Cellular Defense Response Research Articles

The white barley mutant albostrians shows a supersusceptible but symptomless interaction phenotype with the hemibiotrophic fungus Bipolaris sorokiniana.

Bipolaris sorokiniana (teleomorph: Cochliobolus sativus) is a cereal pathogen of increasing global concern, with most significance in Asiatic cropping systems. In order to gain insight into the mechanism of host resistance, we studied fungal development on the supersusceptible barley mutant albostrians and its parent cv. Haisa. A microscopic dissection of early fungal growth on Haisa and green albostrians leaves revealed a distinct epidermis-localized biotrophic and a mesophyll-based necrotrophic phase. White, green, and striped white-green albostrians leaves showed extreme differences in disease development. When comparing cellular defense responses, we found restriction of fungal spreading after successful infection of host mesophyll tissue to be the most important mechanism limiting outbreak of the disease. Colonization of susceptible green leaves, but not extreme colonization of supersusceptible white albostrians leaves, was associated with macroscopically visible lesion formation and mesophyll accumulation of hydrogen peroxide (H2O2), implying a symptomless growth of the pathogen in supersusceptible host tissue. In contrast, early epidermal papilla-based resistance was closely linked to H2O2 accumulation in all leaf types. In white leaves, ascorbate peroxidase (APX), glutathione-S-transferase (GST), and the cell death regulator Bax-inhibitor-1 (BI-1) showed a stronger constitutive or pathogen responsive activation, whereas glycolate oxidase (GLOX) and catalase (CAT2) expression was stronger in green leaves. We discuss supersusceptibility and symptomless growth on the basis of the histochemical and the gene expression data.

Gene expression analysis in cucumber leaves primed by root colonization with Pseudomonas chlororaphis O6 upon challenge-inoculation with Corynespora cassiicola.

Root colonization by Pseudomonas chlororaphis O6, a non-pathogenic rhizobacterium, induced systemic resistance in cucumber against target leaf spot caused by Corynespora cassiicola. A cDNA library was constructed using mRNA extracted from cucumber leaves 12 h after inoculation with C. cassiicola, using plants colonized by O6. To identify genes involved in O6-mediated induced systemic resistance (ISR), we employed a subtractive hybridization method using mRNAs extracted from pathogen-challenged cucumber leaves of plants lacking colonization. Differential screening of the cDNA library led to the isolation of six distinct genes encoding a GTP binding protein, a 60S ribosomal protein, a hypersensitive-induced reaction protein, a ubiquitin extension protein, a pyridine nucleotide-disulfide oxidoreductase, and a signal recognition particle receptor. Expression of these genes was not induced by O6 colonization alone. Rather, transcript accumulation of these genes increased significantly faster and stronger in the O6 colonized than in non-colonized plants after challenge infection. Therefore, O6-mediated ISR may be associated with an enhanced capacity for the rapid and effective activation of cellular defence responses after challenge inoculation.

Overexpressed protein disulfide isomerase in brains of patients with sporadic Creutzfeldt–Jakob disease

Earlier studies have failed to detect covalent modifications in β-sheet-rich scrapie isoform prion protein (PrPSc) and have concluded that the conversion of α-helix-rich cellular form prion protein (PrPC) to PrPSc represents purely conformational transition not involving chemical reactions. However, recent studies have shown that the intradisulfide bond of PrPC can play an important role for instability and conformational change to PrPSc. Interestingly, we found overexpressed protein disufide isomerase (PDI) in brains of sporadic Creutzfeldt–Jakob disease (sCJD, human prion disease) patients using two dimensional electrophoresis and Western blot analysis but not in other neurodegenerative disorders as Down Syndrome and Alzheimer's disease. However, proteinase K digestion and plasminogen binding assay of brain homogenates incubated with PDI suggest that PDI has no effect on either proteinase resistance or conformational change of PrP. Overexpression of PDI protein in sCJD brain may simply reflect a cellular defense response against the altered prion protein.

