Staying alive – is cell death dispensable for plant disease resistance during the hypersensitive response?

Probably the most known and best studied type of plant resistance to pathogenic infections is the hypersensitive response (HR), a form of localized programmed cell death associated with restriction or killing of pathogens that often leads to macroscopically visible localized tissue necrosis. It is generally assumed that cell death and resistance within the HR are physiologically and genetically linked. However, there has been considerable speculation about whether cell death is an absolute requirement for resistance conditioned by the HR. This review discusses the relation of cell death and resistance in the HR, in particular, the importance of cell death in this process. We intend to focus on the increasing amount of research evidence showing that in several plant-pathogen interactions, the two main components of the HR – resistance and cell death – can be physiologically, genetically and temporally uncoupled. In other words, HR should be considered as a combination of resistance and cell death responses, where cell death may be dispensable for plant disease resistance. The varying contribution of these two components (i.e. cell death and resistance) generates an array of defense strategies differing in efficiency. Thus, a very early and rapid defense response seems to contribute to the development of macroscopically symptomless (extreme) resistance, while a moderately early defense response results in resistance with the concomitant development of controlled and limited cell and tissue death (HR). Accordingly, a delayed and failed attempt by the host to elicit resistance responses would result in massively stressed plant tissues (e.g. “systemic HR”) and a partial or almost complete loss of control over pathogen invasion. The dynamic nature of resistance responses in plants implies that resistance can be effective with or without cell death but its outcome and efficiency may depend primarily on the timing and speed of the host response.     

Bacterial Lipopolysaccharides And Plant—pathogen Interactions

Lipopolysaccharides are amphipathic molecules forming the outermost layer of the cell surface of Gram-negative bacteria. They are essential for protecting the cell from hostile environments and, in the case of pathogens, they play a direct role in interactions with eukaryotic host cells. Mutants with altered lipopolysaccharide structure have been obtained with several plant pathogenic bacteria; such mutants generally show reduced virulence. Purified lipopolysaccharide has several effects on plants, notably suppression of the hypersensitive response to subsequently inoculated avirulent pathogens. The suppression is strictly localized and is observed within a time window of, typically, 10–30 h. Although infiltration of lipopolysaccharide into leaves produces no macroscopic symptoms, characteristic changes in plant gene expression can be observed. One effect is to sensitize the plant tissue to subsequent bacterial inoculation so that the sensitized tissue responds more rapidly and intensely, giving partial inhibition of bacterial growth. The synthesis of antimicrobial hydroxycinnamoyl tyramine conjugates is one facet of the process which provides an excellent biochemical model for analysing the phenomenon. Lipopolysaccharide induces the synthesis of two enzymes involved in conjugate production (tyrosine decarboxylase and tyramine-hydroxycinnamoyl transferase), but the conjugates themselves are not produced until bacteria are subsequently inoculated. Using this and other examples we discuss the mechanisms of lipopolysaccharide action on plants in the context of plant disease.     

Depolymerization of the actin cytoskeleton induces defense responses in tobacco plants

Tobacco leaf sections were treated with actin inhibitors, i.e., cytochalasins, to determine the effects of actin depolymerization on tobacco defense responses. Inoculation of the leaf sections with the pathogen Erysiphe cichoracearum, depolymerized the actin cytoskeleton, priming the cells for a hypersensitive response-like cell death. Further, expression of the acidic PR1 and PR2 genes were induced in cytochalasin-treated leaf sections. The intensity of the cytochalasin effects on the defense responses was closely correlated with the extent of actin depolymerization. This suggests that plant cells may perceive perturbation of the actin cytoskeleton, and this stimulus may trigger plant defense responses.     

<Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-dimethylsphingosine,an inhibitor of sphingosine kinase,induces phytoalexin production and hypersensitive cell death of Solanaceae plants without generation of reactive oxygen species

Plant recognition of elicitors derived from pathogens induces various resistant reactions, including production of reactive oxygen species, hypersensitive cell death and accumulation of phytoalexins. Previously, we isolated a ceramide elicitor from Phytophthora infestans, which activates O₂ ⁻ production of potato suspension-cultured cells. In this study, we employed nine ceramide-related chemicals to test their elicitor activity. Although, none of the tested chemicals induced O₂ ⁻ production, N,N-dimethylsphingosine (DMS) induced accumulation of phytoalexin in potato tubers. In potato, tobacco and Nicotiana benthamiana, DMS also induced rapid cell death. DMS-treated potato cells stained with 4′,6-diamidino-2-phenylindole (DAPI) showed chromatin condensation, and isolated DNA from DMS-treated cells had ladder pattern, confirming that DMS-induced plant cell death is a hypersensitive reaction-like programmed cell death. Involvement of ceramide signaling in induction of plant defense reactions is discussed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Engineered Resistance Against Fungal Plant Pathogens

Development of genetic engineering technology and molecular characterization of plant defense responses have provided strategies for controlling plant diseases additional to those based on chemical control or classical breeding programs. Most of these alternative strategies are based on the overproduction of one component of the plant's own defense response. Some strategies exploit the hypersensitive response, a rapid, localized death of tissue surrounding the infection site, which is observed in many resistant plants upon unsuccessful pathogen attack. Most approaches to increase resistance to fungi have been described to be successful under laboratory conditions. Incorporation of these successful, alternative strategies in resistance breeding programs of agriculturally important crops will depend on the results obtained from field experiments.     

Involvement of nitric oxide generation in hypersensitive cell death induced by elicitin in tobacco cell suspension culture

Recent studies suggest that nitric oxide (NO), an important signaling and defense molecule in mammals, plays a key role in activating disease resistance in plants. We characterized NO production by tobacco Bright Yellow-2 cells pharmacologically after treatment with INF1, the major elicitin secreted by the late blight pathogen Phytophthora infestans, prepared from Escherichia coli. NO production rapidly occurred within 1h and reached a maximum level 3–6h after the addition of INF1. Carboxy-PTIO, a NO-specific scavenger, abolished INF1-induced NO production in a dose-dependent manner. Pretreatment of protein synthesis inhibitor cycloheximide and protein kinase inhibitor K252a blocked NO production 3–12h after INF1 treatment, indicating that NO production requires de novo protein synthesis and protein phosphorylation. In an investigation of the relations between NO generation and several defense responses induced by INF1, carboxy-PTIO completely suppressed activation of a 41-kDa protein kinase and cell death by INF1. Carboxy-PTIO also suppressed the induction of hypersensitive-related (hsr) genes HSR515 and HSR203J, the expression of which is strongly correlated with the hypersensitive response in plants. The results suggest that NO plays a crucial role in the induction of hypersensitive cell death.     

A New Cell Wall Located N-rich Protein is Strongly Induced During the Hypersensitive Response in Glycine Max L.

Soybean (Glycine max (L.) Merill, cv. Williams 82) plants and cell cultures respond to avirulent pathogens with a hypersensitive reaction. After inoculation of soybean with Pseudomonas syringae pv. glycinea, carrying the avirulence gene avrA, or zoospores from the fungus Phytophthora sojae Race 1, a resistance-gene-dependent cell death programme is activated. A new gene was identified by differential display of mRNAs that is specifically activated during the early phase of incompatible pathogen-soybean interactions but does not respond to compatible pathogens. The gene is strongly induced within 2h after addition of P. sojae zoospores. A similar kinetic pattern was observed for P. syringae (avrA) inoculated soybean cell cultures. The gene encodes a deduced protein of 368 amino acids with a very high content of asparagine and was therefore termed N-rich protein (NRP). The protein is composed of two distinct domains, of which only the C-terminal domain has striking homology to proteins of unknown function from other plants. An antibody raised against the recombinant NRP recognizes a protein of 42kDa. The protein is located in the cell wall as indicated by cell fractionation studies. Comparison of the genomic DNA-sequence with the cDNA, identified two introns within the open reading frame. The NRP-gene is not directly induced by salicylic acid or hydrogen peroxide, indicating a distinct and specific signal transduction pathway which is only activated during programmed cell death. The NRP-gene appears to be a new marker in soybean activated early in plant disease resistance.     

