Involvement of anion channel in signal transduction of early defense responses elicited by harpinPss in tobacco]

No matter when anion channel inhibitors, DIDS (4, 4'-diisothiocyanatostilbene-2, 2'-disulfonic acid) and A9C (anthracene-9-carboxylic acid) added (before, at the same time of or after harpinPss treatment), they can inhibit harpinPss-induced hypersensitive response in tobacco seedlings and release of active oxygen and extracellular alkalinization in tobacco suspension cells. DIDS and A9C also inhibit harpinPss-induced Ca2+ influx. In all these cases, DIDS is more efficient than A9C. It is postulated that anion channel positively regulates calcium channel in plasma membrane, and harpinPss may function through signal transduction mediated by anion channel and calcium channel to regulate cellular Ca2+ concentration and defense responses.     

Peroxynitrite generation and tyrosine nitration in defense responses in tobacco BY-2 cells

Peroxynitrite (ONOO(-)) is a compound formed by reaction of superoxide (O(2) (-)) with nitric oxide (NO) and is expected to possess characteristics of both O(2) (-) reactivity and NO mobility in order to function as a signal molecule. Although there are several reports that describe the role of ONOO(-) in defense responses in plants, it has been very difficult to detect ONOO(-) in bioimaging due to its short half-life or paucity of methods for ONOO(-)-specific detection among reactive oxygen species or free radicals. Aminophenyl fluorescein (APF), a recently developed novel fluorophore for direct detection of ONOO(-) in bioimaging, was used for intracellular ONOO(-) detection. ONOO(-) generation in tobacco BY-2 cells treated with INF1, the major elicitin secreted by the late blight pathogen Phytophthora infestans, occurred within 1 h and reached a maximum level at 6-12 h after INF1 treatment. Urate, a ONOO(-) scavenger, abolished INF1-induced ONOO(-) generation. It is well known that ONOO(-) reacts with tyrosine residues in proteins to form nitrotyrosine in a nitration reaction as an ONOO(-)-specific reaction. Western blot analysis using anti-nitrotyrosine antibodies recognized nitrotyrosine-containing proteins in 20 and 50 kDa bands in BY-2 protein extract containing SIN-1 [3-(4-morpholinyl) sydnonimine hydrochloride; an ONOO(-) donor]. These bands were also recognized in INF1-treated BY-2 cells and were found to be slightly suppressed by urate. Our study is the first to report ONOO(-) detection and tyrosine nitration in defense responses in plants.     

Antimicrobial peptaibols induce defense responses and systemic resistance in tobacco against tobacco mosaic virus

Trichoderma spp. are well-known biocontrol agents because of their antimicrobial activity against bacterial and fungal phytopathogens. However, the biochemical mechanism of their antiviral activity remains largely unknown. In this study, we found that Trichokonins, antimicrobial peptaibols isolated from Trichoderma pseudokoningii SMF2, could induce defense responses and systemic resistance in tobacco (Nicotiana tabacum var. Samsun NN) against tobacco mosaic virus (TMV) infection. Local Trichokonin (100 nM) treatment led to 54% lesion inhibition, 57% reduction in average lesion diameter and 30% reduction in average lesion area in systemic tissue of tobacco compared with control, indicating that Trichokonins induced resistance in tobacco against TMV infection. Trichokonin treatment increased the production of reactive oxygen species and phenolic compounds in tobacco. Additionally, application of Trichokonins significantly increased activities of pathogenesis-related enzymes PAL and POD, and upregulated the expression of several plant defense genes. These results suggested that multiple defense pathways in tobacco were involved in Trichokonin-mediated TMV resistance. We report on the antivirus mechanism of peptaibols, which sheds light on the potential of peptaibols in plant viral disease control.     

Nitric oxide production and its functional link with OIPK in tobacco defense response elicited by chitooligosaccharide

Chitooligosaccharide (COS) or oligochitosan has been shown to induce tobacco defense responses which are connected with nitric oxide (NO) and OIPK (oligochitosan-induced Ser/Thr protein kinase). The aim of this study was to reveal the relationship between NO production and OIPK pathway in the defense response of tobacco elicited by COS. NO generation was investigated by epidermal strip bioassay and fluorophore microscope using fluorophore diaminofluorescein diacetate (DAF-2DA). Tobacco epidermal cells treated with COS resulted in production of NO, which was first present in chloroplast, then in nucleus, finally in the whole cell; this NO production was sensitive to NO scavenger cPTIO and the mammalian NO synthase (NOS) inhibitor l-NAME, suggesting that NOS-like enzyme maybe involved in NO generation in tobacco epidermal cells. However, NOS and nitrate reductase (NR, EC 1.6.6.1) inhibitors reduced NO content in tobacco leaves by using NO Assay Kit, suggesting both NOS and NR were involved in NO production in tobacco leaves. Using a pharmacological approach and western blotting, we provide evidence that NO acts upstream of OIPK expression. NO scavenger, NOS inhibitor partly blocked the activation of OIPK and the activities of several defense-related enzymes induced by COS; treatment with NO donor sodium nitroprusside (SNP) induced the activation of OIPK and enhanced the defense systems. The results suggest that COS is able to induce NO generation, which results in up-regulation the activities of some defense-related enzymes through an OIPK-dependent or independent pathway.     