Pre-treatment of cucumber plants with acibenzolar-S-methyl systemically primes a phenylalanine ammonia lyase gene (PAL1) for enhanced expression upon attack with a pathogenic fungus

Systemic acquired resistance (SAR) of plants induced upon infection with a necrotizing pathogen, is characterised by an enhanced capacity to induce cellular defence responses to subsequent pathogen attack. Pre-treating the first leaves of cucumber plants with the synthetic SAR activator acibenzolar-S-methyl (ASM; S -methylbenzo[1,2,3] thiadiazole-7-carbothiate) protects whole plants from infection with the virulent fungal pathogen Colletotrichum orbiculare. Using a sensitive fluorescence-based RT-PCR system to follow the expression of three defence-related genes in the third leaves of treated cucumber plants, we have demonstrated, in non-inoculated plants, that ASM treatment alone systemically induces the expression of an acidic peroxidase-encoding gene (POX) and pathogenesis-related protein 1 gene homologue (PR1-1a). In contrast, expression of a gene encoding a phenylalanine ammonia lyase homologue (PAL1) is not directly responsive to ASM treatment. However, expression of POX, PR1-1a, andPAL1 is primed for ASM-dependent enhancement upon subsequent inoculation with C. orbiculare. The results suggest the systemic potentiation of defence-related gene expression is a key component of SAR.

Dietary docosahexaenoic acid is more optimal than eicosapentaenoic acid affecting the level of cellular defence responses of the juvenile grouper Epinephelus malabaricus

The combined effects of dietary docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids on phagocytic, respiratory burst, and leucocyte proliferative activities of the juvenile grouper, Epinephelus malabaricus, were investigated. The test fish were fed for 12wk on test diets containing 1g 100g−1 diet of DHA and EPA in combinations (DHA/EPA: 3/1, 2/1, 1/1, 0·7/1, 0·3/1). In addition to promoting fish growth, high dietary DHA/EPA ratio significantly enhanced phagocytic and respiratory burst activities of grouper head-kidney leucocytes compared with low ratio. Significant correlations were found between leucocyte phagocytic or respiratory burst activities and concentrations of 20:3(n-3), DHA and EPA in fish liver and muscle tissues. Leucocyte proliferation was significantly higher (P< 0·05) when the diets were high in DHA/EPA ratio than low in DHA/EPA ratio, when stimulated by Con A and PHA-P, but not by LPS. Tissue DHA concentrations and leucocyte proliferation were significantly and positively correlated. Fortification of dietary DHA, thus increased T-cell proliferation and phagocytic function of grouper leucocytes. DHA is the only member in the (n-3) highly unsaturated fatty acid family that stimulated phagocytic functions of leucocytes and T-cell proliferation, and is more optimal than EPA affecting the cellular defence responses of the E. malabaricus juveniles.

Insect hemocytes and their role in immunity

The innate immune system of insects is divided into humoral and cellular defense responses. Humoral defenses include antimicrobial peptides, the cascades that regulate coagulation and melanization of hemolymph, and the production of reactive intermediates of oxygen and nitrogen. Cellular defenses refer to hemocyte-mediated responses like phagocytosis and encapsulation. In this review, we discuss the cellular immune responses of insects with emphasis on studies in Lepidoptera and Diptera. Insect hemocytes originate from mesodermally derived stem cells that differentiate into specific lineages identified by morphology, function, and molecular markers. In Lepidoptera, most cellular defense responses involve granular cells and plasmatocytes, whereas in Drosophila they involve primarily plasmatocytes and lamellocytes. Insect hemocytes recognize a variety of foreign targets as well as alterations to self. Both humoral and cell surface receptors are involved in these recognition events. Once a target is recognized as foreign, hemocyte-mediated defense responses are regulated by signaling factors and effector molecules that control cell adhesion and cytotoxicity. Several lines of evidence indicate that humoral and cellular defense responses are well-coordinated with one another. Cross-talk between the immune and nervous system may also play a role in regulating inflammation-like responses in insects during infection.

