Induction Of Hypersensitive Cell Death Research Articles

Silencing of phosphoinositide dependent protein kinase orthologs reduces hypersensitive cell death in Nicotiana benthamiana.

Phosphatidic acid plays an important role in plant immune responses against phytopathogenic bacteria in Nicotiana benthamiana. Here we focused on phosphoinositide dependent protein kinases (PDKs) as a candidate required for phosphatidic acid signaling. Based on Arabidopsis PDK sequences, we identified four putative PDK orthologs in N. benthamiana genome. To address the role of PDKs in plant defense responses, we created all four NbPDKs-silenced plants by virus-induced gene silencing. the NbPDKs-silenced plants showed a moderately reduced growth phenotype. Induction of hypersensitive cell death was compromised in the NbPDKs-silenced plants challenged with Ralstonia solanacearum. The hypersensitive cell death induced by bacterial effectors was also reduced in the NbPDKs-silenced plants. the NbPDKs-silenced plants showed decreased production of salicylic acid, jasmonic acid and jasmonoyl-L-isoleucine, as well as hydrogen peroxide after inoculation with R. solanacearum. These results suggest that NbPDKs might have an important role in the regulation of the hypersensitive cell death via plant hormone signaling and oxidative burst.

The tomato Arp2/3 complex is required for resistance to the powdery mildew fungus Oidium neolycopersici.

The actin-related protein 2/3 complex (Arp2/3 complex), a key regulator of actin cytoskeletal dynamics, has been linked to multiple cellular processes, including those associated with response to stress. Herein, the Solanum habrochaites ARPC3 gene, encoding a subunit protein of the Arp2/3 complex, was identified and characterized. ShARPC3 encodes a 174-amino acid protein possessing a conserved P21-Arc domain. Silencing of ShARPC3 resulted in enhanced susceptibility to the powdery mildew pathogen Oidium neolycopersici (On-Lz), demonstrating a role for ShARPC3 in defence signalling. Interestingly, a loss of ShARPC3 coincided with enhanced susceptibility to On-Lz, a process that we hypothesize is the result of a block in the activity of SA-mediated defence signalling. Conversely, overexpression of ShARPC3 in Arabidopsis thaliana, followed by inoculation with On-Lz, showed enhanced resistance, including the rapid induction of hypersensitive cell death and the generation of reactive oxygen. Heterologous expression of ShARPC3 in the arc18 mutant of Saccharomyces cerevisiae (i.e., ∆arc18) resulted in complementation of stress-induced phenotypes, including high-temperature tolerance. Taken together, these data support a role for ShARPC3 in tomato through positive regulation of plant immunity in response to O.neolycopersici pathogenesis.

Human GLTP and mutant forms of ACD11 suppress cell death in the Arabidopsis acd11 mutant.

The Arabidopsis acd11 mutant exhibits runaway, programmed cell death due to the loss of a putative sphingosine transfer protein (ACD11) with homology to mammalian GLTP. We demonstrate that transgenic expression in Arabidopsis thaliana of human GLTP partially suppressed the phenotype of the acd11 null mutant, resulting in delayed programmed cell death development and plant survival. Surprisingly, a GLTP mutant form impaired in glycolipid transfer activity also complemented the acd11 mutants. To understand the relationship between functional complementarity and transfer activity, we generated site-specific mutants in ACD11 based on homologous GLTP residues required for glycolipid transfer. We show that these ACD11 mutant forms are impaired in their in vitro transfer activity of sphingolipids. However, transgenic expression of these mutant forms fully complemented acd11 mutant cell death, and transgenic plants showed normal induction of hypersensitive cell death upon infection with avirulent strains of Pseudomonas syringae. The significance of these findings with respect to the function(s) of ACD11 in sphingolipid transport and cell death regulation is discussed.

