant nodules without Lbs showed accumulation of ROS and NO and alterations of antioxidants and senescence markers. NO accumulation occurred by a nitrate-independent pathway, probably due to the virtual disappearance of Glb1-1 and the deficiency of Lbs. We conclude that hemoglobins are regulated in a gene-specific manner during nodule development and in response to nitrate and dark stress. Mutant analyses reveal that nodules lacking Lbs experience nitro-oxidative stress and that there is compensation of expression between Lb1 and Lb2. They also show modulation of hemoglobin expression by NLP4 and NAC094.
Subject(s)
Lotus , Nitrates , Nitrates/metabolism , Lotus/physiology , Reactive Oxygen Species/metabolism , Hemoglobins/genetics , Hemoglobins/metabolism , Leghemoglobin/metabolism , Nitric Oxide/metabolism , Symbiosis , Root Nodules, Plant/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism , Gene Expression Regulation, PlantABSTRACT
Multidrug-resistant bacteria are spreading at alarming rates, and despite extensive efforts no new class of antibiotic with activity against Gram-negative bacteria has been approved in over fifty years. Natural products and their derivatives have a key role in combating Gram-negative pathogens. Here we report chemical optimization of the arylomycins-a class of natural products with weak activity and limited spectrum-to obtain G0775, a molecule with potent, broad-spectrum activity against Gram-negative bacteria. G0775 inhibits the essential bacterial type I signal peptidase, a new antibiotic target, through an unprecedented molecular mechanism. It circumvents existing antibiotic resistance mechanisms and retains activity against contemporary multidrug-resistant Gram-negative clinical isolates in vitro and in several in vivo infection models. These findings demonstrate that optimized arylomycin analogues such as G0775 could translate into new therapies to address the growing threat of multidrug-resistant Gram-negative infections.
Subject(s)
Anti-Bacterial Agents/classification , Anti-Bacterial Agents/pharmacology , Gram-Negative Bacteria/drug effects , Peptides, Cyclic/pharmacology , Biocatalysis/drug effects , Biological Products/classification , Biological Products/pharmacology , Drug Resistance, Multiple, Bacterial/drug effects , Escherichia coli/enzymology , Gram-Negative Bacteria/enzymology , Gram-Negative Bacteria/pathogenicity , Gram-Negative Bacterial Infections/drug therapy , Gram-Negative Bacterial Infections/microbiology , Klebsiella pneumoniae/drug effects , Klebsiella pneumoniae/enzymology , Klebsiella pneumoniae/pathogenicity , Lysine/metabolism , Membrane Proteins/antagonists & inhibitors , Microbial Sensitivity Tests , Peptides, Cyclic/chemistry , Porins , Protein Binding , Protein Domains , Serine Endopeptidases , Substrate SpecificityABSTRACT
Nitrogen (N) and phosphorus (P) are the most important macronutrients required for plant growth and development. To cope with the limited and uneven distribution of N and P in complicated soil environments, plants have evolved intricate molecular strategies to improve nutrient acquisition that involve adaptive root development, production of root exudates, and the assistance of microbes. Recently, great advances have been made in understanding the regulation of N and P uptake and utilization and how plants balance the direct uptake of nutrients from the soil with the nutrient acquisition from beneficial microbes such as arbuscular mycorrhiza. Here, we summarize the major advances in these areas and highlight plant responses to changes in nutrient availability in the external environment through local and systemic signals.
ABSTRACT
The ubiquitin system regulates essential cellular processes in eukaryotes. Ubiquitin is ligated to substrate proteins as monomers or chains and the topology of ubiquitin modifications regulates substrate interactions with specific proteins. Thus ubiquitination directs a variety of substrate fates including proteasomal degradation. Deubiquitinase enzymes cleave ubiquitin from substrates and are implicated in disease; for example, ubiquitin-specific protease-7 (USP7) regulates stability of the p53 tumour suppressor and other proteins critical for tumour cell survival. However, developing selective deubiquitinase inhibitors has been challenging and no co-crystal structures have been solved with small-molecule inhibitors. Here, using nuclear magnetic resonance-based screening and structure-based design, we describe the development of selective USP7 inhibitors GNE-6640 and GNE-6776. These compounds induce tumour cell death and enhance cytotoxicity with chemotherapeutic agents and targeted compounds, including PIM kinase inhibitors. Structural studies reveal that GNE-6640 and GNE-6776 non-covalently target USP7 12 Å distant from the catalytic cysteine. The compounds attenuate ubiquitin binding and thus inhibit USP7 deubiquitinase activity. GNE-6640 and GNE-6776 interact with acidic residues that mediate hydrogen-bond interactions with the ubiquitin Lys48 side chain, suggesting that USP7 preferentially interacts with and cleaves ubiquitin moieties that have free Lys48 side chains. We investigated this idea by engineering di-ubiquitin chains containing differential proximal and distal isotopic labels and measuring USP7 binding by nuclear magnetic resonance. This preferential binding protracted the depolymerization kinetics of Lys48-linked ubiquitin chains relative to Lys63-linked chains. In summary, engineering compounds that inhibit USP7 activity by attenuating ubiquitin binding suggests opportunities for developing other deubiquitinase inhibitors and may be a strategy more broadly applicable to inhibiting proteins that require ubiquitin binding for full functional activity.
