Miklós Pogány

Dual Roles of Reactive Oxygen Species and NADPH Oxidase RBOHD in an Arabidopsis-Alternaria Pathosystem

Arabidopsis (Arabidopsis thaliana) NADPH oxidases have been reported to suppress the spread of pathogen- and salicylic acid-induced cell death. Here, we present dual roles of RBOHD (for respiratory burst oxidase homolog D) in an Arabidopsis-Alternaria pathosystem, suggesting either initiation or prevention of cell death dependent on the distance from pathogen attack. Our data demonstrate that a rbohD knockout mutant exhibits increased spread of cell death at the macroscopic level upon inoculation with the fungus Alternaria brassicicola. However, the cellular patterns of reactive oxygen species accumulation and cell death are fundamentally different in the AtrbohD mutant compared with the wild type. Functional RBOHD causes marked extracellular hydrogen peroxide accumulation as well as cell death in distinct, single cells of A. brassicicola-infected wild-type plants. This single cell response is missing in the AtrbohD mutant, where infection triggers spreading-type necrosis preceded by less distinct chloroplastic hydrogen peroxide accumulation in large clusters of cells. While the salicylic acid analog benzothiadiazole induces the action of RBOHD and the development of cell death in infected tissues, the ethylene inhibitor aminoethoxyvinylglycine inhibits cell death, indicating that both salicylic acid and ethylene positively regulate RBOHD and cell death. Moreover, A. brassicicola-infected AtrbohD plants hyperaccumulate ethylene and free salicylic acid compared with the wild type, suggesting negative feedback regulation of salicylic acid and ethylene by RBOHD. We propose that functional RBOHD triggers death in cells that are damaged by fungal infection but simultaneously inhibits death in neighboring cells through the suppression of free salicylic acid and ethylene levels.

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.

Immunoassays for plant cytokinins as tools for the assessment of environmental stress and disease resistance.

The level of cytokinin type hormones present in plant tissues, such as N 6 -(2-isopentenyl)-adenosine (IPA), N 6 -(2isopentenyl)-adenosine (2-iP), trans-zeatin riboside (ZR) and trans-zeatin (Z) is a good indicator of the resistance of plants to abiotic environmental stresses and to necrotic pathogens. Hapten-homologous and hapten-heterologous competitive indirect enzyme-linked immunosorbent assays (ELISAs) were developed, allowing the use of minute amounts of plant extracts for cytokinin analysis. These assays were used for the detection of members of the cytokinin plant hormone family including IPA and ZR types. The assays, in optimized formats, readily detected these plant hormones at concentration levels of 2‐5 ng ml 1 , and showed high specificity for selected cytokinins. Certain assay parameters (e.g. the type of tracer enzyme, incubation and preincubation time, etc.) had a strong influence on detection sensitivity. Nonetheless, the assays appear robust showing tolerance to pH and to several water-miscible organic solvents. The described ELISA systems were sensitive enough to detect endogenous hormone levels in crude plant extracts without intense purification. In vitro selected transgenic tobacco and tomato lines showing tolerance to several stress factors proved to have higher levels of cytokinins than the corresponding control plants indicating that the developed immunoassay is suitable for the determination of stress resistance of plants by monitoring their cytokinin content.

Regulatory Proteolysis in Arabidopsis-Pathogen Interactions.

Approximately two and a half percent of protein coding genes in Arabidopsis encode enzymes with known or putative proteolytic activity. Proteases possess not only common housekeeping functions by recycling nonfunctional proteins. By irreversibly cleaving other proteins, they regulate crucial developmental processes and control responses to environmental changes. Regulatory proteolysis is also indispensable in interactions between plants and their microbial pathogens. Proteolytic cleavage is simultaneously used both by plant cells, to recognize and inactivate invading pathogens, and by microbes, to overcome the immune system of the plant and successfully colonize host cells. In this review, we present available results on the group of proteases in the model plant Arabidopsis thaliana whose functions in microbial pathogenesis were confirmed. Pathogen-derived proteolytic factors are also discussed when they are involved in the cleavage of host metabolites. Considering the wealth of review papers available in the field of the ubiquitin-26S proteasome system results on the ubiquitin cascade are not presented. Arabidopsis and its pathogens are conferred with abundant sets of proteases. This review compiles a list of those that are apparently involved in an interaction between the plant and its pathogens, also presenting their molecular partners when available.