Linear beta-1,3 glucans are elicitors of defense responses in tobacco

Laminarin, a linear beta-1,3 glucan (mean degree of polymerization of 33) was extracted and purified from the brown alga Laminaria digitata. Its elicitor activity on tobacco (Nicotiana tabacum) was compared to that of oligogalacturonides with a mean degree of polymerization of 10. The two oligosaccharides were perceived by suspension-cultured cells as distinct chemical stimuli but triggered a similar and broad spectrum of defense responses. A dose of 200 microg mL(-1) laminarin or oligogalacturonides induced within a few minutes a 1.9-pH-units alkalinization of the extracellular medium and a transient release of H(2)O(2). After a few hours, a strong stimulation of Phe ammonia-lyase, caffeic acid O-methyltransferase, and lipoxygenase activities occurred, as well as accumulation of salicylic acid. Neither of the two oligosaccharides induced tissue damage or cell death nor did they induce accumulation of the typical tobacco phytoalexin capsidiol, in contrast with the effects of the proteinaceous elicitor beta-megaspermin. Structure activity studies with laminarin, laminarin oligomers, high molecular weight beta-1, 3-1,6 glucans from fungal cell walls, and the beta-1,6-1,3 heptaglucan showed that the elicitor effects observed in tobacco with beta-glucans are specific to linear beta-1,3 linkages, with laminaripentaose being the smallest elicitor-active structure. In accordance with its strong stimulating effect on defense responses in tobacco cells, infiltration of 200 microg mL(-1) laminarin in tobacco leaves triggered accumulation within 48 h of the four families of antimicrobial pathogenesis-related proteins investigated. Challenge of the laminarin-infiltrated leaves 5 d after treatment with the soft rot pathogen Erwinia carotovora subsp. carotovora resulted in a strong reduction of the infection when compared with water-treated leaves.     

Dexamethasone-induced killing of neoplastic cells of lymphoid derivation: lack of early calcium involvement

The role of calcium influx in dexamethasone-induced fragmentation of DNA was studied in the glucocorticoid-sensitive human lymphoid line of T cell derivation (CEM-C7). Reduction of calcium content in the medium or the use of EGTA increased DNA fragmentation and appeared to slightly enhance the effect of dexamethasone. Incubation of isolated nuclei in the presence of high concentrations of calcium did not bring about significant DNA fragmentation. Calmidazolium, an antagonist of calmodulin dependent reactions did not reduce the sensitivity of CEM-C7 cells to dexamethasone nor did it modify the response to dexamethasone of the resistant CEM-C1 line. It appears that in contrast to rodent thymocytes, massive calcium influx is not per se responsible for the initiation of directed cell killing (apoptosis).     

Sulfated fucan oligosaccharides elicit defense responses in tobacco and local and systemic resistance against tobacco mosaic virus

Sulfated fucans are common structural components of the cell walls of marine brown algae. Using a fucan-degrading hydrolase isolated from a marine bacterium, we prepared sulfated fucan oligosaccharides made of mono- and disulfated fucose units alternatively bound by alpha-1,4 and alpha-1,3 glycosidic linkages, respectively. Here, we report on the elicitor activity of such fucan oligosaccharide preparations in tobacco. In suspension cell cultures, oligofucans at the dose of 200 microg ml(-1) rapidly induced a marked alkalinization of the extracellular medium and the release of hydrogen peroxide. This was followed within a few hours by a strong stimulation of phenylalanine ammonia-lyase and lipoxygenase activities. Tobacco leaves treated with oligofucans locally accumulated salicylic acid (SA) and the phytoalexin scopoletin and expressed several pathogenesis-related (PR) proteins, but they displayed no symptoms of cell death. Fucan oligosaccharides also induced the systemic accumulation of SA and the acidic PR protein PR-1, two markers of systemic acquired resistance (SAR). Consistently, fucan oligosaccharides strongly stimulated both local and systemic resistance to tobacco mosaic virus (TMV). The use of transgenic plants unable to accumulate SA indicated that, as in the SAR primed by TMV, SA is required for the establishment of oligofucan-induced resistance.     

