József Fodor

Salt tolerance of barley induced by the root endophyte Piriformospora indica is associated with a strong increase in antioxidants.

The root endophytic basidiomycete Piriformospora indica has been shown to increase resistance against biotic stress and tolerance to abiotic stress in many plants. Biochemical mechanisms underlying P. indica-mediated salt tolerance were studied in barley (Hordeum vulgare) with special focus on antioxidants. Physiological markers for salt stress, such as metabolic activity, fatty acid composition, lipid peroxidation, ascorbate concentration and activities of catalase, ascorbate peroxidase, dehydroascorbate reductase, monodehydroascorbate reductase and glutathione reductase enzymes were assessed. Root colonization by P. indica increased plant growth and attenuated the NaCl-induced lipid peroxidation, metabolic heat efflux and fatty acid desaturation in leaves of the salt-sensitive barley cultivar Ingrid. The endophyte significantly elevated the amount of ascorbic acid and increased the activities of antioxidant enzymes in barley roots under salt stress conditions. Likewise, a sustained up-regulation of the antioxidative system was demonstrated in NaCl-treated roots of the salt-tolerant barley cultivar California Mariout, irrespective of plant colonization by P. indica. These findings suggest that antioxidants might play a role in both inherited and endophyte-mediated plant tolerance to salinity.

Local and Systemic Responses of Antioxidants to Tobacco Mosaic Virus Infection and to Salicylic Acid in Tobacco (Role in Systemic Acquired Resistance).

Changes in ascorbate and glutathione levels and in activities of ascorbate peroxidase, catalase, dehydroascorbate reductase (DHAR), glutathione reductase (GR), glutathione S-transferase (GST), and superoxide dismutase (SOD) were investigated in tobacco mosaic virus (TMV)-inoculated lower leaves and in non-inoculated upper leaves of Nicotiana tabacum L. cv Xanthi-nc. In separate experiments the effects of exogenous salicylic acid (SA) were also studied. Symptom appearance after TMV inoculation was preceded by a slight, transient decline of ascorbate peroxidase, GR, GST, and SOD activities in the inoculated lower leaves, but after the onset of necrosis these activities and the glutathione level substantially increased. Ascorbic acid level and DHAR activity declined and dehydroascorbate accumulated in the inoculated leaves. In upper leaves, the glutathione level and the activities of GR, GST, and SOD increased 10 to 14 d after TMV inoculation of the lower leaves, concomitantly with the development of systemic acquired resistance. From the six distinct SOD isoenzymes found in tobacco leaves, only the activities of Cu,Zn-SOD isoenzymes were affected by TMV. SA injection induced DHAR, GR, GST, and SOD activities. Catalase activities were not modified by TMV infection or SA treatment. It is supposed that stimulated antioxidative processes contribute to the suppression of necrotic symptom development in leaves with systemic acquired resistance.

The Janus face of reactive oxygen species in resistance and susceptibility of plants to necrotrophic and biotrophic pathogens

Plant pathogens can be divided into biotrophs and necrotrophs according to their different life styles; biotrophs prefer living, while necrotrophs prefer dead cells for nutritional purposes. Therefore tissue necrosis caused by reactive oxygen species (ROS) during pathogen infection increases host susceptibility to necrotrophic, but resistance to biotrophic pathogen. Consequently, elevation of antioxidant capacity of plants enhances their tolerance to development of necroses caused by necrotrophic pathogens. Plant hormones can strongly influence induction of ROS and antioxidants, thereby influencing susceptibility or resistance of plants to pathogens. Pathogen-induced ROS themselves are considered as signaling molecules. Generally, salicylic acid (SA) signaling induces defense against biotrophic pathogens, whereas jasmonic acid (JA) against necrotrophic pathogens. Furthermore pathogens can modify plants defense signaling network for their own benefit by changing phytohormone homeostasis. On the other hand, ROS are harmful also to the pathogens, consequently they try to defend themselves by elevating antioxidant activity and secreting ROS scavengers in the infected tissue. The Janus face nature of ROS and plant cell death on biotrophic and on necrotrophic pathogens is also supported by the experiments with BAX inhibitor-1 and the mlo mutation of Mlo gene in barley. It was found that ROS and elevated plant antioxidant activity play an important role in systemic acquired resistance (SAR) and induced systemic resistance (ISR), as well as in mycorrhiza induced abiotic and biotic stress tolerance of plants.