A strobilurin fungicide enhances the resistance of tobacco against tobacco mosaic virus and Pseudomonas syringae pv tabaci.

The strobilurin class of fungicides comprises a variety of synthetic plant-protecting compounds with broad-spectrum antifungal activity. In the present study, we demonstrate that a strobilurin fungicide, F 500 (Pyraclostrobin), enhances the resistance of tobacco (Nicotiana tabacum cv Xanthi nc) against infection by either tobacco mosaic virus (TMV) or the wildfire pathogen Pseudomonas syringae pv tabaci. F 500 was also active at enhancing TMV resistance in NahG transgenic tobacco plants unable to accumulate significant amounts of the endogenous inducer of enhanced disease resistance, salicylic acid (SA). This finding suggests that F 500 enhances TMV resistance in tobacco either by acting downstream of SA in the SA signaling mechanism or by functioning independently of SA. The latter assumption is the more likely because in infiltrated leaves, F 500 did not cause the accumulation of SA-inducible pathogenesis-related (PR)-1 proteins that often are used as conventional molecular markers for SA-induced disease resistance. However, accumulation of PR-1 proteins and the associated activation of the PR-1 genes were elicited upon TMV infection of tobacco leaves and both these responses were induced more rapidly in F 500-pretreated plants than in the water-pretreated controls. Taken together, our results suggest that F 500, in addition to exerting direct antifungal activity, may also protect plants by priming them for potentiated activation of subsequently pathogen-induced cellular defense responses.

Influence of calcium on Manduca sexta plasmatocyte spreading and network formation.

Plasmatocytes are a class of insect hemocytes important in the cellular defense response. In some species, they are phagocytic, protecting the insect from smaller pathogens. In many insects, they work in concert with other hemocytes (particularly other plasmatocytes and granular cells) to form nodules and to encapsulate foreign material. To perform these functions, plasmatocytes attach to, spread on, and surround suitable targets. Because of their importance, because we had previously observed that prolonged incubation of hemocytes in solutions containing the divalent cation chelator ethylenediaminetetraacetic acid (EDTA) inhibited plasmatocyte spreading, and because of the importance of divalent cations in many immune-related functions, we investigated the effect of calcium and magnesium on spreading of plasmatocytes from fifth instar Manduca sexta larvae. On glass slides, plasmatocytes spread more quickly and elongated in Grace's medium containing 5 mM calcium, compared to calcium-free medium. In the presence of calcium, plasmatocyte adhesion, spreading, and network formation were not visibly different in magnesium-free and magnesium-containing Grace's medium. Using immunomicroscopy with a monoclonal antibody specific for plasmatocytes, we measured the length and width of plasmatocytes incubated with several different concentrations of calcium. Plasmatocyte length positively correlated with calcium concentration to 5 mM (maximum concentration tested and approximately the hemolymph concentration). Mean plasmatocyte width was less in 0 and 5 mM calcium than in 0.05 or 0.5 mM calcium. On plastic, hemocytes survived longer than on glass (they survived beyond 24 h) and, in 5 mM calcium, formed an extensive network readily visible by phase-contrast microscopy. This network was never as extensive in the absence of calcium. Network formation in the absence of magnesium, but presence of calcium, resembled network formation in standard Grace's medium.

Zinc and Manduca sexta hemocyte functions

Two metalloproteases have recently been linked to the immune response in Lepidoptera. In addition, zinc is highly important in many mammalian immune-related functions. Because of these, we investigated the effect of zinc and two zinc-protease inhibitors on Manduca sexta hemocyte behavior in vitro. Plasmatocytes were significantly more elongated in Grace's medium supplemented with 100 µm zinc chloride than in the absence of zinc. To test whether zinc-dependent proteases were responsible for the increased length seen in the presence of zinc, we tested two zinc-protease inhibitors, phosphoramidon and bestatin. Each resulted in decreased plasmatocyte length compared to the control, but the distributions of lengths differed with each inhibitor. Each inhibitor also affected plasmatocyte network formation in vitro. This work suggests (1) that at least two different zinc proteases are involved in the cellular defense response of M. sexta, and (2) that zinc should be included in media used for in vitro studies of the immune response.

