HSR203 antisense suppression in tobacco accelerates development of hypersensitive cell death

Activation of the tobacco gene hsr203 is rapid, highly localized, specific for incompatible plant-pathogen interactions, and strongly correlated with programmed cell death occurring in response to diverse pathogens. Functional characterization of hsr203 gene product has shown that HSR203 is a serine hydrolase that displays esterase activity. We show here that transgenic tobacco plants deficient in HSR203 protein exhibit an accelerated hypersensitive response when inoculated with an avirulent strain of Ralstonia solanacearum. This response was accompanied by a maximal level of cell death and a drastic inhibition of in planta bacterial growth. Transgenic plants deficient in HSR203 were also found to show increased resistance in a dosage-dependent manner to Pseudomonas syringae pv. pisi, another avirulent bacterial pathogen, and to virulent and avirulent races of Phytophthora parasitica, a fungal pathogen of tobacco, but not to different virulent bacteria. Surprisingly, expression of another hsr gene, hsr515, and that of the defence genes PR1-a and PR5, was strongly reduced in the transgenic lines. Our results suggest that hsr203 antisense suppression in tobacco can have pleiotropic effects on HR cell death and defence mechanisms, and induces increased resistance to different pathogens.     

Cell death by necrosis, a regulated way to go

Apoptosis is a programmed form of cell death with well-defined morphological traits that are often associated with activation of caspases. More recently evidence has become available demonstrating that upon caspase inhibition alternative programs of cell death are executed, including ones with features characteristic of necrosis. These findings have changed our view of necrosis as a passive and essentially accidental form of cell death to that of an active, regulated and controllable process. Also necrosis has now been observed in parallel with, rather than as an alternative pathway to, apoptosis. Thus, cell death responses are extremely flexible despite being programmed. In this review, some of the hallmarks of different programmed cell death modes have been highlighted before focusing the discussion on necrosis. Obligatory events associated with this form of cell death include uncompensated cell swelling and related changes at the plasma membrane. In this context, representatives of the transient receptor channel family and their regulation are discussed. Also mechanisms that lead to execution of the necrotic cell death program are highlighted. Emphasis is laid on summarizing our understanding of events that permit switching between cell death modes and how they connect to necrosis. Finally, potential implications for the treatment of some disease states are mentioned.     

Red light imaging for programmed cell death visualization and quantification in plant–pathogen interactions

Studies on plant–pathogen interactions often involve monitoring disease symptoms or responses of the host plant to pathogen-derived immunogenic patterns, either visually or by staining the plant tissue. Both these methods have limitations with respect to resolution, reproducibility, and the ability to quantify the results. In this study we show that red light detection by the red fluorescent protein (RFP) channel of a multipurpose fluorescence imaging system that is probably available in many laboratories can be used to visualize plant tissue undergoing cell death. Red light emission is the result of chlorophyll fluorescence on thylakoid membrane disassembly during the development of a programmed cell death process. The activation of programmed cell death can occur during either a hypersensitive response to a biotrophic pathogen or an apoptotic cell death triggered by a necrotrophic pathogen. Quantifying the intensity of the red light signal enables the magnitude of programmed cell death to be evaluated and provides a readout of the plant immune response in a faster, safer, and nondestructive manner when compared to previously developed chemical staining methodologies. This application can be implemented to screen for differences in symptom severity in plant–pathogen interactions, and to visualize and quantify in a more sensitive and objective manner the intensity of the plant response on perception of a given immunological pattern. We illustrate the utility and versatility of the method using diverse immunogenic patterns and pathogens.     

