Plant-induced cell death in the oomycete pathogen Phytophthora parasitica

The activation of programmed cell death in the host during plant-pathogen interactions is an important component of the plant disease resistance mechanism. In this study we show that activation of programmed cell death in microorganisms also regulates plant-pathogen interactions. We found that a form of vacuolar cell death is induced in the oomycete Phytophthora parasitica--the agent that causes black shank disease in Nicotiana tabacum--by extracellular stimuli from resistant tobacco. The single-celled zoospores underwent cell death characterized by dynamic membrane rearrangements, cell shrinkage, formation of numerous large vacuoles in the cytoplasm and degradation of cytoplasmic components before plasma membrane disruption. Phytophthora cell death required protein synthesis but not caspase activation, and was associated with the production of intracellular reactive oxygen species. This characterization of plant-mediated cell death signalling in pathogens will enhance our understanding of the biological processes regulating plant-pathogen interactions, and improve our ability to control crop diseases.     

Nitric oxide signalling functions in plant-pathogen interactions

Nitric oxide (NO) is a highly reactive molecule that rapidly diffuses and permeates cell membranes. During the last few years NO has been detected in several plant species, and the increasing number of reports on its function in plants have implicated NO as a key molecular signal that participates in the regulation of several physiological processes; in particular, it has a significant role in plant resistance to pathogens by triggering resistance-associated cell death and by contributing to the local and systemic induction of defence genes. NO stimulates signal transduction pathways through protein kinases, cytosolic Ca2+ mobilization and protein modification (i.e. nitrosylation and nitration). In this review we will examine the synthesis of NO, its effects, functions and signalling giving rise to the hypersensitive response and systemic acquired resistance during plant-pathogen interactions.     

Regulators of cell death in disease resistance

Cell death and disease resistance are intimately connected in plants. Plant disease resistance genes (R genes) are key components in pathogen perception and have a potential to activate cell death pathways. Analysis of R proteins suggests common molecular mechanisms for pathogen recognition and signal emission whereas the subsequent signalling unexpectedly involves a network of pathways of parallel, branching and converging action. Disease resistance signalling mutants have revealed novel types of regulatory proteins whose biochemical functions are still unknown. Accumulation of small molecules such as salicylic acid, reactive oxygen intermediates, and nitric oxide amplifies resistance responses and directs cells to initiate cell death programs. Genetic analyses of lesion mimic mutants provide a glimpse of how cell death thresholds are set via an interplay of positive and negative regulatory components.     

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.     

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.     

Very long chain fatty acid and lipid signaling in the response of plants to pathogens

Recent findings indicate that lipid signaling is essential for plant resistance to pathogens. Besides oxylipins and unsaturated fatty acids known to play important signaling functions during plant-pathogen interactions, the very long chain fatty acid (VLCFA) biosynthesis pathway has been recently associated to plant defense through different aspects. VLCFAs are indeed required for the biosynthesis of the plant cuticle and the generation of sphingolipids. Elucidation of the roles of these lipids in biotic stress responses is the result of the use of genetic approaches together with the identification of the genes/proteins involved in their biosynthesis. This review focuses on recent observations which revealed the complex function of the cuticle and cuticle-derived signals, and the key role of sphingolipids as bioactive molecules involved in signal transduction and cell death regulation during plant-pathogen interactions.Key words: very long chain fatty acids (VLCFAs), plant-pathogen interactions, lipid signaling, sphingolipids, epicuticular waxes, lipid rafts, cuticle, plant defense     

ABA, hydrogen peroxide and nitric oxide signalling in stomatal guard cells

Increased synthesis and redistribution of the phytohormone abscisic acid (ABA) in response to water deficit stress initiates an intricate network of signalling pathways in guard cells leading to stomatal closure. Despite the large number of ABA signalling intermediates that are known in guard cells, new discoveries are still being made. Recently, the reactive oxygen species hydrogen peroxide (H2O2) and the reactive nitrogen species nitric oxide (NO) have been identified as key molecules regulating ABA-induced stomatal closure in various species. As with many other physiological responses in which H2O2 and NO are involved, stomatal closure in response to ABA also appears to require the tandem synthesis and action of both these signalling molecules. Recent pharmacological and genetic data have identified NADPH oxidase as a source of H2O2, whilst nitrate reductase has been identified as a source of NO in Arabidopsis guard cells. Some signalling components positioned downstream of H2O2 and NO are calcium, protein kinases and cyclic GMP. However, the exact interaction between the various signalling components in response to H2O2 and NO in guard cells remains to be established.     