N-terminal domain including conserved flg22 is required for flagellin-induced hypersensitive cell death in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Flagellin in Pseudomonas syringae is a potent elicitor of defense responses including hypersensitive cell death in dicot plants. The oligopeptides flg22 consisting of 22 conserved amino acids near the N-terminus of flagellins is reported to induce plant defense responses. Because glycosylation of the central domain of flagellin affects its elicitor activity, we investigated whether any peptide sequence in addition to flg22 is required for flagellin-induced hypersensitive reaction. A study of recombinant flagellin polypeptides indicated that the N-terminal domain including the conserved flg22 is required for flagellin-induced hypersensitive cell death in Arabidopsis thaliana.     

Selbst oder Nicht-Selbst �C die Rezeptoren des pflanzlichen Immunsystems

Plants recognize conserved molecular structures from microorganisms, which triggers active immune responses. Successful pathogens have to overcome this level of immunity; however, plants in turn can adapt their immune system, thus plants and pathogens are in an evolutionary arms race. As being sessile organisms, plants need to integrate and adapt to changing environmental conditions such as light, temperature, drought, or microorganisms. Plants protect themselves against diseases through sensitive recognition of potential pathogens and effective defense systems. The first level of the plant immune system provides recognition of a broad spectrum of microorganisms leading to defense activation (Bittel and Robatzek 2007). The second level of the plant immunity allows certain plant cultivars to detect of specific pathogen strains??a phenomenon also referred to as ??gene-for-gene resistance?? (Jones and Dangl 2006). The first level of immunity occurs rapidly and triggers active defenses normally without harm to the plant cell. The second level of plant immunity develops over days and deploys a local cell death, which prevents pathogens from further spread into tissues. In addition to these cell-autonomous defense systems, plants have also evolved strategies of systemic immunity.     

<Emphasis Type="Italic">N</Emphasis>-acyl-homoserine lactone-mediated quorum sensing blockage,a novel strategy for attenuating pathogenicity of Gram-negative bacterial plant pathogens

Quorum sensing is a bacterial communication mechanism by which bacteria sense their own population size and couple specific gene expression to cell density. In Gram-negative bacteria, the most commonly used quorum sensing signals are N-acyl homoserine lactones (AHLs). It is now apparent that many pathogenic bacteria employ quorum sensing to control premature expression of virulence factors. This control is thought to decrease the likelihood that the plant host would detect the pathogens presence and activate its defense system. Novel strategies that target bacterial quorum sensing systems in order to control plant bacterial diseases are discussed.     

The oligopeptide elicitor Pep-13 induces salicylic acid-dependent and -independent defense reactions in potato

The Phytophthora-derived oligopeptide elicitor, Pep-13, originally identified as an inducer of plant defense in the nonhost–pathogen interaction of parsley and Phytophthora sojae, triggers defense responses in potato. In cultured potato cells, Pep-13 treatment results in an oxidative burst and activation of defense genes. Infiltration of Pep-13 into leaves of potato plants induces the accumulation of hydrogen peroxide, defense gene expression and the accumulation of jasmonic and salicylic acids. Derivatives of Pep-13 show similar elicitor activity in parsley and potato, suggesting a receptor-mediated induction of defense response in potato similar to that observed in parsley. However, unlike in parsley, infiltration of Pep-13 into leaves leads to the development of hypersensitive response-like cell death in potato. Interestingly, Pep-13-induced necrosis formation, hydrogen peroxide formation and accumulation of jasmonic acid, but not activation of a subset of defense genes, is dependent on salicylic acid, as shown by infiltration of Pep-13 into leaves of potato plants unable to accumulate salicylic acid. Thus, in a host plant of Phytophthora infestans, Pep-13 is able to elicit salicylic acid-dependent and -independent defense responses.     

