Superoxide Generation in Chemically Activated Resistance of Barley in Response to Inoculation with the Powdery Mildew Fungus

In a previous work, a phenotype-specific accumulation of superoxide radical anions (O^??₂) after attack of the powdery mildew fungus (Blumeria [syn. Erysiphe] graminis f.sp. hordei) in near-isogenic barley (Hordeum vulgare L.) lines bearing different Mlx genes for resistance was described (Hückelhoven and Kogel, 1998). We have now a histochemical study of the pathogenesis-related O^??₂ generation in the systemic activated resistance (SAR) response induced in barley cv Pallas by the plant activator 2,6-dichloroisonicotinic acid (DCINA). SAR-specific defence was conducted prevalently characterized by penetration resistance. Fungal arrest was observed before haustorium formation by a highly localized cell wall reinforcement (effective papillae) and, in most cases, by a subsequent hypersensitive cell death (HR). No O^??₂ generation was found in association with these plant defence responses. However, a strong O^??₂ burst in the attacked epidermal cells was detected in the control plants which were not activated by DCINA. This burst coincided with cell wall penetration and subsequent contact of the pathogen with the host plasma membrane. A strong SAR-related O^??₂ burst was induced in the mesophyll tissue beneath the attacked and hypersensitively reacting epidermal cells in plants treated with DCINA. The accumulation of O^??₂ was confined to chloroplasts. The remarkable burst in mesophyll tissue was not followed by mesophyll-HR indicating that chloroplastic O^??₂ generation is not sufficient for the hypersensitive cell death. Since the same pattern of pathogenesis-related O^??₂ accumulation was identified for race-specific response mediated by the Mlg gene for powdery mildew resistance, the present data are consistent with the hypothesis that the SAR phenotype is a phenocopy of the Mlg-type resistance (Kogel et al., 1994).     

Early H(2)O(2) accumulation in mesophyll cells leads to induction of glutathione during the hyper-sensitive response in the barley-powdery mildew interaction

H(2)O(2) production and changes in glutathione, catalase, and peroxidase were followed in whole-leaf extracts from the susceptible (AlgS [Algerian/4* (F14) Man.(S)]; ml-a1 allele) and resistant (AlgR [Algerian/4* (F14) Man.(R)]; Ml-a1 allele) barley (Hordeum vulgare) isolines between 12 and 24 h after inoculation with powdery mildew (Blumeria graminis [DC]. Speer [syn. Erysiphe graminis DC] f.sp hordei Marchal). Localized papilla responses and cell death hypersensitive responses were not observed within the same cell. In hypersensitive response sites, H(2)O(2) accumulation first occurred in the mesophyll underlying the attacked epidermal cell. Subsequently, H(2)O(2) disappeared from the mesophyll and accumulated around attacked epidermal cells. In AlgR, transient glutathione oxidation coincided with H(2)O(2) accumulation in the mesophyll. Subsequently, total foliar glutathione and catalase activities transiently increased in AlgR. These changes, absent from AlgS, preceded inoculation-dependent increases in peroxidase activity that were observed in both AlgR and AlgS at 18 h. An early intercellular signal precedes H(2)O(2), and this elicits anti-oxidant responses in leaves prior to events leading to death of attacked cells.     

Functional analysis and RFLP-mediated mapping of the Mlg resistance locus in barley

The semi-dominantly acting allele of the Mlg locus in barley confers a race-specific resistance to the causal agent of powdery mildew, Erysiphe graminis f.sp. hordel . High resolution genetic mapping via RFLP analysis enabled us to identify a set of markers on chromosome 4 which are either tightly linked or co-segregate with the resistance function. Comparison of marker distances in segregating F₂ populations originating from different genotypes revealed an unexpected variation of recombination frequencies in the vicinity of the locus. Based on near-isogenic lines carrying either resistance gene Mlg or Mla12 and a cell-type specific analysis it was demonstrated that Mlg acts at an earlier stage of fungal development than Mla12 . A time-course analysis of the early infection process combined with gene dosage experiments provided strong evidence that attacked cells retain viability after the defence response and that hypersensitive cell death (HR) is a secondary consequence, but not causally required for Mlg -mediated arrest of fungal growth. It is speculated that gene dosage experiments may provide a means to separate primary from secondary defence reactions.     

