Inter-organellar communication is vital for successful innate immune responses that confer defense against pathogens. However, little is known about how chloroplasts, which are a major production ...site of pro-defense molecules, communicate and coordinate with other organelles during defense. Here we show that chloroplasts send out dynamic tubular extensions called stromules during innate immunity or exogenous application of the pro-defense signals, hydrogen peroxide (H2O2) and salicylic acid. Interestingly, numerous stromules surround nuclei during defense response, and these connections correlate with an accumulation of chloroplast-localized NRIP1 defense protein and H2O2 in the nucleus. Furthermore, silencing and knockout of chloroplast unusual positioning 1 (CHUP1) that encodes a chloroplast outer envelope protein constitutively induces stromules in the absence of pathogen infection and enhances programmed cell death. These results support a model in which stromules aid in the amplification and/or transport of pro-defense signals into the nucleus and other subcellular compartments during immunity.
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•Chloroplast stromules are induced during plant immune responses•Pro-PCD signals such as SA and H2O2 induce stromules•Stromules form dynamic connections with nucleus during immune responses•Constitutively induced stromules enhance PCD during plant immune responses
Caplan et al. show that dynamic tubular projections from chloroplasts called stromules are induced during a plant immune response. Stromules form connections with the nucleus and may aid in the transport of pro-defense signals. Stromules enhance programmed cell death and may facilitate its progression during an immune response.
Bacterial cells are surrounded by a polymer known as peptidoglycan (PG), which protects the cell from changes in osmotic pressure and small molecule insults. A component of this material, ...N-acetyl-muramic acid (NAM), serves as a core structural element for innate immune recognition of PG fragments. We report the synthesis of modifiable NAM carbohydrate derivatives and the installation of these building blocks into the backbone of Gram-positive and Gram-negative bacterial PG utilizing metabolic cell wall recycling and biosynthetic machineries. Whole cells are labelled via click chemistry and visualized using super-resolution microscopy, revealing higher resolution PG structural details and allowing the cell wall biosynthesis, as well as its destruction in immune cells, to be tracked. This study will assist in the future identification of mechanisms that the immune system uses to recognize bacteria, glean information about fundamental cell wall architecture and aid in the design of novel antibiotics.
The origin and early evolution of sex chromosomes have been hypothesized to involve the linkage of factors with antagonistic effects on male and female function. Garden asparagus (
) is an ideal ...species to investigate this hypothesis, as the X and Y chromosomes are cytologically homomorphic and evolved from an ancestral autosome pair in association with a shift from hermaphroditism to dioecy. Mutagenesis screens paired with single-molecule fluorescence in situ hybridization directly implicate Y-specific genes that respectively suppress female (pistil) development and are necessary for male (anther) development. Comparison of contiguous X and Y chromosome assemblies shows that hemizygosity underlies the loss of recombination between the genes suppressing female organogenesis (
) and promoting male function (
). We also experimentally demonstrate the function of
These findings provide direct evidence that sex chromosomes can function through linkage of two sex determination genes.
During plant-pathogen interactions, the plant may mount several types of defense responses to either block the pathogen completely or ameliorate the amount of disease. Such responses include release ...of reactive oxygen species (ROS) to attack the pathogen, as well as formation of cell wall appositions (CWAs) to physically block pathogen penetration. A successful pathogen will likely have its own ROS detoxification mechanisms to cope with this inhospitable environment. Here, we report one such candidate mechanism in the rice blast fungus, Magnaporthe oryzae, governed by a gene we refer to as MoHYR1. This gene (MGG_07460) encodes a glutathione peroxidase (GSHPx) domain, and its homologue in yeast was reported to specifically detoxify phospholipid peroxides. To characterize this gene in M. oryzae, we generated a deletion mutantΔhyr1 which showed growth inhibition with increased amounts of hydrogen peroxide (H₂O₂). Moreover, we observed that the fungal mutants had a decreased ability to tolerate ROS generated by a susceptible plant, including ROS found associated with CWAs. Ultimately, this resulted in significantly smaller lesion sizes on both barley and rice. In order to determine how this gene interacts with other (ROS) scavenging-related genes in M. oryzae, we compared expression levels of ten genes in mutant versus wild type with and without H₂O₂. Our results indicated that the HYR1 gene was important for allowing the fungus to tolerate H₂O₂ in vitro and in planta and that this ability was directly related to fungal virulence.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Plant small RNAs are 21-24 nucleotide, noncoding RNAs that function as regulators in plant growth and development. Colorimetric detection of plant small RNAs was made possible with the introduction ...of locked nucleic acid probes. However, fluorescent detection of plant small RNAs has been challenging due to the high autofluorescence from plant tissue. Here we report a fluorescent in situ detection method for plant small RNAs. This method can be applied to most plant samples and tissue types and also can be adapted for single-molecule detection of small RNAs with super-resolution microscopy.