Comparative expression analysis of genes induced during development of bacterial rot and induction of hypersensitive cell death in lettuce

The development of bacterial rot disease caused by Pseudomonas cichorii is closely associated with programmed cell death. To investigate the molecular events occurring during the development of bacterial rot, we isolated 20 P. cichorii-responsive genes (PcRGs) in lettuce by differential display. Among these PcRGs, signal transduction-, transcription/translation- and defense/stress responses-related PcRGs were subjected to a comparative expression study. We used RNA samples isolated from lettuce leaves inoculated with P. cichorii and hypersensitive response-inducing Pseudomonas syringae pv. syringae. Expression of PcRG1-5-5 (spliceosomal protein), 2-9-2 (protein kinase) and 1-6-2 (ACC oxidase), 7-5 (alternative oxidase) and BI-I (bax inhibitor I) significantly increased in lettuce leaves inoculated with both P. cichorii and P. syringae pv. syringae. Intriguingly, PcRG 1-2-6 (protein phosphatase 2C) and 4-D-5 (protein kinase) were only up-regulated in P. cichorii-inoculated lettuce, whereas expression of PcRG1-3-2 (ribonucleoprotein) was only enhanced in P. syringae pv. syringae-inoculated lettuce. Expressions of PcRG1-3-2, 1-5-5, 1-6-2, 2-9-2, 7-5 and BI-I were induced by treatments with salicylic acid and/or methyl jasmonate. However, expression of PcRG1-2-6 and 4-D-5, which were specifically up-regulated by P. cichorii, were scarcely affected by these chemicals. Pharmacological studies suggested that ethylene and alternative oxidase were commonly related to disease development and hypersensitive responses. By contrast, there may be a different role for protein synthesis and protein kinase during disease development and in hypersensitive responses. These results suggested the overall similarity of genes expressed during disease development and in hypersensitive responses. However, there were differences not only in induction kinetics and the level of gene expression but also in the signal transduction pathway between hypersensitive responses and disease development.

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 1 h and reached a maximum level 3–6 h 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 3–12 h 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.

Induction of hypersensitive cell death by hydrogen peroxide produced through polyamine degradation in tobacco plants.

Screening immediate-early responding genes during the hypersensitive response (HR) against tobacco mosaic virus infection in tobacco (Nicotiana tabacum) plants, we identified a gene encoding ornithine decarboxylase. Subsequent analyses showed that other genes involved in polyamine biosynthesis were also up-regulated, resulting in the accumulation of polyamines in apoplasts of tobacco mosaic virus-infected leaves. Inhibitors of polyamine biosynthesis, alpha-difluoromethyl-ornithine, however, suppressed accumulation of polyamines, and the rate of HR was reduced. In contrast, polyamine infiltration into a healthy leaf induced the generation of hydrogen peroxide and simultaneously caused HR-like cell death. Polyamine oxidase activity in the apoplast increased up to 3-fold that of the basal level during the HR, and its suppression with a specific inhibitor, guazatine, resulted in reduced HR. Because it is established that hydrogen peroxide is one of the degradation products of polyamines, these results indicate that one of the biochemical events in the HR is production of polyamines, whose degradation induces hydrogen peroxide, eventually resulting in hypersensitive cell death.

Lipoxygenase in pea roots subjected to biotic stress

Cyst nematode Heterodera goettingiana is a major pest for pea crop. Sources of resistance to this disease have been found in germplasm pea, although the mechanism of host resistance is still obscure. Lipoxygenases (LOX) have been proposed to be involved in the defence of host-parasite interactions. Resistant accession displayed at 24–72 h after nematode infection an incompatible interaction with H. goettingiana characterised by the induction of hypersensitive cell death and induction of LOX activity, mRNA and fatty acid hydroperoxides. In contrast, in susceptible pea roots infected with nematodes the induction of LOX activity, mRNA and fatty acid hydroperoxides was less consistent and delayed and no cell death was observed. A single polypeptide, with an apparent molecular mass of 96 kDa, was identified in both resistant and susceptible either uninfected or infected pea roots. Subcellular localisation showed that immunogold labelling was present in the cytoplasm, vacuoles, and plastids of differentiating parenchyma cells of uninfected and infected roots. Labelling increased weakly in differentiating cells of the feeding sites (syncytia) in susceptible pea at 72 h after infection, and was of greater magnitude in resistant tissues. Our results suggest that LOXs might have a role in root-nematode interaction and in resistance mechanisms.

