Nodulation of Aeschynomene afraspera and A. indica by Photosynthetic Bradyrhizobium Sp. Strain ORS285: The Nod-Dependent Versus the Nod-Independent Symbiotic Interaction

Here, we present a comparative analysis of the nodulation processes of Aeschynomene afraspera and A. indica that differ in their requirement for Nod factors (NF) to initiate symbiosis with photosynthetic bradyrhizobia. The infection process and nodule organogenesis was examined using the green fluorescent protein–labeled Bradyrhizobium sp. strain ORS285 able to nodulate both species. In A. indica, when the NF-independent strategy is used, bacteria penetrated the root intercellularly between axillary root hairs and invaded the subepidermal cortical cells by invagination of the host cell wall. Whereas the first infected cortical cells collapsed, the infected ones immediately beneath kept their integrity and divided repeatedly to form the nodule. In A. afraspera, when the NF-dependent strategy is used, bacteria entered the plant through epidermal fissures generated by the emergence of lateral roots and spread deeper intercellularly in the root cortex, infecting some cortical cells during their progression. Whereas the infected cells of the lower cortical layers divided rapidly to form the nodule, the infected cells of the upper layers gave rise to an outgrowth in which the bacteria remained enclosed in large tubular structures. Together, two distinct modes of infection and nodule organogenesis coexist in Aeschynomene legumes, each displaying original features.

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Transformed Hairy Roots of Arachis hypogea: A Tool for Studying Root Nodule Symbiosis in a Non–Infection Thread Legume of the Aeschynomeneae Tribe

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-22-2-0132 ◽

2009 ◽

Vol 22 (2) ◽

pp. 132-142 ◽

Cited By ~ 37

Author(s):

Senjuti Sinharoy ◽

Sudip Saha ◽

Susanta Roy Chaudhury ◽

Maitrayee DasGupta

Keyword(s):

Hairy Roots ◽

Fluorescent Protein ◽

Root Hairs ◽

Infection Thread ◽

Natural Soil ◽

35S Promoter ◽

Wild Type ◽

Cortical Cells ◽

Arachis Hypogea ◽

Green Fluorescent

Arachis hypogea is a non–“infection thread” (IT) legume where rhizobial entry or dissemination in the nodules never involves IT. Rhizobia invade through epidermal “cracks” and directly access the cortical cells to develop the characteristic aeschynomenoid nodules. For investigating these nonclassical nodulation features in Arachis spp., we developed an efficient procedure for Agrobacterium rhizogenes R1000-mediated transformation of this plant. In this study, we optimized the induction of hairy roots and nodulation of composite Arachis hypogea plants in the presence of Bradyrhizobium sp. (Arachis) strain NC92. 35S promoter-driven green fluorescent protein and β-glucuronidase expression indicated transformation frequency to be above 80%. The transformed roots had the characteristic rosette-type root hairs and had normal level of expression of symbiosis-related genes SymRK and CCaMK. The transgenic nodules resembled the wild-type nodules with an exception of 2 to 3%, where they structurally deviated from the wild-type nodules to form nodular roots. A 16S rRNA profile of an infected-zone metagenome indicated that identical populations of bradyrhizobia invaded both composite wild-type plants grown in natural soil. Our results demonstrate that Arachis hairy root is an attractive system for undertaking investigations of the nonclassical features associated with its nitrogen-fixing symbiotic interactions.

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Invasion by Invitation: Rhizobial Infection in Legumes

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-08-10-0181 ◽

2011 ◽

Vol 24 (6) ◽

pp. 631-639 ◽

Cited By ~ 116

Author(s):

Jeremy D. Murray

Keyword(s):