Influence of an arbuscular mycorrhizal fungus on the interaction of a binucleate Rhizoctonia species with Myc+ and Myc- pea roots.

A binucleate Rhizoctonia (BNR) species was isolated from a clay loam soil on the Epoisses experimental station of INRA, Dijon and identified as belonging to the anastomosis Group A (AG-A). The BNR was inoculated to a Myc- Pisum sativum mutant (P53, sym30 locus) and its wild-type parent (cv Frisson) in the presence or absence of the arbuscular mycorrhizal fungus Glomus mosseae. The BNR had no significant effect on plant weight. Myc+ and Myc- roots were equally susceptible towards BNR and showed no localized cellular defense responses. The presence of BNR decreased significantly the percentage of root length colonized by G. mosseae and, inversely, G. mosseae reduced the number of BNR monilioid chains formed in root epidermal cells of the two pea genotypes. The pisatin concentration was increased significantly by BNR in both Myc+ and Myc- roots and by G. mosseae in the wild-type pea plants. The highest accumulation of pisatin was observed in Myc+ roots when both fungi were present.

Zinc and Manduca sexta hemocyte functions.

Two metalloproteases have recently been linked to the immune response in Lepidoptera. In addition, zinc is highly important in many mammalian immune-related functions. Because of these, we investigated the effect of zinc and two zinc-protease inhibitors on Manduca sexta hemocyte behavior in vitro. Plasmatocytes were significantly more elongated in Grace's medium supplemented with 100 micro m zinc chloride than in the absence of zinc. To test whether zinc-dependent proteases were responsible for the increased length seen in the presence of zinc, we tested two zinc-protease inhibitors, phosphoramidon and bestatin. Each resulted in decreased plasmatocyte length compared to the control, but the distributions of lengths differed with each inhibitor. Each inhibitor also affected plasmatocyte network formation in vitro. This work suggests (1) that at least two different zinc proteases are involved in the cellular defense response of M. sexta, and (2) that zinc should be included in media used for in vitro studies of the immune response.

Effect of the entomopathogenic fungus Metarhizium anisopliae (Metsch.) Sorokin on cellular defence response and oxygen uptake of the wax moth Galleria mellonella L. (Lep., Pyralidae)

Remarkable changes in the cellular response and oxygen uptake in the wax moth Galleria mellonella were observed when larvae were infected with the entomopathogenic fungus Metarhizium anisopliae. The number of haemocytes increased 48, 72 and 96 h after infection with fungal conidiospores. Extensive blebbing of the cell membrane in the haemocytes and large numbers of vacuoles were noticed as a result of fungal infection. The study showed a significant difference in oxygen uptake between healthy and infected larvae and the greatest uptake was recorded 72 h after fungal infection. The results suggested that the rate of cellular reaction and oxygen uptake were associated with the development of fungal infection.

Identification of soybean elicitation competency factor, CF-1, as the soybean Kunitz trypsin inhibitor

The soybean defence responses that occur in cells immediately adjacent (proximal) to the hypersensitive response (HR) can be induced by the cell wall glucan elicitor (WGE) from Phytophthora sojae, but in some cultivars only in cells that have been pre-conditioned by endogenous competency factors associated with wounded or HR-dying cells. Competency factor, CF-1, causes a general amplification of phenylpropanoid responses to elicitor and greatly enhances the phenolic polymer response to elicitor. CF-1 activity, which is heat-labile and associated with a low speed centrifugal pellet of soybean tissues, can be partially mimicked by reduced glutathione (GSH). Here it is reported that GSH releases CF-1 activity into intercellular washing fluids, suggesting it is associated with the apoplast. Likewise, GSH can be used to enrich CF-1 activity greatly in extracts from low speed pellets. This enrichment allowed the purification of CF-1 to near homogeneity and its subjection to mass spectral and sequence analysis. CF-1 was identified as the soybean Kunitz trypsin inhibitor (SKTI). Although plant protease inhibitors have been implicated in insect and pathogen resistance, to the authors' knowledge this is the first suggestion of a regulatory role in activation of cellular defence responses. Protease inhibitors play a role in many signaling processes in eukaryotic systems, and SKTI has intriguing three-dimensional homologies to interleukin 1-α and 1-β and fibroblast growth factor.