Non-host resistance of barley is associated with a hydrogen peroxide burst at sites of attempted penetration by wheat powdery mildew fungus

In barley, non-host resistance against the wheat powdery mildew fungus (Blumeria graminis f.sp. tritici, Bgt) is associated with the formation of cell wall appositions and a hypersensitive reaction in which epidermal cells die rapidly in response to fungal attack. In the interaction of barley with the pathogenic barley powdery mildew fungus (Blumeria graminis f.sp. hordei, Bgh), these defence reactions are also associated with accumulation of H₂O₂. To elucidate the mechanism of non-host resistance, the accumulation of H₂O₂ in response to Bgt was studied in situ by histochemical staining with diaminobenzidine. H₂O₂ accumulation was found in cell wall appositions under appressoria from Bgt and in cells undergoing a hypersensitive reaction. A mutation (mlo5) at the barley Mlo locus, that confers broad spectrum resistance to Bgh, did not influence the barley defence phenotype to Bgt. Significantly, Bgt triggered cell death on mlo5-barley while Bgh did not.     

Hypersensitive cell death and papilla formation in barley attacked by the powdery mildew fungus are associated with hydrogen peroxide but not with salicylic acid accumulation

We analyzed the pathogenesis-related generation of H₂O₂ using the microscopic detection of 3,3-diaminobenzidine polymerization in near-isogenic barley (Hordeum vulgare L.) lines carrying different powdery mildew (Blumeria graminis f.sp. hordei) resistance genes, and in a line expressing chemically activated resistance after treatment with 2,6-dichloroisonicotinic acid (DCINA). Hypersensitive cell death in Mla12 and Mlg genotypes or after chemical activation by DCINA was associated with H₂O₂ accumulation throughout attacked cells. Formation of cell wall appositions (papillae) mediated in Mlg and mlo5 genotypes and in DCINA-activated plants was paralleled by H₂O₂ accumulation in effective papillae and in cytosolic vesicles of up to 2 μm in diameter near the papillae. H₂O₂ was not detected in ineffective papillae of cells that had been successfully penetrated by the fungus. These findings support the hypothesis that H₂O₂ may play a substantial role in plant defense against the powdery mildew fungus. We did not detect any accumulation of salicylic acid in primary leaves after inoculation of the different barley genotypes, indicating that these defense responses neither relied on nor provoked salicylic acid accumulation in barley.     

Involvement of superoxide generation in salicylic acid-induced stomatal closure in Vicia faba.

Salicylic acid (SA), the known mediator of systemic acquired resistance, induced stomatal closure of Vicia faba L. Application of SA to the epidermal peels evoked an elevation of chemiluminescence of Cripridina lucigenin-derived chemiluminescent reagent (CLA) which is sensitive to superoxide anion (O(2)(.-)). The SA-induced generation of chemiluminescence was suppressed by O(2)(.-)-specific scavengers superoxide dismutase (SOD) and 4,5-dihydroxy-1,3-benzenedisulfonic acid (Tiron). These results suggest that O(2)(.-) was generated in epidermal peels by SA-treatment. A peroxidase inhibitor salicylhydroxamic acid (SHAM) inhibited guaiacol peroxidase activity and suppressed the SA-induced CLA chemiluminescence in the epidermal peels, suggesting that O(2)(.-) generation occurred by the peroxidase-catalyzed reaction as proposed for SA-treated tobacco cell suspension culture [Kawano et al. (1998) Plant Cell Physiol. 39: 721]. SOD, Tiron or SHAM suppressed the SA-induced stomatal closure. Moreover, application of superoxide-generating system also induced stomatal closure. These results support the concept of involvement of reactive oxygen species in signal transduction in SA-induced stomatal closure.     