Suppression of tobacco mosaic virus-induced hypersensitive-type necrotization in tobacco at high temperature is associated with downregulation of NADPH oxidase and superoxide and stimulation of dehydroascorbate reductase

Tissue necroses and resistance during the hypersensitive response (HR) of tobacco to tobacco mosaic virus (TMV) are overcome at temperatures above 28 degrees C and the virus multiplies to high levels in the originally resistant N-gene expressing plants. We have demonstrated that chemical compounds that generate reactive oxygen species (ROS) or directly applied hydrogen peroxide (H(2)O(2)) are able to induce HR-type necroses in TMV-inoculated Xanthi-nc tobacco even at high temperatures (e.g. 30 degrees C). The amount of superoxide (O(2)(*-)) decreased, while H(2)O(2) slightly increased in TMV- and mock-inoculated leaves at 30 degrees C, as compared with 20 degrees C. Activity of NADPH oxidase and mRNA levels of genes that encode NADPH oxidase and an alternative oxidase, respectively, were significantly lower, while activity of dehydroascorbate reductase was significantly higher at 30 degrees C, as compared with 20 degrees C. It was possible to reverse or suppress the chemically induced HR-type necrotization at 30 degrees C by the application of antioxidants, such as superoxide dismutase and catalase, demonstrating that the development of HR-type necroses indeed depends on a certain level of superoxide and other ROS. Importantly, high TMV levels at 30 degrees C were similar in infected plants, whether the HR-type necrotization developed or not. Suppression of virus multiplication in resistant, HR-producing tobacco at lower temperatures seems to be independent of the appearance of necroses but is associated with temperatures below 28 degrees C.

Barley Mla and Rar mutants compromised in the hypersensitive cell death response against Blumeria graminis f.sp. hordei are modified in their ability to accumulate reactive oxygen intermediates at sites of fungal invasion

Abstract. The pathogenesis-related accumulation of superoxide radical anions (O·−2) and hydrogen peroxide (H2O2) was comparatively analyzed in a barley line (Hordeum vulgare L. cv Sultan-5) carrying the powdery mildew (Blumeria graminis f.sp. hordei, Speer, Bgh) resistance gene Mla12, and in susceptible mutants defective in Mla12 or in genes “required for Mla12-specified disease resistance” (Rar1 and Rar2). In-situ localization of reactive oxygen intermediates was performed both by microscopic detection of azide-insensitive nitroblue tetrazolium (NBT) reduction or diaminobenzidine (DAB) polymerization, and by an NBT-DAB double-staining procedure. The Mla12-mediated hypersensitive cell death occurred either in attacked epidermal cells or adjacent mesophyll cells of wild-type plants. Whole-cell H2O2 accumulation was detected in dying cells, while O·−2 emerged in adjacent cells. Importantly, all susceptible mutants lacked these reactions. An oxalate oxidase, which is known to generate H2O2 and has been implicated in barley resistance against the powdery mildew fungus, was not differentially expressed between the wild type and all mutants. The results demonstrate that the Rar1 and Rar2 gene products, which are control elements of R-gene-mediated programmed cell death, also control accumulation of reactive oxygen intermediates but not the pathogenesis-related expression of oxalate oxidase.

Elevation of glutathione level and activation of glutathione-related enzymes affect virus infection in tobacco.

The effects of two chemicals, L-2-oxothiazolidine-4-carboxylic acid (OTC) and (S)-carvone, were investigated on the development of necrotic symptoms and on the virus concentration in tobacco mosaic virus (TMV)-infected tobacco plants. OTC treatments markedly increased the cellular glutathione (GSH) levels in tobacco leaf discs. In addition, OTC pretreatment considerably decreased both the number of necrotic lesions and the virus content in TMV-infected leaf discs. The monoterpene (S)-carvone increased only slightly the GSH content of leaf tissues and caused lipid peroxidation. (S)-carvone dramatically induced the activity of glutathione S-transferase and to a lesser extent elevated also the activities of ascorbate peroxidase and glutathione reductase. Treatments with (S)-carvone strongly reduced the number and size of necrotic lesions, but did not influence the virus concentration. The results show that increased levels of GSH and activities of GSH-related enzymes by OTC and (S)-carvone reduce necrotization of virus-infected tissues. However, virus multiplication and lesion formation do not necessarily correlate: virus multiplication is suppressed only by substantially elevated GSH contents.

Down-regulation of antioxidative capacity in a transgenic tobacco which fails to develop acquired resistance to necrotization caused by TMV.

Antioxidant status was assayed in leaves of two local lesion hosts of tobacco mosaic virus (TMV), namely in wild-type Xanthi-nc tobacco and in NahG transgenic tobacco, the latter of which is not able to accumulate salicylic acid (SA) and therefore is unable to develop systemic acquired resistance (SAR). Activities of several enzymes related to antioxidative defense, and the levels of glutathione, chlorogenic acid and rutin were studied. The majority of antioxidant enzymes were less active in uninfected NahG tobacco than in Xanthi-nc. Furthermore, important enzymatic and non-enzymatic antioxidants were down-regulated in TMV-infected NahG plants, as compared to Xanthi-nc. Correspondingly, SA pretreatment primed the leaves for stronger induction of antioxidants in infected Xanthi-nc, but not in NahG tobaccos. The antioxidant status of NahG tobacco even decreased after an attempted induction of SAR, while the antioxidative level increased in Xanthi-nc leaves in which the SAR was successfully induced. After infection, a greater accumulation of superoxide and H 2 O 2, and a more intensive necrotization was positively correlated with the reduced capability of NahG leaf tissue to detoxify reactive oxygen species.