Streptococcus pyogenes induces oncosis in macrophages through the activation of an inflammatory programmed cell death pathway

Macrophages are crucial components of the host defence against Streptococcus pyogenes . Here, we demonstrate the ability of S. pyogenes to kill macrophages through the activation of an inflammatory programmed cell death pathway. Macrophages exposed to S. pyogenes exhibited extensive cytoplasmic vacuolization, cellular and organelle swelling and rupture of the plasma membrane typical of oncosis. The cytotoxic effect of S. pyogenes on macrophages is mediated by the streptococcal cytolysins streptolysin S and streptolysin O and does not require bacterial internalization. S. pyogenes -induced death of macrophages was not affected by the addition of osmoprotectant, implicating the activation of an orchestrated cell death pathway rather than a simple osmotic lysis. This programme cell death pathway involves the loss of mitochondria transmembrane potential (Δ ψ _m) and was inhibited by the addition of exogenous glycine, which has been shown to prevent necrotic cell death by blocking the opening of death channels in the plasma membrane. The production of reactive oxygen species and activation of calpains were identified as mediators of the cell death process. We conclude that activation of the inflammatory programmed cell death pathway in macrophages could constitute an important pathogenic mechanism by which S. pyogenes evades host immune defences and causes disease.     

Hypersensitive response-related death

The hypersensitive response (HR) of plants resistant to microbial pathogens involves a complex form of programmed cell death (PCD) that differs from developmental PCD in its consistent association with the induction of local and systemic defence responses. Hypersensitive cell death is commonly controlled by direct or indirect interactions between pathogen avirulence gene products and those of plant resistance genes and it can be the result of multiple signalling pathways. Ion fluxes and the generation of reactive oxygen species commonly precede cell death, but a direct involvement of the latter seems to vary with the plant-pathogen combination. Protein synthesis, an intact actin cytoskeleton and salicylic acid also seem necessary for cell death induction. Cytological studies suggest that the actual mode and sequence of dismantling the cell contents varies among plant-parasite systems although there may be a universal involvement of cysteine proteases. It seems likely that cell death within the HR acts more as a signal to the rest of the plant rather than as a direct defence mechanism.     

Cell death mediated by MAPK is associated with hydrogen peroxide production in Arabidopsis.

Rapid and localized programmed cell death, known as the hypersensitive response (HR) is frequently associated with plant disease resistance. In contrast to our knowledge about the regulation and execution of apoptosis in animal system, information about plant HR is limited. Recent studies implicated the mitogen-activated protein kinase (MAPK) cascade in regulating plant HR cell death as well as several other defense responses during incompatible interactions between plants and pathogens. Here, we report the generation of transgenic Arabidopsis plants that express the active mutants of AtMEK4 and AtMEK5, two closely related MAPK kinases under the control of a steroid-inducible promoter. Induction of the transgene expression by the application of dexamethasone, a steroid, leads to HR-like cell death, which is preceded by the activation of endogenous MAPKs and the generation of hydrogen peroxide. Both prolonged MAPK activation and reactive oxygen species generation have been implicated in the regulation of HR cell death induced by incompatible pathogens. As a result, we speculate that the prolonged activation of the MAPK pathway in cells could disrupt the redox balance, which leads to the generation of reactive oxygen species and eventually HR cell death.     

Nitric oxide function and signalling in plant disease resistance

Nitric oxide (NO) is one of only a handful of gaseous signalling molecules. Its discovery as the endothelium-derived relaxing factor (EDRF) by Ignarro revolutionized how NO and cognate reactive nitrogen intermediates, which were previously considered to be toxic molecules, are viewed. NO is now emerging as a key signalling molecule in plants, where it orchestrates a plethora of cellular activities associated with growth, development, and environmental interactions. Prominent among these is its function in plant hypersensitive cell death and disease resistance. While a number of sources for NO biosynthesis have been proposed, robust and biologically relevant routes for NO production largely remain to be defined. To elaborate cell death during an incompatible plant-pathogen interaction NO functions in combination with reactive oxygen intermediates. Furthermore, NO has been shown to regulate the activity of metacaspases, evolutionary conserved proteases that may be intimately associated with pathogen-triggered cell death. NO is also thought to function in multiple modes of plant disease resistance by regulating, through S-nitrosylation, multiple nodes of the salicylic acid (SA) signalling pathway. These findings underscore the key role of NO in plant-pathogen interactions.     