Subject(s)
Aminopyridines/chemistry , Aminopyridines/pharmacology , Indazoles/chemistry , Indazoles/pharmacology , Phenols/chemistry , Phenols/pharmacology , Pyridines/chemistry , Pyridines/pharmacology , Ubiquitin-Specific Peptidase 7/antagonists & inhibitors , Ubiquitin/metabolism , Animals , Binding, Competitive , Cell Line, Tumor , Drug Synergism , Female , Humans , Mice , Mice, SCID , Models, Molecular , Neoplasms/drug therapy , Neoplasms/enzymology , Neoplasms/pathology , Protein Binding , Proto-Oncogene Proteins c-mdm2/metabolism , Proto-Oncogene Proteins c-pim-1/antagonists & inhibitors , Substrate Specificity , Tumor Suppressor Protein p53/deficiency , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Ubiquitin/chemistry , Ubiquitin-Specific Peptidase 7/chemistry , Ubiquitin-Specific Peptidase 7/deficiency , Ubiquitin-Specific Peptidase 7/metabolismABSTRACT
Visualization of root colonization by arbuscular mycorrhizal fungi (AMF) is the most elementary experiment in the field of mycorrhizal symbiosis. The most widely used approach for evaluating levels of AMF colonization is staining with trypan blue or ink, which is scored using the time-consuming grid intersection method. Here we demonstrate the use of an anthocyanin-based visual marker system for visualizing AMF colonization of Medicago truncatula roots. Expression of MtLAP1, a transcription factor which regulates the production of anthocyanins, from the AMF-induced Kunitz Protease Inhibitor 106 promoter, allowed the visualization of arbuscules in live plant tissues without microscopy or staining. This marker system allowed straightforward qualitative evaluation of the ram1, vpy and dmi3 AMF phenotypes using Agrobacterium rhizogenes hairy-root transformation. For the strigolactone biosynthesis mutant carotenoid cleavage dioxygenase 8a and a novel mutant scooby, which show quantitative AMF symbiotic phenotypes, the amount of anthocyanins in the roots estimated by spectrophotometry correlated very well with colonization levels estimated by staining and scoring using the grid intersection method. The LAP1-based marker system therefore provides a highly efficient approach for mutant screening and monitoring of AMF colonization in live tissues by eye, or for quantitative assessment using a simple and quick photometric assay.
Subject(s)
Medicago truncatula , Mycorrhizae , Medicago truncatula/microbiology , Mycorrhizae/physiology , Anthocyanins/metabolism , Plant Roots/metabolism , Symbiosis/physiology , PigmentationABSTRACT
Most land plants live in close contact with beneficial soil microbes: the majority of land plant species establish symbiosis with arbuscular mycorrhizal fungi, while most legumes, the third largest plant family, can form a symbiosis with nitrogen-fixing rhizobia. These microbes contribute to plant nutrition via endosymbiotic processes that require modulating the expression and function of plant transporter systems. The efficient contribution of these symbionts involves precisely controlled integration of transport, which is enabled by the adaptability and plasticity of their transporters. Advances in our understanding of these systems, driven by functional genomics research, are rapidly filling the gap in knowledge about plant membrane transport involved in these plant-microbe interactions. In this review, we synthesize recent findings associated with different stages of these symbioses, from the pre-symbiotic stage to nutrient exchange, and describe the role of host transport systems in both mycorrhizal and legume-rhizobia symbioses.