低聚糖素诱导橡胶树抗白粉病作用机制初探

通过比较喷洒低聚糖素后橡胶树叶片表皮细胞结构、过氧化物酶活性和酚类物质含量的变化表明,低聚糖素可诱导橡胶树的组织结构及生理生化过程发生变化。这些因素是低聚糖素提高橡胶树对白粉病抗病性的重要作用机制。     

Superoxide and hydrogen peroxide play different roles in the nonhost interaction of barley and wheat with inappropriate formae speciales of Blumeria graminis

Nonhost resistance of cereals to inappropriate formae speciales of Blumeria graminis is little understood. However, on the microscopic level, nonhost defense to B. graminis is reminiscent of host defense preventing fungal development by penetration resistance and the hypersensitive cell death response (HR). We analyzed histochemically the accumulation of superoxide anion radicals (O2*-) and hydrogen peroxide (H2O2) at sites of B. graminis attack in nonhost barley and wheat. Superoxide visualized by subcellular reduction of nitroblue tetrazolium accumulated in association with successful fungal penetration in attacked cells and in cells neighboring HR. In contrast, H2O2 accumulated in cell wall appositions beneath fungal penetration attempts or in the entire epidermal cell during HR. The data provide evidence for different roles and sources of superoxide and H2O2 in the nonhost interaction of cereals with inappropriate formae speciales of B. graminis.     

An Early Indicator of Resistance in Barley to Russian Wheat Aphid

During early stages of infestation by Russian wheat aphids (Diuraphis noxia [Mordvilko]; RWAs), barley (Hordeum vulgare L.) leaf cells collapsed and showed autofluorescence in the mesophyll and bundle sheath adjacent to the RWA stylet sheath. The response was visually similar to the hypersensitive cell death response, typical of resistance to microbial pathogens. Resistant barley produced significantly more collapsed, autofluorescent cells (CAC) than did susceptible barley. RWA stylet entry sites and sheath paths also fluoresced, making them easy to observe in whole leaf sections. The number of CAC increased with the number of RWAs and with the number of days of feeding in resistant plants. The CAC could be observed 1 d following infestation, making this the most rapid plant response toward the RWAs known to date. The response may be useful in screening for resistant plants and may provide insight into resistance mechanisms in barley.     

Separate Assays Specific for Ascorbate Peroxidase and Guaiacol Peroxidase and for the Chloroplastic and Cytosolic Isozymes of Ascorbate Peroxidase in Plants

Ascorbate (AsA) peroxidase can be inactivated both by p-chloromercuribenzoateand by the depletion of AsA but guaiacol peroxidases, such ashorseradish peroxidase, cannot. The cytosolic isozymes of AsAperoxidase are less sensitive to depletion of AsA than the chloroplasticisozymes, which include stromal [Chen and Asada (1989) PlantCell Physiol. 30: 987] and thyla-koid-bound [Miyake and Asada(1992) Plant Cell Physiol. 33: 541] enzymes. Exploring theseproperties, we established simple methods for separate assaysof AsA peroxidase and guaiacol peroxidase and of the three isozymesof AsA peroxidase in plant extracts. These methods were usedto characterize the guaiacol peroxidases and isozymes of AsAperoxidase in plants and algae. (Received October 20, 1993; Accepted February 7, 1994)     

Enhanced-peroxidatic activity in specific catalase isozymes of tobacco, barley, and maize