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 signalling following plant/pathogen interactions involves the generation of reactive oxygen and reactive nitrogen species

It is now clear that reactive oxygen species (ROS), such as hydrogen peroxide (H₂O₂), and reactive nitrogen species, such as nitric oxide (NO), are produced by plant cells in response to a variety of stresses, including pathogen challenge. Such molecules may be involved in direct defence mechanisms, such as cross-linking of plant cell walls, or as antimicrobial agents. However, it is also apparent that cells generate such reactive species as signalling molecules, produced at controlled levels, and leading to defined responses. Signalling responses to ROS and NO include the activation of mitogen-activated protein kinases, and the up- and down-regulation of gene expression, often leading to localised programmed cell death, characteristic of the hypersensitive response. Therefore, ROS and NO are key molecules which may help to orchestrate events following pathogen challenge. Here we review the generation and role of both reactive oxygen and reactive nitrogen species in plant cells.     

Defence signalling pathways in cereals.

The combination of mutational and molecular studies has shed light on the role of reactive oxygen intermediates and programmed cell death in cereal disease resistance mechanisms. Rice Rac1 and barley Rar1 represent conserved disease resistance signalling genes, which may have related functions in animals. The analysis of non-pathogenic Magnaporthe grisea mutants may provide novel tools to study host defence pathways.     

Signalling in Rhizobacteria-Induced Systemic Resistance in Arabidopsis thaliana

Abstract: To protect themselves from disease, plants have evolved sophisticated defence mechanisms in which the signal molecules salicylic acid, jasmonic acid and ethylene often play crucial roles. Elucidation of signalling pathways controlling disease resistance is a major objective in research on plant-pathogen interactions. The capacity of a plant to develop a broad spectrum, systemic acquired resistance (SAR) after primary infection with a necrotizing pathogen is well-known and its signal transduction pathway extensively studied. Plants of which the roots have been colonized by specific strains of non-pathogenic fluorescent Pseudomonas spp. develop a phenotypically similar form of protection that is called rhizobacteria-mediated induced systemic resistance (ISR). In contrast to pathogen-induced SAR, which is regulated by salicylic acid, rhizobacteria-mediated ISR is controlled by a signalling pathway in which jasmonic acid and ethylene play key roles. In the past eight years, the model plant species Arabidopsis thaliana was explored to study the molecular basis of rhizobacteria-mediated ISR. Here we review current knowledge of the signal transduction steps involved in the ISR pathway that leads from recognition of the rhizobacteria in the roots to systemic expression of broad-spectrum disease resistance in aboveground foliar tissues.     

Reactive Oxygen Species during Plant-microorganism Early Interactions

Reactive Oxygen Species (ROS) are continuously produced as a result of aerobic metabolism or in response to biotic and abiotic stresses. ROS are not only toxic by-products of aerobic metabolism, but are also signalling molecules involved in several developmental processes in all organisms. Previous studies have clearly shown that an oxidative burst often takes place at the site of attempted invasion during the early stages of most plant-pathogen interac-tions. Moreover, a second ROS production can be observed during certain types of plant-pathogen interactions, which triggers hyper-sensitive cell death (HR). This second ROS wave seems absent during symbiotic interactions. This difference between these two responses is thought to play an important signalling role leading to the establishment of plant defense. In order to cope with the deleterious effects of ROS, plants are fitted with a large panel of enzymatic and non-enzymatic antioxidant mechanisms. Thus, increasing numbers of publications report the characterisation of ROS producing and scavenging systems from plants and from microorganisms during interactions. In this review, we present the current knowledge on the ROS signals and their role during plant-microorganism interactions.     

NO way back: nitric oxide and programmed cell death in Arabidopsis thaliana suspension cultures

Recent research has implicated nitric oxide (NO) in the induction of the hypersensitive response (HR) during plant-pathogen interactions. Here we demonstrate that Arabidopsis suspension cultures generate elevated levels of NO in response to challenge by avirulent bacteria, and, using NO donors, show that these elevated levels of NO are sufficient to induce cell death in Arabidopsis cells independently of reactive oxygen species (ROS). We also provide evidence that NO-induced cell death is a form of programmed cell death (PCD), requiring gene expression, and has a number of characteristics of PCD of mammalian cells: NO induced chromatin condensation and caspase-like activity in Arabidopsis cells, while the caspase-1 inhibitor, Ac-YVAD-CMK, blocked NO-induced cell death. A well-established second messenger mediating NO responses in mammalian cells is cGMP, produced by the enzyme guanylate cyclase. A specific inhibitor of guanylate cyclase blocked NO-induced cell death in Arabidopsis cells, and this inhibition was reversed by the cell-permeable cGMP analogue, 8Br-cGMP, although 8Br-cGMP alone did not induce cell death or potentiate NO-induced cell death. This suggests that cGMP synthesis is required but not sufficient for NO-induced cell death in Arabidopsis. In-gel protein kinase assays showed that NO activates a potential mitogen-activated protein kinase (MAPK), although a specific inhibitor of mammalian MAPK activation, PD98059, which blocked H2O2-induced cell death, did not inhibit the effects of NO.     