Root Hair ◽

Root Hairs ◽

Infection Process ◽

Nodule Development ◽

Nod Factors ◽

Cortical Cells ◽

Root Cortex ◽

Root Hair Cell ◽

Cytoplasmic Bridges ◽

Cytoskeletal Rearrangements

Nodulation of legume roots typically begins with rhizobia attaching to the tip of a growing root-hair cell. The attached rhizobia secrete Nod factors (NF), which are perceived by the plant. This initiates a series of preinfection events that include cytoskeletal rearrangements, curling at the root-hair tip, and formation of radially aligned cytoplasmic bridges called preinfection threads (PIT) in outer cortical cells. Within the root-hair curl, an infection pocket filled with bacteria forms, from which originates a tubular invagination of cell wall and membrane called an infection thread (IT). IT formation is coordinated with nodule development in the underlying root cortex tissues. The IT extends from the infection pocket down through the root hair and into the root cortex, where it passes through PIT and eventually reaches the nascent nodule. As the IT grows, it is colonized by rhizobia that are eventually released into cells within the nodule, where they fix nitrogen. NF can also induce cortical root hairs that appear to originate from PIT and can become infected like normal root hairs. Several genes involved in NF signaling and some of the downstream transcription factors required for infection have been characterized. More recently, several genes with direct roles in infection have been identified, some with roles in actin rearrangement and others with possible roles in protein turnover and secretion. This article provides an overview of the infection process, including the roles of NF signaling, actin, and calcium and the influence of the hormones ethylene and cytokinin.

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Vaccinia Virus F13L Protein with a Conserved Phospholipase Catalytic Motif Induces Colocalization of the B5R Envelope Glycoprotein in Post-Golgi Vesicles

Journal of Virology ◽

10.1128/jvi.75.16.7528-7542.2001 ◽

2001 ◽

Vol 75 (16) ◽

pp. 7528-7542 ◽

Cited By ~ 35

Author(s):

Matloob Husain ◽

Bernard Moss

Keyword(s):

Vaccinia Virus ◽

Fluorescent Protein ◽

Plasmid Vector ◽

Recombinant Vaccinia Virus ◽

Open Reading Frames ◽

Type I ◽

Golgi Vesicles ◽

Infected Cells ◽

Catalytic Motif ◽

Green Fluorescent

ABSTRACT The wrapping of intracellular mature vaccinia virions by modifiedtrans-Golgi or endosomal cisternae to form intracellular enveloped virions is dependent on at least two viral proteins encoded by the B5R and F13L open reading frames. B5R is a type I integral membrane glycoprotein, whereas F13L is an unglycosylated, palmitylated protein with a motif that is conserved in a superfamily of phospholipid-metabolizing enzymes. Microscopic visualization of the F13L protein was achieved by fusing it to the enhanced green fluorescent protein (GFP). F13L-GFP was functional when expressed by a recombinant vaccinia virus in which it replaced the wild-type F13L gene or by transfection of uninfected cells with a plasmid vector followed by infection with an F13L deletion mutant. In uninfected or infected cells, F13L-GFP was associated with Golgi cisternae and post-Golgi vesicles containing the LAMP 2 late endosomal-lysosomal marker. Association of F13L-GFP with vesicles was dependent on an intact phospholipase catalytic motif and sites of palmitylation. The B5R protein was also associated with LAMP2-containing vesicles when F13L-GFP was coexpressed, but was largely restricted to Golgi cisternae in the absence of F13L-GFP or when the F13L moiety was mutated. We suggest that the F13L protein, like its human phospholipase D homolog, regulates vesicle formation and that this process is involved in intracellular enveloped virion membrane formation.

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Confocal Analysis of the Distribution and Persistence of Sindbis Virus (TaV-GFP) Infection in Midguts of Aedes aegypti Mosquitoes

Microscopy and Microanalysis ◽

10.1017/s1431927620001270 ◽

2020 ◽

Vol 26 (2) ◽

pp. 267-274

Author(s):

Jason J. Saredy ◽

Florence Y. Chim ◽

Zoë L. Lyski ◽

Yani P. Ahearn ◽

Doria F. Bowers

Keyword(s):