Functional characterization of interferon regulatory factor 3a (IRF-3a), an alternative splice isoform of IRF-3.

Virus infection of numerous cell types results in the transcriptional induction of a subset of virus- and interferon (IFN)-stimulated genes. The beta IFN (IFN-beta) gene is one of these rapidly induced genes; it serves as a fundamental component of the cellular defense response in eliciting potent antiviral, immunomodulatory, and antiproliferative effects. One of the transcription factors involved in the stringent regulation of IFN-beta production following virus infection is interferon regulatory factor (IRF) 3 (IRF-3). We have characterized an alternatively spliced isoform of IRF-3 that we have called IRF-3a. IRF-3a can selectively and potently inhibit virus-induced activation of the IFN-beta promoter. IRF-3a lacks half of the DNA binding domain found in IRF-3 and is unable to bind to the classical IRF binding elements, IFN-stimulated response elements. These studies suggest that IRF-3a may act as a modulator of IRF-3.

Priming as a mechanism in induced systemic resistance of plants

Induced systemic resistance is a plant defence state that is associated with an enhanced ability – the so-called priming – to resist pathogen attack by stronger activation of cellular defence responses. So far, however, priming has not been widely appreciated when studying induced plant disease resistance. During the past several years, it has been demonstrated that pre-treatment of cultured parsley cells with inducers of systemic resistance, salicylic acid or a benzothiadiazole, leads to the direct activation of a set of defence-related genes and also primes the cells for stronger elicitation of another set of defence genes including those encoding phenylalanine ammonia-lyase. From these results, it was concluded that the resistance inducers have at least a dual role in plant defence-gene activation. When elucidating whether priming plays a role in induced systemic resistance of Arabidopsis, pre-treating plants with benzothiadiazole was found to augment the subsequent activation of phenylalanine ammonia-lyase genes by Pseudomonas infection, wounding and osmotic stress and also to enhance wound/osmotic stress-induced callose production. The augmentation of phenylalanine ammonia-lyase gene activation or/and callose deposition was not seen in the Arabidopsis non-expresser of pathogenesis-related genes1 mutant which is compromised in induced resistance, while it was present, without benzothiadiazole pre-treatment, in the constitutive expresser of pr genes1 and 5 mutants in which induced resistance is constitutive. Together these studies point to priming as an important cellular mechanism in induced systemic resistance of plants which requires the intact non-expresser of pathogenesis-related genes1 gene.

The scientific basis of immunonutrition.

Substrates with immune-modulating actions have been identified among both macro- and micronutrients. Currently, the modes of action of individual immune-modulating substrates, and their effects on clinical outcomes, are being examined. At present, some enteral formulas are available for the clinical setting which are enriched with selected immune-modulating nutrients. The purpose of the present paper is to review the scientific rationale of enteral immunonutrition. The major aspects considered are mucosal barrier structure and function, cellular defence function and local or systemic inflammatory response. It is notable that in critical illness the mucosal barrier and cellular defence are impaired and a reinforcement with enteral immunonutrition is desirable, while local or systemic inflammatory response should be down regulated by nutritional interventions. The results available from clinical trials are conflicting. Meta-analyses of recent trials show improvements such as reduced risk of infection, fewer days on a ventilator, and reduced length of intensive care unit and hospital stay. Thus, a grade A recommendation was proclaimed for the clinical use of enteral immune-modulating diets. Improvement in outcome was only seen when critical amounts of the immune-modulating formula were tolerated in patients classified as being malnourished. However, in other patients with severe sepsis, shock and organ failure, no benefit or even disadvantages from immunonutrition were reported. In such severe conditions we hypothesize that systemic inflammation might be undesirably intensified by arginine and unsaturated fatty acids, directly affecting cellular defence and inflammatory response. We therefore recommend that in patients suffering from systemic inflammatory response syndrome great caution should be exercised when immune-enhancing substrates are involved which may aggravate systemic inflammation.