Plant innate immunity is mediated by Resistance (R) proteins, which bear a striking resemblance to animal molecules of similar function. Tobacco N is a TIR-NB-LRR R gene that confers resistance to ...Tobacco mosaic virus, specifically the p50 helicase domain. An intriguing question is how plant R proteins recognize the presence of pathogen-derived Avirulence (Avr) elicitor proteins. We have used biochemical cell fraction and immunoprecipitation in addition to confocal fluorescence microscopy of living tissue to examine the association between N and p50. Surprisingly, both N and p50 are cytoplasmic and nuclear proteins, and N's nuclear localization is required for its function. We also demonstrate an in planta association between N and p50. Further, we show that N's TIR domain is critical for this association, and indeed, it alone can associate with p50. Our results differ from current models for plant innate immunity that propose detection is mediated solely through the LRR domains of these molecules. The data we present support an intricate process of pathogen elicitor recognition by R proteins involving multiple subcellular compartments and the formation of multiple protein complexes.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Plant–microbe interactions Park, Eunsook; Nedo, Alexander; Caplan, Jeffrey L. ...
The New phytologist,
02/2018, Letnik:
217, Številka:
3
Journal Article
Recenzirano
Odprti dostop
Plants have evolved a multilayered immune system with well-orchestrated defense strategies against pathogen attack. Multiple immune signaling pathways, coordinated by several subcellular compartments ...and interactions between these compartments, play important roles in a successful immune response. Pathogens use various strategies to either directly attack the plant’s immune system or to indirectly manipulate the physiological status of the plant to inhibit an immune response. Microscopy-based approaches have allowed the direct visualization of membrane trafficking events, cytoskeleton reorganization, subcellular dynamics and interorganellar communication during the immune response. Here, we discuss the contributions of organelles and the cytoskeleton to the plant’s defense response against microbial pathogens, as well as the mechanisms used by pathogens to target these compartments to overcome the plant’s defense barrier.
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•Interface-scale colloid mobilization was studied theoretically and experimentally.•Conditions for mobilization via lifting, sliding, and rolling were developed.•Colloid and substrate ...contact angles were used as mobilization criteria.•Drainage and imbibition have intrinsic differences affecting colloid mobilization.•Trailing films and colloid aggregates affect accuracy of theoretical analysis.
Understanding colloid mobilization during transient flow in soil is important for addressing colloid and contaminant transport issues. While theoretical descriptions of colloid detachment exist for saturated systems, corresponding mechanisms of colloid mobilization during drainage and imbibition have not been considered in detail. In this work, theoretical force and torque analyses were performed to examine the interactive effects of adhesion, drag, friction, and surface tension forces on colloid mobilization and to outline conditions corresponding to the mobilization mechanisms such as lifting, sliding, and rolling. Colloid and substrate contact angles were used as variables to determine theoretical criteria for colloid mobilization mechanisms during drainage and imbibition. Experimental mobilization of hydrophilic and hydrophobic microspheres with drainage and imbibition fronts was investigated in hydrophilic and hydrophobic channels using a confocal microscope. Colloid mobilization differed between drainage and imbibition due to different dynamic contact angles and interfacial geometries on the contact line. Experimental results did not fully follow the theoretical criteria in all cases, which was explained with additional factors not included in the theory such as presence of aggregates and trailing films. Theoretical force and torque analyses resulted in similar mobilization predictions and suggested that all mobilization mechanisms contributed to the observed colloid mobilization.