Pyroptosis in Kidney Disease

In the last several decades, apoptosis interference has been considered clinically irrelevant in the context of renal injury. Recent discovery of programmed necrotic cell death, including necroptosis, ferroptosis, and pyroptosis refreshed our understanding of the role of cell death in kidney disease. Pyroptosis is characterized by a lytic pro- inflammatory type of cell death resulting from gasdermin-induced membrane permeabilization via activation of inflammatory caspases and inflammasomes. The danger-associated molecular patterns (DAMPs), alarmins and pro-inflammatory cytokines are released from pyroptotic cells in an uncontrolled manner, which provoke inflammation, resulting in secondary organ or tissue injuries. The caspases and inflammasome activation-related proteins and pore-forming effector proteins known as GSDMD and GSDME have been implicated in a variety of acute and chronic microbial and non-microbial kidney diseases. Here, we review the recent advances in pathological mechanisms of pyroptosis in kidney disease and highlight the potential therapeutic strategies in future.     

GSDMD介导的细胞焦亡在感染性疾病中的研究进展

细胞焦亡是细胞感染时由炎症小体介导,以裂解细胞为特点的程序性死亡形式。其激活途径分为依赖半胱氨酸蛋白酶-1或半胱氨酸蛋白酶-4/5/11活化的经典与非经典途径。目前的研究表明细胞焦亡过程中主要效应蛋白是具有膜成孔活性的gasdermin(也作GSDM)家族成员。因此,细胞焦亡也被称为gasdermin介导的程序性坏死。当宿主受到感染时,细胞焦亡与宿主自身其他免疫防御机制存在互相调节机制,保证宿主在清除感染的同时降低自身损伤程度。本文笔者将从研究最为广泛的GSDMD在细胞焦亡途径中的作用机制、细胞焦亡在感染性疾病中的研究进展以及细胞焦亡与其他程序性死亡在感染性疾病中的相互作用这三个方面作系统叙述,期望为今后研究如何通过细胞焦亡途径治疗感染性疾病提供理论基础。     

VPEgamma exhibits a caspase-like activity that contributes to defense against pathogens

BACKGROUND: Caspases are a family of aspartate-specific cysteine proteases that play an essential role in initiating and executing programmed cell death (PCD) in metazoans. Caspase-like activities have been shown to be required for the initiation of PCD in plants, but the genes encoding those activities have not been identified. VPEgamma, a cysteine protease, is induced during senescence, a form of PCD in plants, and is localized in precursor protease vesicles and vacuoles, compartments associated with PCD processes in plants. RESULTS: We show that VPEgamma binds in vivo to a general caspase inhibitor and to caspase-1-specific inhibitors, which block the activity of VPEgamma. A cysteine protease inhibitor, cystatin, accumulates to 20-fold higher levels in vpegamma mutants. Homologs of cystatin are known to suppress hypersensitive cell death in plant and animal systems. We also report that infection with an avirulent strain of Pseudomonas syringae results in an increase of caspase-1 activity, and this increase is partially suppressed in vpegamma mutants. Plants overexpressing VPEgamma exhibit a greater amount of ion leakage during infection with P. syringae, suggesting that VPEgamma may regulate cell death progression during plant-pathogen interaction. VPEgamma expression is induced after infection with P. syringae, Botrytis cinerea, and turnip mosaic virus, and knockout of VPEgamma results in increased susceptibility to these pathogens. CONCLUSIONS: We conclude that VPEgamma is a caspase-like enzyme that has been recruited in plants to regulate vacuole-mediated cell dismantling during cell death, a process that has significant influence in the outcome of a diverse set of plant-pathogen interactions.     

Hyperosmotic stress induces metacaspase- and mitochondria-dependent apoptosis in Saccharomyces cerevisiae

During the last years, several reports described an apoptosis-like programmed cell death process in yeast in response to different environmental aggressions. Here, evidence is presented that hyperosmotic stress caused by high glucose or sorbitol concentrations in culture medium induces in Saccharomyces cerevisiae a cell death process accompanied by morphological and biochemical indicators of apoptotic programmed cell death, namely chromatin condensation along the nuclear envelope, mitochondrial swelling and reduction of cristae number, production of reactive oxygen species and DNA strand breaks, with maintenance of plasma membrane integrity. Disruption of AIF1 had no effect on cell survival, but lack of Yca1p drastically reduced metacaspase activation and decreased cell death indicating that this death process was associated to activation of this protease. Supporting the involvement of mitochondria and cytochrome c in caspase activation, the mutant strains cyc1Deltacyc7Delta and cyc3Delta, both lacking mature cytochrome c, displayed a decrease in caspase activation associated to increased cell survival when exposed to hyperosmotic stress. These findings indicate that hyperosmotic stress triggers S. cerevisiae into an apoptosis-like programmed cell death that is mediated by a caspase-dependent mitochondrial pathway partially dependent on cytochrome c.     