The mutualistic fungus Piriformospora indica protects barley roots from a loss of antioxidant capacity caused by the necrotrophic pathogen Fusarium culmorum.

Fusarium culmorum causes root rot in barley (Hordeum vulgare), resulting in severely reduced plant growth and yield. Pretreatment of roots with chlamydospores of the mutualistic root-colonizing basidiomycete Piriformospora indica (subdivision Agaricomycotina) prevented necrotization of root tissues and plant growth retardation commonly associated with Fusarium root rot. Quantification of Fusarium infections with a real-time polymerase chain reaction assay revealed a correlation between root rot symptoms and the relative amount of fungal DNA. Fusarium-infected roots showed reduced levels of ascorbate and glutathione (GSH), along with reduced activities of antioxidant enzymes such as superoxide dismutase, ascorbate peroxidase, GSH reductase, dehydroascorbate reductase, and monodehydroascorbate reductase. Consistent with this, Fusarium-infected roots showed elevated levels of lipid hydroperoxides and decreased ratios of reduced to oxidized forms of ascorbate and GSH. In clear contrast, roots treated with P. indica prior to inoculation with F. culmorum showed levels of ascorbate and GSH that were similar to controls. Likewise, lipid peroxidation and the overall reduction in antioxidant enzyme activities were largely attenuated by P. indica in roots challenged by F. culmorum. These results suggest that P. indica protects roots from necrotrophic pathogens, at least partly, through activating the plants antioxidant capacity.

Functional assessment of the pathogenesis-related protein PR-1b in barley

Abstract The pathogenesis-related protein 1 (PR-1b) of barley (Hordeum vulgare L.) is a marker for the attack by the powdery mildew fungus (Blumeria graminis f.sp. hordei, Bgh) and other pathogens. PR-1b consists of 164 amino acids and has a potential signal peptide for export into the cell wall. Here, we show that PR-1b is differentially expressed in near-isogenic barley lines exhibiting various forms of defence phenotypes including papilla formation and the hypersensitive cell death. To elucidate PR-1b function, we transiently silenced PR-1 expression by double stranded RNA (dsRNA) interference in the moderately susceptible barley double mutant line A89 (genotype: mlo5–ror1), which shows a papillae-based defence phenotype. Upon bombardment of leaf segments with PR-1b dsRNA and a GFP marker gene construct, Bgh slightly more frequently penetrated the plant cell wall of transformed epidermal cells relative to cells bombarded with human control dsRNA. We conclude that PR-1b contributes to penetration resistance to the powdery mildew fungus in barley. We also observed that PR-1b expression correlates with the production of H2O2 in responses to Bgh and Bipolaris sorokiniana and was induced upon infiltration of the H2O2 producing mixture of glucose and glucose oxidase.

The regulation of δ11-desaturase gene expression in the pheromone gland of Mamestra brassicae (Lepidoptera; Noctuidae) during pheromonogenesis

Cabbage moth (Mamestra brassicae) females produce sex pheromones to attract conspecific males. In our M. brassicae colony, the pheromone blend is composed of Z11-hexadecenyl acetate (Z11-16Ac) and hexadecyl acetate (16Ac) in a 93:7 ratio. A fatty acyl Δ11-desaturase is involved in the production of the main pheromone component. The release of Pheromone Biosynthesis Activating Neuropeptide (PBAN) regulates the pheromone production in the pheromone gland (PG). We cloned a cDNA encoding the MambrΔ11-desaturase and analyzed its expression profile over time in M. brassicae tissues. Transcript levels of the Δ11-desaturase in larvae, pupal PGs, fat body, brain and muscle tissues were <0.1% of that in female PGs, whereas expression in male genitalia was 2%. In the PGs of virgin females the expression level increased continuously from eclosion to the end of the 1st day when it reached a plateau without further significant fluctuation up to the 8th day. In contrast, we recorded a characteristic daily rhythmicity in pheromone production with a maximum around 200 ng Z11-16Ac/PG. In some experiments, females were decapitated to prevent PBAN release and thereby inhibit pheromone production, which remarkably increased after treatment with Mambr-Pheromonotropin. Further experiments revealed that mating resulted in a significant suppression of pheromone production. However, expression of the Δ11-desaturase was not affected by any of these interventions, suggesting that its not regulated by PBAN. Fluorescent microscopy was used to study the potential role of lipid droplets during pheromone production, however, no lipid droplets were identified indicating that pheromonogenesis is regulated via de novo fatty acid synthesis.