Described is the spatiotemporally controlled labeling and patterning of biomolecules in live cells through the catalytic activation of bioorthogonal chemistry with light, referred to as “CABL”. Here, ...an unreactive dihydrotetrazine (DHTz) is photocatalytically oxidized in the intracellular environment by ambient O2 to produce a tetrazine that immediately reacts with a trans-cyclooctene (TCO) dienophile. 6-(2-Pyridyl)dihydrotetrazine-3-carboxamides were developed as stable, cell permeable DHTz reagents that upon oxidation produce the most reactive tetrazines ever used in live cells with Diels–Alder kinetics exceeding k 2 of 106 M–1 s–1. CABL photocatalysts are based on fluorescein or silarhodamine dyes with activation at 470 or 660 nm. Strategies for limiting extracellular production of singlet oxygen are described that increase the cytocompatibility of photocatalysis. The HaloTag self-labeling platform was used to introduce DHTz tags to proteins localized in the nucleus, mitochondria, actin, or cytoplasm, and high-yielding subcellular activation and labeling with a TCO-fluorophore were demonstrated. CABL is light-dose dependent, and two-photon excitation promotes CABL at the suborganelle level to selectively pattern live cells under no-wash conditions. CABL was also applied to spatially resolved live-cell labeling of an endogenous protein target by using TIRF microscopy to selectively activate intracellular monoacylglycerol lipase tagged with DHTz-labeled small molecule covalent inhibitor. Beyond spatiotemporally controlled labeling, CABL also improves the efficiency of “ordinary” tetrazine ligations by rescuing the reactivity of commonly used 3-aryl-6-methyltetrazine reporters that become partially reduced to DHTzs inside cells. The spatiotemporal control and fast rates of photoactivation and labeling of CABL should enable a range of biomolecular labeling applications in living systems.
We report that two different bacterial organisms engage in heterologous cell fusion that leads to massive exchange of cellular material, including proteins and RNA, and the formation of persistent ...hybrid cells. The interspecies cell fusion observed here involves a syntrophic microbial system, but these heterologous cell fusions were observed even under nonstrict syntrophic conditions, leaving open the possibility that strict syntrophy may not be necessary for interspecies cell fusion and cellular material exchange. Formation of hybrid cells that contain proteins and RNA from both organisms is unexpected and unprecedented. Such fusion events are likely widely distributed in nature, but have gone undetected. The implications are profound and may shed light onto many unexplained phenomena in human health, natural environments, evolutionary biology, and biotechnology.
ABSTRACT
Microbial syntrophy is universal in nature, profoundly affecting the composition and function of microbiomes. We have recently reported data suggesting direct cell-to-cell interactions leading to electron and material exchange between the two microbes in the syntrophy between
Clostridium ljungdahlii
and
C. acetobutylicum
. Here, transmission electron microscopy and electron tomography demonstrated cell wall and membrane fusions between the two organisms, whereby
C. ljungdahlii
appears to invade
C. acetobutylicum
pole to pole. Correlative fluorescence transmission electron microscopy demonstrated large-scale exchange of proteins. Flow cytometry analysis captured the extent and dynamic persistence of these interactions. Dividing hybrid cells were identified containing stained proteins from both organisms, thus demonstrating persistence of cells with exchanged cellular components. Fluorescence microscopy and flow cytometry of one species with stained RNA and the other tagged with a fluorescent protein demonstrated extensive RNA exchange and identified hybrid cells, some of which continued to divide, while some were in an advanced
C. acetobutylicum
sporulation form. These data demonstrate that cell fusion enables large-scale cellular material exchange between the two organisms. Although unanticipated and never previously reported, these phenomena are likely widely distributed in nature, have profound implications for species evolution and the function of microbial communities, and could find utility in biotechnology. They may shed new light onto little-understood phenomena, such as antibiotic heteroresistance of pathogens, pathogen invasion of human tissues, and the evolutionary trajectory and persistence of unculturable bacteria.
IMPORTANCE
We report that two different bacterial organisms engage in heterologous cell fusion that leads to massive exchange of cellular material, including proteins and RNA, and the formation of persistent hybrid cells. The interspecies cell fusion observed here involves a syntrophic microbial system, but these heterologous cell fusions were observed even under nonstrict syntrophic conditions, leaving open the possibility that strict syntrophy may not be necessary for interspecies cell fusion and cellular material exchange. Formation of hybrid cells that contain proteins and RNA from both organisms is unexpected and unprecedented. Such fusion events are likely widely distributed in nature, but have gone undetected. The implications are profound and may shed light onto many unexplained phenomena in human health, natural environments, evolutionary biology, and biotechnology.
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