Characterisation of the saponin hydrolysing enzyme avenacoside-α-l-rhamnosidase from the fungal pathogen of cereals, Stagonospora avenae

The fungal pathogen Stagonospora avenae f. sp. avenaria infects oat leaves, which contain the saponins avenacoside A and B. The avenacosides are glycosylated steroidal saponins that occur within oat leaves in a non-fungitoxic form and are converted upon damage or pathogen invasion to their antifungal form by a plant enzyme. It has previously been shown that oat-attacking isolates of S. avenae are able to hydrolyse the sugar chain at the C3 position of the avenacosides. This carbohydrate moiety is a branched chain that consists of one -{scL}-rhamnose and two or three -{scD}-glucose residues in avenacosides A and B, respectively. Removal of the -{scL}-rhamnose residue is sufficient to detoxify the avenacosides. This work describes the purification of the avenacoside-degrading -{scL}-rhamnosidase–and determination of peptide sequence from the protein which represents the first -{scL}-rhamnosidase thus characterised in a fungal plant pathogen.     

Expression of harpin(xoo) in transgenic tobacco induces pathogen defense in the absence of hypersensitive cell death

ABSTRACT Harpin(Xoo), encoded by the hpaG(Xoo) gene of Xanthomonas oryzae pv. oryzae, is a member of the harpin group of proteins that induce pathogen resistance and hypersensitive cell death (HCD) in plants. We elaborated whether both processes are correlated in hpaG(Xoo)-expressing tobacco (HARTOB) plants, which produced harpin(Xoo) intracellularly. Resistance to fungal, bacterial, and viral pathogens increased in HARTOB, in correlation with the expression of hpaG(Xoo), the gene NPR1 that regulates several resistance pathways, and defense genes GST1, Chia5, PR-1a, and PR-1b that are mediated by different signals. However, reactive oxygen intermediate burst, the expression of HCD marker genes hsr203 and hin1, and cell death did not occur spontaneously in HARTOB, though they did in untransformed and HARTOB plants treated exogenously with harpin(Xoo). Thus, the transgenic expression of harpin(Xoo) confers nonspecific pathogen defense in the absence of HCD.     

Changes in susceptibility of bean leaves (Phaseolus vulgaris) to Sclerotinia sclerotiorum and Botrytis cinerea by pre-inoculative ozone exposures

The effects of ozone on the susceptibility of leaves ofPhaseolus vulgaris toSclerotinia sclerotiorum andBotrytis cinerea have been investigated. Seedlings of one ozone-sensitive (Pros) and five relatively ozone-insensitive cultivars (Gamin, Precores, Groffy, Narda, Berna) were exposed to different ozone concentrations (0, 120, 180 and 270 g m^–3) for 8 h. One day after the exposures, primary leaves were detached and immediately inoculated with spores of either pathogen suspended in water or in a 62.5 mM KH₂PO₄ (Pi) solution. Visible ozone injury differed between the cultivars and increased with increasing ozone concentration. On the leaves of non-exposed plants, spores of the pathogens suspended in water caused very few lesions, whereas fungal pathogenicity was stimulated by addition of Pi to the inoculum. Ozone-injured leaves of all cultivars exhibited lesions after inoculation of the leaves with the pathogens suspended in water, and the number of lesions was positively correlated with the level of ozone injury for either pathogen and cultivar. The increase in susceptibility of bean leaves in response to increasing ozone concentrations was greater forB. cinerea than forS. sclerotiorum when spores were suspended in water, but was similar when the spores were suspended in Pi.In general, the number of lesions following inoculation with spores in Pi increased with increasing ozone concentration. However, the number of lesions in the ozone-insensitive Groffy was reduced by an exposure to 120 g m^–3 but increased with higher concentrations. This pattern of susceptibility response to the pathogens was not found in the other ozone-insensitive cultivars and, thus, did not appear to be related to the inherent ozone-insensitivity in bean.