Cytological indication of the involvement of calcium and calcium-related proteins in the early responses ofBryonia dioica to mechanical stimulus

Summary The distribution of membrane-bound calium, activated calmodulin, and callose synthesis was visualized inBryonia dioica internodes before and after mechanical stimulus, using fluorescent probes, respectively, chlorotetracycline, fluphenazine, and aniline blue. Bright chlorotetracycline fluorescence remains localized in the plasma membrane of control cells, 30 s after stimulation calcium left the plasmalemma. A delocalization of activated calmodulin was observed after wounding and deposition of callose, which could not be detected before, appeared in the same times in most cells. The callose formation and the decrease in membrane-associated calcium suggest a rapid influx of calcium in the cytosol and an intervention of this ion in the cascade of the early events underlyingBryonia dioica thigmomorphogenesis.Abbreviation CTC chlorotetracycline     

Active oxygen species production in tobacco cells elicited by cryptogein*

We analyse the relationship between active oxygen species (AOS) production and pH changes induced in tobacco cells by cryptogein, a fungal proteinaceous elicitor of defence mechanisms in plants. When tobacco cells were treated with cryptogein, an intracellular acidification, an alkalinization of the extracellular medium and a transient burst of AOS (H₂O₂) were observed. Treatment of elicited cells with either diphenyleneiodonium (DPI), an inhibitor of the neutrophil NADPH oxidase, or Tiron, which scavenges O₂^˙? abolished AOS production. These data suggest the involvement of a NADPH oxidase-like enzyme leading to H₂O₂ production through O₂^˙? dismutation. Although H₂O₂ production could be, per se, the origin of the pH changes observed, we showed that it was not the main cause, since DPI and Tiron did not inhibit extracellular alkalinization. On the other hand, cryptogein-induced changes in pH could be abolished using fusicoccin (FC), which is known to stimulate the plasmalemma H⁺ ATPase. Consequently, the observed changes in pH induced by cryptogein could be mainly due to the inhibition of the plasmalemma H⁺-ATPase activity. Furthermore, changes in extracellular pH were shown to modulate the intensity of AOS production by elicited cells. The possible regulation of the NAD(P)H oxidase activity of plant cells by changes in pH is further discussed.     

A novel elicitor identified from Magnaporthe oryzae triggers defense responses in tobacco and rice

Key message

Our studies indicate a potential important elicitor candidate which can aid in the fight against a worldwide disease, rice blast.

Abstract

In this study, we report the purification, identification, characterization, and gene cloning of a novel hypersensitive response-inducing protein elicitor (MoHrip2) secreted from an important pathogenic fungus, Magnaporthe oryzae. The protein fraction was isolated from the culture filtrate of M. oryzae and identified by de novo sequencing. The elicitor-encoding gene mohrip2 was cloned following sequence comparison and PCR amplification. This 459-bp gene encodes a 152-residue polypeptide that contains an 18-residue signal peptide and exhibits a pI of 4.72 and an apparent molecular mass of 16 kDa. The hypothetical protein, MoHrip2, was expressed in Escherichia coli, and both the recombinant and the endogenous protein caused necrotic lesions in tobacco leaves. In addition to phenolic compound deposition and alkalization of the extracellular medium, MoHrip2 also induced hydrogen peroxide production and nitric oxide accumulation in tobacco cells. Moreover, rice seedlings treated with MoHrip2 exhibited pronounced resistance to M. oryzae compared with control seedlings.     

Regulation of Arabidopsis defense responses against Spodoptera littoralis by CPK-mediated calcium signaling

Background

Genetic markers and linkage mapping are basic prerequisites for marker-assisted selection and map-based cloning. In the case of the key grassland species Lolium spp., numerous mapping populations have been developed and characterised for various traits. Although some genetic linkage maps of these populations have been aligned with each other using publicly available DNA markers, the number of common markers among genetic maps is still low, limiting the ability to compare candidate gene and QTL locations across germplasm.

Results

A set of 204 expressed sequence tag (EST)-derived simple sequence repeat (SSR) markers has been assigned to map positions using eight different ryegrass mapping populations. Marker properties of a subset of 64 EST-SSRs were assessed in six to eight individuals of each mapping population and revealed 83% of the markers to be polymorphic in at least one population and an average number of alleles of 4.88. EST-SSR markers polymorphic in multiple populations served as anchor markers and allowed the construction of the first comprehensive consensus map for ryegrass. The integrated map was complemented with 97 SSRs from previously published linkage maps and finally contained 284 EST-derived and genomic SSR markers. The total map length was 742 centiMorgan (cM), ranging for individual chromosomes from 70 cM of linkage group (LG) 6 to 171 cM of LG 2.