Aedes Aegypti ◽

Sindbis Virus ◽

Fluorescent Protein ◽

Blood Feeding ◽

Target Tissue ◽

Secondary Target ◽

Posterior Midgut ◽

Infected Cells ◽

Green Fluorescent ◽

Global Threat

AbstractBiological transmission of arthropod-borne viruses (arboviruses) to vertebrate hosts by hematophagous insects poses a global threat because such arboviruses can result in a range of serious public health infectious diseases. Sindbis virus (SINV), the prototype Alphavirus, was used to track infections in the posterior midgut (PMG) of Aedes aegypti adult mosquitoes. Females were fed viremic blood containing a virus reporter, SINV [Thosea asigna virus-green fluorescent protein (TaV-GFP)], that leaves a fluorescent signal in infected cells. We assessed whole-mount PMGs to identify primary foci, secondary target tissues, distribution, and virus persistence. Following a viremic blood meal, PMGs were dissected and analyzed at various days of post blood-feeding. We report that virus foci indicated by GFP in midgut epithelial cells resulted in a 9.8% PMG infection and a 10.8% dissemination from these infected guts. The number of virus foci ranged from 1 to 3 per individual PMG and was more prevalent in the PMG-middle > PMG-frontal > PMG-caudal regions. SINV TaV-GFP was first observed in the PMG (primary target tissue) at 3 days post blood-feeding, was sequestered in circumscribed foci, replicated in PMG peristaltic muscles (secondary target tissue) following dissemination, and GFP was observed to persist in PMGs for 30 days postinfection.

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Nature and consequences of interactions between Salmonella enterica serovar Dublin and host cells in cattle

Veterinary Research ◽

10.1186/s13567-019-0720-5 ◽

2019 ◽

Vol 50 (1) ◽

Cited By ~ 1

Author(s):

Prerna Vohra ◽

Christina Vrettou ◽

Jayne C. Hope ◽

John Hopkins ◽

Mark P. Stevens

Keyword(s):

Lymph Nodes ◽

Salmonella Enterica ◽

Fluorescent Protein ◽

Host Cells ◽

Large Animal ◽

Ileal Mucosa ◽

Zoonotic Infections ◽

Infected Cells ◽

Green Fluorescent ◽

Salmonella Enterica Serovar Dublin

AbstractSalmonella enterica is a veterinary and zoonotic pathogen of global importance. While murine and cell-based models of infection have provided considerable knowledge about the molecular basis of virulence of Salmonella, relatively little is known about salmonellosis in naturally-affected large animal hosts such as cattle, which are a reservoir of human salmonellosis. As in humans, Salmonella causes bovine disease ranging from self-limiting enteritis to systemic typhoid-like disease and exerts significant economic and welfare costs. Understanding the nature and consequences of Salmonella interactions with bovine cells will inform the design of effective vaccines and interventions to control animal and zoonotic infections. In calves challenged orally with S. Dublin expressing green fluorescent protein (GFP) we observed that the bacteria were predominantly extracellular in the distal ileal mucosa and within gut-associated lymph nodes 48 h post-infection. Intracellular bacteria, identified by flow cytometry using the GFP signal, were predominantly within MHCII+ macrophage-like cells. In contrast to observations from murine models, these S. Dublin-infected cells had elevated levels of MHCII and CD40 compared to both uninfected cells from the same tissue and cells from the cognate tissue of uninfected animals. Moreover, no gross changes of the architecture of infected lymph nodes were observed as was described previously in a mouse model. In order to further investigate Salmonella-macrophage interactions, net replication of S. enterica serovars that differ in virulence in cattle was measured in bovine blood-derived macrophages by enumeration of gentamicin-protected bacteria and fluorescence dilution, but did not correlate with host-specificity.

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High Physiological Levels of LMP1 Result in Phosphorylation of eIF2α in Epstein-Barr Virus-Infected Cells

Journal of Virology ◽

10.1128/jvi.78.4.1657-1664.2004 ◽

2004 ◽

Vol 78 (4) ◽

pp. 1657-1664 ◽

Cited By ~ 48

Author(s):

Ngan Lam ◽

Mark L. Sandberg ◽

Bill Sugden

Keyword(s):