Infra-red thermography revealed a role for mitochondria in pre-symptomatic cooling during harpin-induced hypersensitive response

The establishment of Erwinia amylovora harpin-induced hypersensitive response (HR) in Nicotiana sylvestris was followed by infra-red thermography (IRT). Three to four hours after elicitation, the temperature decreased in the harpin-infiltrated zone associated to stomatal opening. The marked drop in temperature which reached 2 degrees C and preceded necrosis symptoms for several hours, is thus likely caused by higher transpiration. Neither of these effects was observed in a respiratory mutant, affected in complex I structure and function and over-expressing alternative oxidase, indicating that they are directly or indirectly mediated by mitochondrial function. However, as the HR establishment was similar in both wild type and mutant, cell death was either uncorrelated with the observed epidermal changes or occurred by a different signalling pathway in the two genotypes. IRT revealed a novel aspect of plant-pathogen interactions and could be applied to screen for mutants affected in elicitor signalling and/or for respiratory mutants.     

Effects of urate, a natural inhibitor of peroxynitrite-mediated toxicity, in the response of Arabidopsis thaliana to the bacterial pathogen Pseudomonas syringae

Urate, a natural peroxynitrite scavenger, has been used to investigate the possible role of peroxynitrite during plant-pathogen interactions. Urate greatly reduced lesion formation in Arabidopsis leaves treated with an abiotic peroxynitrite-generating system or with a peroxynitrite solution, indicating that it can act as an effective scavenger in planta. In the interaction with the avirulent Pseudomonas syringae pv. phaseolicola (avrRPM1+), cell death in the inoculated area was strongly reduced by urate, without compromising disease resistance. In contrast, urate promoted discrete cell death in response to an isogenic Pseudomonas syringae (avrRPM1-), which did not trigger an HR when inoculated alone, and it induced resistance and arrest of pathogen growth. Scavenging of peroxynitrite did not modify the response of Arabidopsis to an avirulent strain of Xanthomonas campestris pv campestris, that showed a high resistance to NO and peroxynitrite. Our data indicate that peroxynitrite plays a significant role in the responses of plants to Pseudomonas syringae.     

Signalling by extracellular nucleotides in health and disease

Nucleotides are released from all cells through regulated pathways or as a result of plasma membrane damage or cell death. Outside the cell, nucleotides act as signalling molecules triggering multiple responses via specific plasma membrane receptors of the P2 family. In the nervous system, purinergic signalling has a key function in neurotransmission. Outside the nervous system, purinergic signalling is one of the major modulators of basal tissue homeostasis, while its dysregulation contributes to the pathogenesis of various disease, including inflammation and cancer. Pre-clinical and clinical evidence shows that selective P2 agonists or antagonists are effective treatments for many pathologies, thus highlighting the relevance of extracellular nucleotides and P2 receptors as therapeutic targets.     

Changes in the antioxidant systems as part of the signaling pathway responsible for the programmed cell death activated by nitric oxide and reactive oxygen species in tobacco Bright-Yellow 2 cells

Nitric oxide (NO) has been postulated to be required, together with reactive oxygen species (ROS), for the activation of the hypersensitive reaction, a defense response induced in the noncompatible plant-pathogen interaction. However, its involvement in activating programmed cell death (PCD) in plant cells has been questioned. In this paper, the involvement of the cellular antioxidant metabolism in the signal transduction triggered by these bioactive molecules has been investigated. NO and ROS levels were singularly or simultaneously increased in tobacco (Nicotiana tabacum cv Bright-Yellow 2) cells by the addition to the culture medium of NO and/or ROS generators. The individual increase in NO or ROS had different effects on the studied parameters than the simultaneous increase in the two reactive species. NO generation did not cause an increase in phenylalanine ammonia-lyase (PAL) activity or induction of cellular death. It only induced minor changes in ascorbate (ASC) and glutathione (GSH) metabolisms. An increase in ROS induced oxidative stress in the cells, causing an oxidation of the ASC and GSH redox pairs; however, it had no effect on PAL activity and did not induce cell death when it was generated at low concentrations. In contrast, the simultaneous increase of NO and ROS activated a process of death with the typical cytological and biochemical features of hypersensitive PCD and a remarkable rise in PAL activity. Under the simultaneous generation of NO and ROS, the cellular antioxidant capabilities were also suppressed. The involvement of ASC and GSH as part of the transduction pathway leading to PCD is discussed.     