肠道病毒71型感染诱导细胞程序性死亡形态学及分子生物学特征的初步研究

肠道病毒 71型(enterovirus type 71,EV71)感染常可引起婴幼儿手足口病(hand,foot and mouth disease,HFMD),还可引起中枢神经系统并发症等重症,甚至死亡。研究认为,EV71诱发重症的原因主要与病毒感染诱导细胞程序性死亡(programmed cell death,PCD)及诱导细胞产生大量炎症因子有关。病毒感染可通过激活不同的信号通路触发细胞程序性死亡,主要包括含半胱氨酸的天冬氨酸蛋白水解酶(cysteinyl aspartate specific proteinase,caspase)依赖的细胞凋亡、细胞焦亡,以及非caspase依赖的细胞坏死性凋亡。本研究旨在探讨EV71感染诱导细胞程序性死亡的形态学和分子生物学特征,利用显微镜和免疫荧光技术检测EV71感染后细胞形态变化,JC-1染色检测感染后细胞线粒体膜电位变化,流式细胞术及Annexin V-FITC/PI双染法、乳酸脱氢酶释放量法检测感染细胞的细胞膜损伤程度,结合蛋白免疫印迹法检测病毒感染后细胞中多聚ADP核糖聚合酶[poly(ADP-ribose) polymerase,PARP]、caspase-9、caspase-3等凋亡因子,以及细胞焦亡关键效应蛋白Gasdermin D、坏死性凋亡效应蛋白MLKL的磷酸化情况。结果显示,EV71感染后细胞主要呈现凋亡特征,并伴随少量细胞坏死。与细胞凋亡相关的PARP被剪切,caspase-9和caspase-3等相关因子被激活。经泛caspase抑制剂处理后,细胞程序性死亡被抑制,但仍有部分细胞坏死。结果提示,EV71感染以细胞凋亡为主,也可能存在非caspase依赖的细胞程序性死亡。     

Genetic elucidation of nitric oxide signaling in incompatible plant-pathogen interactions

Recent experiments indicate that nitric oxide (NO) plays a pivotal role in disease resistance and several other physiological processes in plants. However, most of the current information about the function of NO in plants is based on pharmacological studies, and additional approaches are therefore required to ascertain the role of NO as an important signaling molecule in plants. We have expressed a bacterial nitric oxide dioxygenase (NOD) in Arabidopsis plants and/or avirulent Pseudomonas syringae pv tomato to study incompatible plant-pathogen interactions impaired in NO signaling. NOD expression in transgenic Arabidopsis resulted in decreased NO levels in planta and attenuated a pathogen-induced NO burst. Moreover, NOD expression in plant cells had very similar effects on plant defenses compared to NOD expression in avirulent Pseudomonas. The defense responses most affected by NO reduction during the incompatible interaction were decreased H(2)O(2) levels during the oxidative burst and a blockage of Phe ammonia lyase expression, the key enzyme in the general phenylpropanoid pathway. Expression of the NOD furthermore blocked UV light-induced Phe ammonia lyase and chalcone synthase gene expression, indicating a general signaling function of NO in the activation of the phenylpropanoid pathway. NO possibly functions in incompatible plant-pathogen interactions by inhibiting the plant antioxidative machinery, and thereby ensuring locally prolonged H(2)O(2) levels. Additionally, albeit to a lesser extent, we observed decreases in salicylic acid production, a diminished development of hypersensitive cell death, and a delay in pathogenesis-related protein 1 expression during these NO-deficient plant-pathogen interactions. Therefore, this genetic approach confirms that NO is an important regulatory component in the signaling network of plant defense responses.     

Apoptosis of human T-cells: induction by glucocorticoids or surface receptor ligation in vitro and ex vivo.