Gene Expression ◽

Epstein Barr Virus ◽

Fluorescent Protein ◽

Transmembrane Domain ◽

Barr Virus ◽

Signaling Domain ◽

Infected Cells ◽

Epstein Barr ◽

Green Fluorescent ◽

Carboxy Terminal

ABSTRACT LMP1 is an Epstein-Barr virus (EBV)-encoded membrane protein essential for the proliferation of EBV-infected lymphoblasts (E. Kilger, A. Kieser, M. Baumann, and W. Hammerschmidt, EMBO J. 17:1700-1709, 1998). LMP1 also inhibits gene expression and induces cytostasis in transfected cells when it is expressed at levels as little as twofold higher than the average for EBV-positive lymphoblasts (M. Sandberg, A. Kaykas, and B. Sugden, J. Virol. 74:9755-9761, 2000; A. Kaykas and B. Sugden, Oncogene 19:1400-1410, 2000). We have found that in three different clones of EBV-infected lymphoblasts the levels of expression of LMP1 in individual cells in each clone ranged over 100-fold. This difference is due to a difference in levels of the LMP1 transcript. In these clones, cells expressing high levels of LMP1 incorporated less BrdU. We also found that induction of expression of LMP1 or of a derivative of LMP1 with its transmembrane domain fused to green fluorescent protein instead of its carboxy-terminal signaling domain resulted in phosphorylation of eIF2α in EBV-negative Burkitt's lymphoma cells. This induction of phosphorylation of eIF2α was also detected in EBV-infected lymphoblasts, in which high levels of LMP1 correlated with high levels of phosphorylation of eIF2α. Our results indicate that inhibition of gene expression and of cell proliferation by LMP1 occurs normally in EBV-infected cells.

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Novel Real-Time Monitoring System for Human Cytomegalovirus- Infected Cells In Vitro That Uses a Green Fluorescent Protein-PML-Expressing Cell Line

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01641-05 ◽

2006 ◽

Vol 50 (8) ◽

pp. 2806-2813 ◽

Cited By ~ 10

Author(s):

T. Ueno ◽

Y. Eizuru ◽

H. Katano ◽

T. Kurata ◽

T. Sata ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Cell Line ◽

Real Time ◽

Human Cytomegalovirus ◽

Hcmv Infection ◽

Fluorescent Protein ◽

Clinical Isolates ◽

Pml Bodies ◽

Infected Cells ◽

Green Fluorescent

ABSTRACT Promyelocytic leukemia (PML) bodies are discrete nuclear foci that are intimately associated with many DNA viruses. In human cytomegalovirus (HCMV) infection, the IE1 (for “immediate-early 1”) protein has a marked effect on PML bodies via de-SUMOylation of PML protein. Here, we report a novel real-time monitoring system for HCMV-infected cells using a newly established cell line (SE/15) that stably expresses green fluorescent protein (GFP)-PML protein. In SE/15 cells, HCMV infection causes specific and efficient dispersion of GFP-PML bodies in an IE1-dependent manner, allowing the infected cells to be monitored by fluorescence microscopy without immunostaining. Since a specific change in the detergent solubility of GFP-PML occurs upon infection, the infected cells can be quantified by GFP fluorescence measurement after extraction. With this assay, the inhibitory effects of heparin and neutralizing antibodies were determined in small-scale cultures, indicating its usefulness for screening inhibitory reagents for laboratory virus strains. Furthermore, we established a sensitive imaging assay by counting the number of nuclei containing dispersed GFP-PML, which is applicable for titration of slow-growing clinical isolates. In all strains tested, the virus titers estimated by the GFP-PML imaging assay were well correlated with the plaque-forming cell numbers determined in human embryonic lung cells. Coculture of SE/15 cells and HCMV-infected fibroblasts permitted a rapid and reliable method for estimating the 50% inhibitory concentration values of drugs for clinical isolates in susceptibility testing. Taken together, these results demonstrate the development of a rapid, sensitive, quantitative, and specific detection system for HCMV-infected cells involving a simple procedure that can be used for titration of low-titer clinical isolates.

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Ontogenèse des nodules caulinaires du Sesbania rostrata (légumineuses)

Canadian Journal of Botany ◽

10.1139/b84-137 ◽

1984 ◽

Vol 62 (5) ◽

pp. 982-994 ◽

Cited By ~ 40

Author(s):

E. Duhoux

Keyword(s):

Sesbania Rostrata ◽

Cortical Cells ◽

Root Cortex ◽

Central Mass ◽

Intracellular Release ◽

Infection Threads ◽

Bacterial Penetration ◽

Infected Cells ◽

Determinate Growth

Stem nodules of the legume Sesbania rostrata are ovoids, contain chlorophyll and have determinate growth. They possess a large central mass of infected cells. Stem mamillae are regularly arranged in vertical files along the stem and develop into nodules when they are infected by a specific Rhizobium. Each nodule arises from the development of an infected region of the incipient root cortex. The infection in S. rostrata has been shown to proceed in four sequential stages. Some of them have never been shown to occur in other legumes: (i) bacterial penetration takes place in degenerated (dead) cortical cells; (ii) proliferation of the bacteria occurs in the intercellular cavities and initiates a meristematic nodule; (iii) protusion of infection threads at first occurs intercellularly and then intracellularly from the cavities; (iv) finally there is an intracellular release of Rhizobia by an endocytotic process.