Activation of Defense Responses to Fusarium Infection in Asparagus densiflorus

Defense responses to Fusarium oxysporum f. sp. asparagi and F. proliferatum were compared after root inoculation of the asparagus fern, Asparagus densiflorus vars. Myersii and Sprengeri, and cultivated asparagus, A. officinalis cv. Guelph Millennium. Both varieties of A. densiflorus exhibited a hypersensitive response with rapid death of epidermal cells within 8–24 h and restricted the fungal growth. In A. officinalis roots, rapid cell death was not found, and necrotic lesions were observed 8–14 d after fungal inoculation. Peroxidase and phenylalanine ammonia-lyase activities increased significantly in inoculated A. densiflorus but not A. officinalis plants. Local and systemic induction of peroxidase activity was detected after pathogen inoculation in root and spear tissues, respectively, of A. densiflorus. POX activity decreased in roots of inoculated A. officinalis by 8 d post-inoculation. Germination and germ tube growth were inhibited when spores of F. oxysporum f. sp. asparagi were incubated in root exudates and on root segment surfaces of inoculated A. densiflorus plants exhibiting hypersensitive cell death. Spore germination of F. proliferatum and three fungi non-pathogenic to cultivated asparagus was inhibited as well. Rapid induction of hypersensitive cell death in A. densiflorus was associated with restriction of fungal growth, and activation of peroxidase and phenylalanine ammonia-lyase, two defense enzymes thought to be important for plant disease resistance.

Nitric oxide functions as a signal in plant disease resistance.

Recognition of an avirulent pathogen triggers the rapid production of the reactive oxygen intermediates superoxide (O2-) and hydrogen peroxide (H2O2). This oxidative burst drives crosslinking of the cell wall, induces several plant genes involved in cellular protection and defence, and is necessary for the initiation of host cell death in the hypersensitive disease-resistance response. However, this burst is not enough to support a strong disease-resistance response. Here we show that nitric oxide, which acts as a signal in the immune, nervous and vascular systems, potentiates the induction of hypersensitive cell death in soybean cells by reactive oxygen intermediates and functions independently of such intermediates to induce genes for the synthesis of protective natural products. Moreover, inhibitors of nitric oxide synthesis compromise the hypersensitive disease-resistance response of Arabidopsis leaves to Pseudomonas syringae, promoting disease and bacterial growth. We conclude that nitric oxide plays a key role in disease resistance in plants.

Induction of Hypersensitive Cell Death by a Fungal Protein in Cultures of Tobacco Cells

Treatment of suspension-cultured tobacco (Nicotiana tabacum cv. Xanthi) cells (line XD6S) with fungal proteinaceous elicitors, namely, xylanase (EC 3.2.1.8) from Trichoderma viride (TvX) and xylanase from T. reesei (TrX), induced shrinkage of the cytoplasm, condensation of the nucleus, and, finally, cell death, which were accompanied by typical defense responses that included an oxidative burst and expression of defense genes. A Ca2+ channel blocker, Gd3+, inhibited the typical response of XD6S cells to TvX, which resembled the hypersensitive reaction (HR). These results suggested that the influx of Ca2+ ions plays an important role as a secondary signal. The HR was not observed in TvX-treated tobacco cells (line BY-2) derived from cv. Bright Yellow 2. This result suggests that key features of cultivar-specific interaction can be observed in cultures of tobacco cells. Xylanase from Bacillus circulans (BcX) and B. subtilis (BsX), which has enzymatic properties similar to those of TvX but an amino acid sequence different from that of TvX, did not induce the HR-like response in XD6S cells. These results suggest that the elicitor action of TvX is not due to its ability to hydrolyze cell walls but requires the TvX-specific recognition factors in plant cells. Thus, TvX-induced cell death was not due to some general toxic effect, but seems to be mediated by the activation of a specific cellular signal-transduction cascade that converges with a pathway that activates the intracellular cell death program.