Molecular mechanisms of nitrogen dioxide induced epithelial injury in the lung

The lung can be exposed to a variety of reactive nitrogen intermediates through the inhalation of environmental oxidants and those produced during inflammation. Reactive nitrogen species (RNS) include, nitrogen dioxide (.NO2) and peroxynitrite (ONOO-). Classically known as a major component of both indoor and outdoor air pollution, .NO2 is a toxic free radical gas. .NO2 can also be formed during inflammation by the decomposition of ONOO- or through peroxidase-catalyzed reactions. Due to their reactive nature, RNS may play an important role in disease pathology. Depending on the dose and the duration of administration, .NO, has been documented to cause pulmonary injury in both animal and human studies. Injury to the lung epithelial cells following exposure to .NO2 is characterized by airway denudation followed by compensatory proliferation. The persistent injury and repair process may contribute to airway remodeling, including the development of fibrosis. To better understand the signaling pathways involved in epithelial cell death by .NO2 or otherRNS, we routinely expose cells in culture to continuous gas-phase .NO2. Studies using the .NO2 exposure system revealed that lung epithelial cell death occurs in a density dependent manner. In wound healing experiments, .NO2 induced cell death is limited to cells localized in the leading edge of the wound. Importantly, .NO2-induced death does not appear to be dependent on oxidative stress per se. Potential cell signaling mechanisms will be discussed, which include the mitogen activated protein kinase, c-Jun N-terminal Kinase and the Fas/Fas ligand pathways. During periods of epithelial loss and regeneration that occur in diseases such as asthma or during lung development, epithelial cells in the lung may be uniquely susceptible to death. Understanding the molecular mechanisms of epithelial cell death associated with the exposure to .NO2 will be important in designing therapeutics aimed at protecting the lung from persistent injury and repair.     

Cellular localization of ROS and NO in olive reproductive tissues during flower development

Background  

Recent studies have shown that reactive oxygen species (ROS) and nitric oxide (NO) are involved in the signalling processes taking place during the interactions pollen-pistil in several plants. The olive tree (Olea europaea L.) is an important crop in Mediterranean countries. It is a dicotyledonous species, with a certain level of self-incompatibility, fertilisation preferentially allogamous, and with an incompatibility system of the gametophytic type not well determined yet. The purpose of the present study was to determine whether relevant ROS and NO are present in the stigmatic surface and other reproductive tissues in the olive over different key developmental stages of the reproductive process. This is a first approach to find out the putative function of these signalling molecules in the regulation of the interaction pollen-stigma.     

Simulation of fungal-mediated cell death by fumonisin B1 and selection of fumonisin B1-resistant (fbr) Arabidopsis mutants

Fumonisin B1 (FB1), a programmed cell death-eliciting toxin produced by the necrotrophic fungal plant pathogen Fusarium moniliforme, was used to simulate pathogen infection in Arabidopsis. Plants infiltrated with 10 microM FB1 and seedlings transferred to agar media containing 1 microM FB1 develop lesions reminiscent of the hypersensitive response, including generation of reactive oxygen intermediates, deposition of phenolic compounds and callose, accumulation of phytoalexin, and expression of pathogenesis-related (PR) genes. Arabidopsis FB1-resistant (fbr) mutants were selected directly by sowing seeds on agar containing 1 microM FB1, on which wild-type seedlings fail to develop. Two mutants chosen for further analyses, fbr1 and fbr2, had altered PR gene expression in response to FB1. fbr1 and fbr2 do not exhibit differential resistance to the avirulent bacterial pathogen Pseudomonas syringae pv maculicola (ES4326) expressing the avirulence gene avrRpt2 but do display enhanced resistance to a virulent isogenic strain that lacks the avirulence gene. Our results demonstrate the utility of FB1 for high-throughput isolation of Arabidopsis defense-related mutants and suggest that pathogen-elicited programmed cell death of host cells may be an important feature of compatible plant-pathogen interactions.