Activated T-cells are susceptible to induction of apoptosis or programmed cell death in response to ligation of several cell surface structures, including CD2, CD3, and CD95/Fas. These mechanisms may be important in the regulation of immune responses and in prevention of autoimmunity. We used flow cytometric quantitation of DNA strand breaks to detect T-cells committed to programmed cell death. Activated human peripheral blood T-lymphocytes, and freshly isolated human thymocytes underwent apoptosis when exposed to dexamethasone or to monoclonal antibodies directed at CD2 or CD3. Interleukin-2 reduced spontaneous or dexamethasone-induced apoptosis, but augmented apoptosis due to ligation of CD2. A neutralizing anti-Fas antibody reduced the amount of DNA strand breakage, not only in T-cells exposed to antibodies to CD2 or CD3, but also in dexamethasone-treated cultures. In vivo activated T-cells, from inflammatory synovial fluids, were sensitive to immediate induction of DNA strand breaks without prior in vitro activation by lectin and IL-2. Taken together, the results indicated that: 1. Human lymphocytes, like murine thymocytes, are sensitive to glucocorticoid-induced apoptosis, as well as to programmed cell death triggered through surface receptors; 2. The effects of IL-2 on T-cell apoptosis depend on the apoptotic stimulus; 3. Fas/Fas ligand interactions may be relevant for both membrane receptor and glucocorticoid-induced cell death; and 4. Induction of T-cell apoptosis may be important in therapeutic effects of glucocorticoids in human disease.     

NO way to live; the various roles of nitric oxide in plant-pathogen interactions

Nitric oxide has attracted considerable interest from plant pathologists due its established role in regulating mammalian anti-microbial defences, particularly via programmed cell death (PCD). Although NO plays a major role in plant PCD elicited in response to certain types of pathogenic challenge, the race-specific hypersensitive response (HR), it is now evident that NO also acts in the regulation of non-specific, papilla-based resistance to penetration by plant cells that survive attack and, possibly, in systemic acquired resistance. Equally, the potential roles of NO signalling/scavenging within the pathogen are being recognized. This review will consider key defensive roles played by NO in living cells during plant-pathogen interactions, as well as in those undergoing PCD.     

Two vacuole-mediated defense strategies in plants

As plants lack immune cells, each cell has to defend itself against invading pathogens. Plant cells have a large central vacuole that accumulates a variety of hydrolytic enzymes and antimicrobial compounds, raising the possibility that vacuoles play a role in plant defense. However, how plants use vacuoles to protect against invading pathogens is poorly understood. Recently, we characterized two vacuole-mediated defense strategies associated with programmed cell death (PCD). In one strategy, vacuolar processing enzyme (VPE) mediated the disruption of the vacuolar membrane, resulting in the release of vacuolar contents into the cytoplasm in response to viral infection. In the other strategy, proteasome-dependent fusion of the central vacuole with the plasma membrane caused the discharge of vacuolar antibacterial protease and cell death-promoting contents from the cell in response to bacterial infection. Intriguingly, both strategies relied on enzymes with caspase-like activities: the vacuolar membrane-collapse system required VPE, which has caspase-1-like activity and the membrane-fusion system required a proteasome that has caspase-3-like activity. Thus, plants may have evolved a cellular immune system that involves vacuolar membrane collapse to prevent the systemic spread of viral pathogens and membrane fusion to inhibit the proliferation of bacterial pathogens.Key words: plant-pathogen interaction, vacuole, hypersensitive cell death, caspase activity, vacuolar processing enzyme, proteasome     

Structural Characterizations of the Fas Receptor and the Fas-Associated Protein with Death Domain Interactions

The Fas receptor is a representative death receptor, and the Fas-associated protein with death domain (FADD) is a crucial adapter protein needed to support the Fas receptor’s activity. The Fas–FADD interactions constitute an important signaling pathway that ultimately induces apoptosis or programmed cell death in biological systems. The interactions responsible for this cell-death process are governed by the binding process of the Fas ligand to the Fas, followed by the caspase cascade activation. Using a computational approach, the present communication explores certain essential structural aspects of the Fas–FADD death domains and their interfacial interactions.