Bacterial-injection-induced syntheses of N-beta-alanyldopamine and Dopa decarboxylase in the hemolymph of coleopteran insect, Tenebrio molitor larvae.

Injection of Escherichia coli into larvae of the coleopteran Tenebrio molitor resulted in the appearance of a dopamine-like substance on the electrochemical detector. To characterize this dopamine-like substance, we purified it to homogeneity from the immunized hemolymph and determined its molecular structure to be N-beta-alanyldopamine using the liquid chromatographic/tandem mass spectrometric method. Chemically synthesized N-beta-alanyldopamine showed the same retention time on HPLC as the purified N-beta-alanyldopamine from immunized larvae. To elucidate the molecular mechanism of N-beta-alanyldopamine synthesis in vivo, we examined the enzyme activity of Dopa decarboxylase against E. coli-injected hemolymph of T. molitor larvae. The enzyme activity of Dopa decarboxylase increased dramatically approximately 8 h after injection; Dopa decarboxylase activity of injected larvae being 10-times higher than naive larvae after 24 h. To evaluate the extent of quantitative changes of Dopa decarboxylase in response to bacterial challenge, Tenebrio Dopa decarboxylase was purified to homogeneity from the whole larvae and a cDNA clone for Tenebrio Dopa decarboxylase was isolated. RNA blot hybridization revealed that expression of the Dopa decarboxylase gene was activated transiently 3-8 h after E. coli challenge. Immunoprecipitation experiments showed that Tenebrio Dopa decarboxylase was detected from 8 to 24 h in E. coli-injected larval extract. Thus, bacterial injection into T. molitor larvae might induce transcriptional activation of a Dopa decarboxylase gene, and then synthesis of N-beta-alanyldopamine. The synthesized N-beta-alanyldopamine might be used as a substrate by phenoloxidase during melanin synthesis in the humoral defense response or the melanotic encapsulation reaction of the cellular defense response.

Mercury induces regional and cell-specific stress protein expression in rat kidney.

Cells respond to physiologic stress by enhancing the expression of specific stress proteins. Heat-shock proteins (hsps) and glucose-regulated proteins (grps) are members of a large superfamily of proteins collectively referred to as stress proteins. This particular stress-protein response has evolved as a cellular strategy to protect, repair, and chaperone other essential cellular proteins. The objective of this study was to evaluate the differential expression of four hsps in the renal cortex and medulla during experimental nephrotoxic injury using HgCl2. Male Sprague-Dawley rats received single injections of HgCl2 (0.25, 0.5, or 1 mg Hg/kg, i.v.). At 4, 8, 16, or 24 h after exposure, kidneys were removed and processed for histopathologic, immunoblot, and immunohistochemical analyses. Nephrosis was characterized as minimal or mild (cytoplasmic condensation, tubular epithelial degeneration, single cell necrosis) at the lower exposures, and progressed to moderate or severe (nuclear pyknosis, necrotic foci, sloughing of the epithelial casts into tubular lumens) at the highest exposures. Western blots of renal proteins were probed with monoclonal antibodies specific for 4 hsps. In whole kidney, Hg(II) induced a time- and dose-related accumulation of hsp72 and grp94. Accumulation of hsp72 was predominantly localized in the cortex and not medulla, while grp94 accumulated primarily in the medulla but not cortex. The high, constitutive expression of hsp73 did not change as a result of Hg(II) exposure, and it was equally localized in cortex and medulla. Hsp90 was not detected in kidneys of control or Hg-treated rats. Since hsp72 has been shown involved in cellular repair and recovery, and since Hg(II) damage occurs primarily in cortex, we investigated the cell-specific expression of this hsp. Hsp72 accumulated primarily in undamaged distal convoluted tubule epithelia, with less accumulation in undamaged proximal convoluted-tubule epithelia. These results demonstrate that expression of specific stress proteins in rat kidney exhibits regional heterogeneity in response to Hg(II) exposure, and a positive correlation exists between accumulation of some stress proteins and acute renal cell injury. While the role of accumulation of hsps and other stress proteins in vivo prior to or concurrent with nephrotoxicity remains to be completely understood, these stress proteins may be part of a cellular defense response to nephrotoxicants. Conversely, renal tubular epithelial cells that do not or are unable to express stress proteins, such as hsp72, may be more susceptible to nephrotoxicity.