Subject(s)
Fabaceae/microbiology , Fabaceae/physiology , Membrane Transport Proteins/drug effects , Mycorrhizae/physiology , Nitrogen Fixation/physiology , Rhizobium/physiology , Symbiosis/physiology , Plant Roots/microbiology , Plant Roots/physiologyABSTRACT
Root hair cells form the primary interface of plants with the soil environment, playing key roles in nutrient uptake and plant defense. In legumes, they are typically the first cells to become infected by nitrogen-fixing soil bacteria during root nodule symbiosis. Here, we report a role for the CELLULOSE SYNTHASE-LIKE D1 (CSLD1) gene in root hair development in the legume species Lotus japonicus. CSLD1 belongs to the cellulose synthase protein family that includes cellulose synthases and cellulose synthase-like proteins, the latter thought to be involved in the biosynthesis of hemicellulose. We describe 11 Ljcsld1 mutant alleles that impose either short (Ljcsld1-1) or variable (Ljcsld1-2 to 11) root hair length phenotypes. Examination of Ljcsld1-1 and one variable-length root hair mutant, Ljcsld1-6, revealed increased root hair cell wall thickness, which in Ljcsld1-1 was significantly more pronounced and also associated with a strong defect in root nodule symbiosis. Lotus japonicus plants heterozygous for Ljcsld1-1 exhibited intermediate root hair lengths, suggesting incomplete dominance. Intragenic complementation was observed between alleles with mutations in different CSLD1 domains, suggesting CSLD1 function is modular and that the protein may operate as a homodimer or multimer during root hair development.
Subject(s)
Glucosyltransferases/genetics , Lotus/genetics , Plant Proteins/genetics , Plant Roots/growth & development , Glucosyltransferases/metabolism , Lotus/enzymology , Lotus/growth & development , Plant Proteins/metabolism , Plant Roots/geneticsABSTRACT
Lipoprotein diacylglyceryl transferase (Lgt) catalyzes the first step in the biogenesis of Gram-negative bacterial lipoproteins which play crucial roles in bacterial growth and pathogenesis. We demonstrate that Lgt depletion in a clinical uropathogenic Escherichia coli strain leads to permeabilization of the outer membrane and increased sensitivity to serum killing and antibiotics. Importantly, we identify G2824 as the first-described Lgt inhibitor that potently inhibits Lgt biochemical activity in vitro and is bactericidal against wild-type Acinetobacter baumannii and E. coli strains. While deletion of a gene encoding a major outer membrane lipoprotein, lpp, leads to rescue of bacterial growth after genetic depletion or pharmacologic inhibition of the downstream type II signal peptidase, LspA, no such rescue of growth is detected after Lgt depletion or treatment with G2824. Inhibition of Lgt does not lead to significant accumulation of peptidoglycan-linked Lpp in the inner membrane. Our data validate Lgt as a novel antibacterial target and suggest that, unlike downstream steps in lipoprotein biosynthesis and transport, inhibition of Lgt may not be sensitive to one of the most common resistance mechanisms that invalidate inhibitors of bacterial lipoprotein biosynthesis and transport. IMPORTANCE As the emerging threat of multidrug-resistant (MDR) bacteria continues to increase, no new classes of antibiotics have been discovered in the last 50 years. While previous attempts to inhibit the lipoprotein biosynthetic (LspA) or transport (LolCDE) pathways have been made, most efforts have been hindered by the emergence of a common mechanism leading to resistance, namely, the deletion of the gene encoding a major Gram-negative outer membrane lipoprotein lpp. Our unexpected finding that inhibition of Lgt is not susceptible to lpp deletion-mediated resistance uncovers the complexity of bacterial lipoprotein biogenesis and the corresponding enzymes involved in this essential outer membrane biogenesis pathway and potentially points to new antibacterial targets in this pathway.
Subject(s)
Escherichia coli/metabolism , Lipoproteins/metabolism , Transferases/metabolism , Animals , Anti-Bacterial Agents/pharmacology , Aspartic Acid Endopeptidases , Bacterial Proteins , Escherichia coli/genetics , Female , Gene Deletion , Gene Expression Regulation, Bacterial/drug effects , Mice , Peptidoglycan/metabolism , Transferases/chemistry , Transferases/genetics , Uropathogenic Escherichia coli/genetics , Uropathogenic Escherichia coli/metabolismABSTRACT
Several ATP-binding cassette (ABC) transporters involved in the arbuscular mycorrhizal symbiosis and nodulation have been identified. We describe three previously unreported ABC subfamily B transporters, named AMN1, AMN2, and AMN3 (ABCB for mycorrhization and nodulation), that are expressed early during infection by rhizobia and arbuscular mycorrhizal fungi. These ABCB transporters are strongly expressed in symbiotically infected tissues, including in root-hair cells with rhizobial infection threads and arbusculated cells. During nodulation, the expression of these genes is highly induced by rhizobia and purified Nod factors and is dependent on DMI3 but is not dependent on other known major regulators of infection, such as NIN, NSP1, or NSP2. During mycorrhization their expression is dependent on DMI3 and RAM1 but not on NSP1 and NSP2. Therefore, they may be commonly regulated through a distinct branch of the common symbiotic pathway. Mutants with exonic Tnt1-transposon insertions were isolated for all three genes. None of the single or double mutants showed any differences in colonization by either rhizobia or mycorrhizal fungi, but the triple amn1 amn2 amn3 mutant showed an increase in nodule number. Further studies are needed to identify potential substrates of these transporters and understand their roles in these beneficial symbioses.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.