Separation of catalase isozymes from leaf extracts of three diverse plant species (Nicotiana sylvestris, Zea mays, Hordeum vulgare L.) revealed a distinct isozyme with enhanced peroxidatic activity (30-, 70-, 28-fold over typical catalase, respectively) which constituted 10 to 20% of the total catalase activity. In maize this isozyme is the product of the Cat3 gene, which is expressed only in mesophyll cells (AS Tsaftaris, AM Bosabalidis, JG Scandalios [1983] Proc Natl Acad Sci USA 80: 4455-4459). A mutation in barley reducing levels of peroxisomal catalase (AC Kendall et al. [1983] Planta 159: 505-511) does not reduce the amount of the isozyme with enhanced peroxidatic activity. Similarly, this isozyme is unaffected in dark-grown barley in spite of a 75% decrease in total catalase activity. These results suggest that this catalase isozyme is under separate genetic control in barley. This may also be the case in tobacco where the catalase isozyme with enhanced peroxidatic activity is an immunologically distinct protein (EA Havir, NA McHale [1989] Plant Physiol 89: 952-957).     

Multivesicular compartments proliferate in susceptible and resistant MLA12-barley leaves in response to infection by the biotrophic powdery mildew fungus

There is growing evidence that multivesicular bodies and cell wall-associated paramural bodies participate in the enhanced vesicle trafficking induced by pathogen attack. Here, we performed transmission electron microscopy in combination with cytochemical localization of H2O2 to investigate multivesicular compartments during establishment of compatible interaction in susceptible barley (Hordeum vulgare) and during hypersensitive response in resistant MLA12-barley infected by the barley powdery mildew fungus (Blumeria graminis f. sp. hordei). Multivesicular bodies, intravacuolar vesicle aggregates and paramural bodies proliferated in the penetrated epidermal cell during development of the fungal haustorium. These vesicular structures also proliferated at the periphery of intact cells, which were adjacent to the hypersensitive dying cells and deposited cell wall appositions associated with H2O2 accumulation. All plasmodesmata between intact cells and hypersensitive cells were constricted or blocked by cell wall appositions. These results suggest that multivesicular compartments participate in secretion of building blocks for cell wall appositions not only to arrest fungal penetration but also to contain hypersensitive cell death through blocking plasmodesmata. They may also participate in internalization of damaged membranes, deleterious materials, nutrients, elicitors and elicitor receptors.     

Biochemical Basis of Resistance of Tobacco Callus Tissue Cultures to Hydroxyphenylethylamines

It has been reported that hydroxyphenylethylamines, such as tyramine and octopamine, are toxic to tobacco (Nicotiana tabacum L.) callus cultures grown in the presence of auxins, whereas calli grown in the presence of cytokinins and crown gall cultures are resistant to these amines (P. Christou and K.A. Barton [1989] Plant Physiol 89: 564-568). In an attempt to understand the underlying mechanism of this resistance, we compared the fates of tyramine in tyramine-sensitive and tyramine-resistant tobacco tissue cultures (cv Xanthi nc). The very rapid formation of black-colored oxidation products from tyramine in sensitive tissues suggested that the toxicity might be caused by the oxidation of tyramine by phenol oxidases present in the tissues or released into the medium after subculture. This was confirmed through many indirect procedures (effect of exogenously added tyrosinase, induction of polyphenol oxidase [PPO] activity by auxin, etc.). The study of tyramine structure-activity relationships further suggested that the toxicity of tyramine might be due to the formation of indolequinones after oxidation by PPO. Subculture of calli grown on 2,4-dichlorophenoxyacetic acid in a medium containing benzyladenine triggered a slow decrease in PPO activity and dramatic increases in peroxidase and tyramine hydroxycinnamoyl transferase (THT) activities. THT was undetectable in calli grown on 2,4-dichlorophenoxyacetic acid but very active in tyramine-resistant crown gall cultures. Moreover, when [3H]tyramine was fed in vivo to tyramine-resistant tissues, it was rapidly integrated into cell walls in the wound periderm formed at the periphery of the calli. Both the conjugation of tyramine and its integration into cell walls could compete with the formation of toxic quinones and therefore play a part in the resistance. Thus, it seems likely that the control of the toxicity of hydroxyphenylethylamines by cytokinins results primarily from changes in the metabolism and the compartmentation of these amines.