Conditioning of cellular defence responses to powdery mildew in cereal leaves by prior attack

Abstract Field-grown plants sequentially encounter many different fungal pathogens and nonpathogens that are capable of triggering an array of responses that may affect the subsequent level of disease they develop following later pathogen attack. These changes, which are induced by prior encounters, may be manifest as increased susceptibility or enhanced resistance to later pathogen attack; they may be expressed systemically or their effects may be localized within a few cells distance of the original encounter site. Here, we review our recent investigations of cellular changes effected by sequential inoculations of cereal leaves with the powdery mildew fungus Blumeria graminis DC. In susceptible barley and oats, a successful B. graminis attack followed by haustorium formation, renders the attacked cell, and to some extent its adjacent cells, highly accessible to later B. graminis attacks. By contrast, a failed attack due to papilla formation by the attacked host cells, renders the attacked cell and its adjacent cells highly inaccessible to later B. graminis attacks. Importantly, barley carrying the mlo5 allele for powdery mildew resistance is also conditioned to accessibility if prior attacks by an mlo-virulent isolate penetrates successfully. In the partial resistant oat cultivar Maldwyn B. graminis attacks either succeeded, failed due to papilla deposition, or failed because the attacked host cells died in response to the attacks. Sequential inoculation of Maldwyn demonstrated the induction of accessibility and inaccessibility, as well as a complete suppression of cell death response to attack where the cells had survived an earlier attack. Furthermore, when a prior attack induced cell death, a later attack on adjacent cells caused greatly increased rate of cell death, demonstrating potentiation of cell death. The importance of the induced cellular changes for plant resistance in the field is discussed.

N-Acylethanolamines in signal transduction of elicitor perception. Attenuation Of alkalinization response and activation of defense gene expression

In a recent study of N-acylphosphatidylethanolamine (NAPE) metabolism in elicitor-treated tobacco (Nicotiana tabacum L.) cells, we identified a rapid release and accumulation of medium-chain N-acylethanolamines (NAEs) (e.g. N-myristoylethanolamine or NAE 14:0) and a compensatory decrease in cellular NAPE (K.D. Chapman, S. Tripathy, B. Venables, A.D. Desouza [1998] Plant Physiol 116: 1163-1168). In the present study, we extend this observation and report a 10- to 50-fold increase in NAE 14:0 content in leaves of tobacco (cv Xanthi) plants treated with xylanase or cryptogein elicitors. Exogenously supplied synthetic NAE species affected characteristic elicitor-induced and short- and long-term defense responses in cell suspensions of tobacco and long-term defense responses in leaves of intact tobacco plants. In general, synthetic NAEs inhibited elicitor-induced medium alkalinization by tobacco cells in a time- and concentration-dependent manner. Exogenous NAE 14:0 induced expression of phenylalanine ammonia lyase in a manner similar to fungal elicitors in both cell suspensions and leaves of tobacco. NAE 14:0, but not myristic acid, activated phenylalanine ammonia lyase expression at submicromolar concentrations, well within the range of NAE 14:0 levels measured in elicitor-treated plants. Collectively, these results suggest that NAPE metabolism, specifically, the accumulation of NAE 14:0, are part of a signal transduction pathway that modulates cellular defense responses following the perception of fungal elicitors.