Subject(s)
Medicago truncatula , Mycorrhizae , ATP-Binding Cassette Transporters/genetics , Gene Expression Regulation, Plant , Medicago truncatula/genetics , Medicago truncatula/metabolism , Mycorrhizae/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism , Plant Roots/metabolism , Signal Transduction , SymbiosisABSTRACT
CERBERUS (also known as LIN) and VAPYRIN (VPY) are essential for infection of legumes by rhizobia and arbuscular mycorrhizal fungi (AMF). Medicago truncatula LIN (MtLIN) was reported to interact with MtVPY, but the significance of this interaction is unclear and the function of VPY in Lotus japonicus has not been studied. We demonstrate that CERBERUS has auto-ubiquitination activity in vitro and is localized within distinct motile puncta in L. japonicus root hairs and in Nicotiana benthamiana leaves. CERBERUS colocalized with the trans-Golgi network/early endosome markers. In L. japonicus, two VPY orthologs (LjVPY1 and LjVPY2) were identified. CERBERUS interacted with and colocalized with both LjVPY1 and LjVPY2. Co-expression of CERBERUS with LjVPY1 or LjVPY2 in N. benthamiana led to increased protein levels of LjVPY1 and LjVPY2, which accumulated as mobile punctate bodies in the cytoplasm. Conversely, LjVPY2 protein levels decreased in cerberus roots after rhizobial inoculation. Mutant analysis indicates that LjVPY1 and LjVPY2 are required for rhizobial infection and colonization by AMF. Our data suggest that CERBERUS stabilizes LjVPY1 and LjVPY2 within the trans-Golgi network/early endosome, where they might function to regulate endocytic trafficking and/or the formation or recycling of signaling complexes during rhizobial and AMF symbiosis.
Subject(s)
Lotus , Rhizobium , Gene Expression Regulation, Plant , Lotus/genetics , Lotus/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism , Root Nodules, Plant/metabolism , SymbiosisABSTRACT
The formation of nitrogen-fixing no dules on legume roots requires the coordination of infection by rhizobia at the root epidermis with the initiation of cell divisions in the root cortex. During infection, rhizobia attach to the tip of elongating root hairs which then curl to entrap the rhizobia. However, the mechanism of root hair deformation and curling in response to symbiotic signals is still elusive. Here, we found that small GTPases (MtRac1/MtROP9 and its homologs) are required for root hair development and rhizobial infection in Medicago truncatula. Our results show that the Nod factor receptor LYK3 phosphorylates the guanine nucleotide exchange factor MtRopGEF2 at S73 which is critical for the polar growth of root hairs. In turn, phosphorylated MtRopGEF2 can activate MtRac1. Activated MtRac1 was found to localize at the tips of root hairs and to strongly interact with LYK3 and NFP. Taken together, our results support the hypothesis that MtRac1, LYK3, and NFP form a polarly localized receptor complex that regulates root hair deformation during rhizobial infection.
Subject(s)
Guanine Nucleotide Exchange Factors/metabolism , Medicago truncatula/enzymology , Plant Proteins/metabolism , Plant Root Nodulation , rac1 GTP-Binding Protein/metabolism , Medicago truncatula/growth & development , Medicago truncatula/microbiology , Phosphorylation , Plant Roots/enzymology , Plant Roots/growth & development , Plant Roots/microbiology , SymbiosisABSTRACT
Loss of barley Mildew Resistance Locus O (MLO) is known to confer durable and robust resistance to powdery mildew (Blumeria graminis), a biotrophic fungal leaf pathogen. Based on the increased expression of MLO in mycorrhizal roots and its presence in a clade of the MLO family that is specific to mycorrhizal-host species, we investigated the potential role of MLO in arbuscular mycorrhizal interactions. Using mutants from barley (Hordeum vulgare), wheat (Triticum aestivum), and Medicago truncatula, we demonstrate a role for MLO in colonization by the arbuscular mycorrhizal fungus Rhizophagus irregularis. Early mycorrhizal colonization was reduced in mlo mutants of barley, wheat, and M. truncatula, and this was accompanied by a pronounced decrease in the expression of many of the key genes required for intracellular accommodation of arbuscular mycorrhizal fungi. These findings show that clade IV MLOs are involved in the establishment of symbiotic associations with beneficial fungi, a role that has been appropriated by powdery mildew.