Conclusions

The consensus linkage map for ryegrass based on eight mapping populations and constructed using a large set of publicly available Lolium EST-SSRs mapped for the first time together with previously mapped SSR markers will allow for consolidating existing mapping and QTL information in ryegrass. Map and markers presented here will prove to be an asset in the development for both molecular breeding of ryegrass as well as comparative genetics and genomics within grass species.     

Role of calcium in serine transport into tobacco cells

The transport of serine into tobacco (Nicotiana tabacum L. var. Xanthi) cells grown in liquid medium was studied. Serine transport was maximal below pH 4.0. A time-dependent stimulation of transport was observed when cells were incubated in medium containing 0.5 mm Ca(2+). Maximum transport rates were achieved after 6 hours preincubation in Ca(2+). The following three distinct roles of Ca(2+) in serine transport were demonstrated: time-dependent stimulation of transport rate, maintenance of high transport rates, and retention of transported material. Stimulation occurred in the presence of either Ca(2+) or Mg(2+) and was inhibited by either La(3+) or K(+). Removal of Ca(2+) from the transport medium caused a rapid decline in the rate of serine uptake. This decline was prevented by addition of La(3+) after Ca(2+) removal. Cells transferred to medium lacking Ca(2+) lost substantial amounts of transported serine, this loss was significantly reduced by either La(3+) or K(+).Cells placed in (45)Ca(2+) rapidly bound more than 3 micromoles of Ca(2+)/gram fresh weight, which was exchangeable within 10 minutes with medium Ca(2+). Seventy-five per cent of the (45)Ca(2+) transported into the cells in 4 hours could be exchanged with medium Ca(2+) in the same period. The amount of net Ca(2+) transport into tobacco cells is insignificant relative to the total exchangeable Ca(2+).It is proposed that serine transport into tobacco cells involves H(+) cotransport and that the stimulation by Ca(2+) is due to an increase in the proton-motive force.     

Nonspecific elicitation of defense reaction in suspension tobacco cells by elicitors fromArmillaria

Ergosterol and chitin oligomers were detected in water extracts from Armillaria gallica, A. cepistipes, A. tabescens, A. ostoyae and A. mellea containing as active components elicitors able to trigger early events of defense reaction in suspension tobacco cells. More virulent strains of A. ostoyae and A. mellea had the same ability of elicitation as weak pathogens A. gallica, A. cepistipes, A. tabescens. The elicitation of the defense reaction early events by chitin oligomers was markedly enhanced by ergosterol probably due to the activation of several signal pathways.     

Protein composition of tobacco leaves with antiviral resistance induced by activators of defense responses and TMV infection

In tobacco (Nicotiana tabacum L.) plants of hypersensitive cv. Samsun NN, a capability of necrosis lesion formation and protein patterns were studied after induction of antiviral resistance by defense responses activators (DRA) (arachidonic acid, ubiquinone 50, and vitamin E) and by infection with tobacco mosaic virus (TMV). DRA and TMV improved both local and systemic leaf resistance to TMV. Native protein electrophoresis demonstrated differences in the composition of leaf proteins extracted under acidic and alkaline conditions. SDS-PAGE revealed proteins accumulated during the development of systemic antiviral resistance after lower leaf treatments with DRA and of local resistance induced by pretreatment with TMV. It was shown that various DRA affected protein patterns similarly, whereas TMV infection resulted in other changes. It is supposed that different pathways function in tobacco plants during induction of systemic resistance by DRA and TMV infection.     

Scanning electron-microscopic study of the involvement of coelomic cells in earthworm antibacterial defense

Summary The first steps of an antibacterial reaction in the earthworm Eisenia fetida andrei were investigated. The main cellular mediators of this activity are the chloragocytes, a class of free coelomocytes existing only in annelids. Our observations using scanning electron microscopy have shown that chloragocytes were able to agglutinate and perhaps to destroy pathogenic bacteria such as Bacillus megaterium in the same way that they agglutinate and lyse vertebrate erythrocytes. Bacteria known to be non-pathogenic for the worm, such as Acinetobacter, were not agglutinated but slowly eliminated by segregation into brown bodies. Chloragocytes maintained in vitro, lost their chloragosomes and exhibited stronger agglutination activity against pathogenic bacteria than chloragocytes in situ. From this increased efficiency of chloragocytes in vitro, we infer that, in normal living conditions, chloragocytes probably intervene in antibacterial defense mainly after their extrusion from the coelomic cavity and their spreading and degranulation at the surface of the integument.