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Ubiquitous Carbohydrate Binding Modules Decorate 936 Lactococcal Siphophage Virions

Viruses ◽

10.3390/v11070631 ◽

2019 ◽

Vol 11 (7) ◽

pp. 631 ◽

Cited By ~ 7

Author(s):

Stephen Hayes ◽

Jennifer Mahony ◽

Renaud Vincentelli ◽

Laurie Ramond ◽

Arjen Nauta ◽

...

Keyword(s):

Fluorescent Protein ◽

Bioinformatic Analysis ◽

Infection Process ◽

Ecological Niches ◽

Carbohydrate Binding ◽

Binding Assays ◽

Protein Fusion ◽

Carbohydrate Binding Modules ◽

Structural Assembly ◽

Green Fluorescent

With the availability of an increasing number of 3D structures of bacteriophage components, combined with powerful in silico predictive tools, it has become possible to decipher the structural assembly and functionality of phage adhesion devices. In the current study, we examined 113 members of the 936 group of lactococcal siphophages, and identified a number of Carbohydrate Binding Modules (CBMs) in the neck passage structure and major tail protein, on top of evolved Dit proteins, as recently reported by us. The binding ability of such CBM-containing proteins was assessed through the construction of green fluorescent protein fusion proteins and subsequent binding assays. Two CBMs, one from the phage tail and another from the neck, demonstrated definite binding to their phage-specific host. Bioinformatic analysis of the structural proteins of 936 phages reveals that they incorporate binding modules which exhibit structural homology to those found in other lactococcal phage groups and beyond, indicating that phages utilize common structural “bricks” to enhance host binding capabilities. The omnipresence of CBMs in Siphophages supports their beneficial role in the infection process, as they can be combined in various ways to form appendages with different shapes and functionalities, ensuring their success in host detection in their respective ecological niches.

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ROMK1 (Kir1.1) Causes Apoptosis and Chronic Silencing of Hippocampal Neurons

Journal of Neurophysiology ◽

10.1152/jn.2000.84.2.1062 ◽

2000 ◽

Vol 84 (2) ◽

pp. 1062-1075 ◽

Cited By ~ 62

Author(s):

H. Nadeau ◽

S. McKinney ◽

D. J. Anderson ◽

H. A. Lester

Keyword(s):

Cell Death ◽

Hippocampal Neurons ◽

Fluorescent Protein ◽

Input Resistance ◽

Firing Frequency ◽

Compensatory Mechanisms ◽

Spike Shape ◽

Infected Cells ◽

Green Fluorescent ◽

Potential Threshold

Lentiviral vectors were constructed to express the weakly rectifying kidney K+ channel ROMK1 (Kir1.1), either fused to enhanced green fluorescent protein (EGFP) or as a bicistronic message (ROMK1-CITE-EGFP). The channel was stably expressed in cultured rat hippocampal neurons. Infected cells were maintained for 2–4 wk without decrease in expression level or evidence of viral toxicity, although 15.4 mM external KCl was required to prevent apoptosis of neurons expressing functional ROMK1. No other trophic agents tested could prevent cell death, which was probably caused by K+loss. This cell death did not occur in glia, which were able to support ROMK1 expression indefinitely. Functional ROMK1, quantified as the nonnative inward current at −144 mV in 5.4 mM external K+blockable by 500 μM Ba2+, ranged from 1 to 40 pA/pF. Infected neurons exhibited a Ba2+-induced depolarization of 7 ± 2 mV relative to matched EGFP-infected controls, as well as a 30% decrease in input resistance and a shift in action potential threshold of 2.6 ± 0.5 mV. This led to a shift in the relation between injected current and firing frequency, without changes in spike shape, size, or timing. This shift, which quantifies silencing as a function of ROMK1 expression, was predicted from Hodgkin-Huxley models. No cellular compensatory mechanisms in response to expression of ROMK1 were identified, making ROMK1 potentially useful for transgenic studies of silencing and neurodegeneration, although its lethality in normal K+ has implications for the use of K+ channels in gene therapy.

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