Overlapping Yet Response-Specific Transcriptome Alterations Characterize the Nature of Tobacco-Pseudomonas syringae Interactions.

In this study transcriptomic alterations of bacterially induced pattern triggered immunity (PTI) were compared with other types of tobacco–Pseudomonas interactions. In addition, using pharmacological agents we blocked some signal transduction pathways (Ca2+ influx, kinases, phospholipases, proteasomic protein degradation) to find out how they contribute to gene expression during PTI. PTI is the first defense response of plant cells to microbes, elicited by their widely conserved molecular patterns. Tobacco is an important model of Solanaceae to study resistance responses, including defense mechanisms against bacteria. In spite of these facts the transcription regulation of tobacco genes during different types of plant bacterial interactions is not well-described. In this paper we compared the tobacco transcriptomic alterations in microarray experiments induced by (i) PTI inducer Pseudomonas syringae pv. syringae type III secretion mutant (hrcC) at earlier (6 h post inoculation) and later (48 hpi) stages of defense, (ii) wild type P. syringae (6 hpi) that causes effector triggered immunity (ETI) and cell death (HR), and (iii) disease-causing P. syringae pv. tabaci (6 hpi). Among the different treatments the highest overlap was between the PTI and ETI at 6 hpi, however, there were groups of genes with specifically altered activity for either type of defenses. Instead of quantitative effects of the virulent P. tabaci on PTI-related genes it influenced transcription qualitatively and blocked the expression changes of a special set of genes including ones involved in signal transduction and transcription regulation. P. tabaci specifically activated or repressed other groups of genes seemingly not related to either PTI or ETI. Kinase and phospholipase A inhibitors had highest impacts on the PTI response and effects of these signal inhibitors on transcription greatly overlapped. Remarkable interactions of phospholipase C-related pathways with the proteasomal system were also observable. Genes specifically affected by virulent P. tabaci belonged to various previously identified signaling routes, suggesting that compatible pathogens may modulate diverse signaling pathways of PTI to overcome plant defense.

Penetration and translocation of Erwinia amylovora-specific bacteriophages in apple - a possibility of enhanced control of fire blight

We have investigated the uptake and delivery of Erwinia amylovora-specific bacteriophages in apple plants. The main aim of this study was to assess the potential of phage application as a means for improving phage persistence and thereby the control of fire blight, the disease caused by E. amylovora. Both phage strains tested (ΦEa104 and H5K) were able to translocate in apple seedlings and were detectable by a modified Adams’ drop test and real-time qPCR in plant parts above ground level following their application to the roots. Conversely, phages were detectable in roots after spraying them onto the stem and leaves. A water suspension of phages effectively decreased symptom severity of E. amylovora infection in apple seedlings following treatment of roots or aerial plant parts and application to the cotyledon, as judged by symptom bonitation. A similar effect was achieved by spraying a phage suspension onto flowering firethorn shoots. Interestingly no significant differences in controlling E. amylovora infection were found among the two phage strains tested. It seems that phages specific to E. amylovora can penetrate plants and exhibit a decrease in severity of symptoms caused by the phytopathogen. Demonstrating in planta translocation of E. amylovora-specific bacteriophages and their effect of reducing fire blight symptoms may significantly contribute to a better control of E. amylovora and promote further investigations on penetration and translocation of phages into plants.

Characterization of Myoviridae and Podoviridae family bacteriophages of Erwinia amylovora from Hungary - potential of application in biological control of fire blight

Twelve bacteriophage isolates of Erwinia amylovora, the causal agent of fire blight, were isolated from blighted apple, pear and quince trees from different sites in Hungary. According to morphological characteristics they were assigned to the order Caudovirales, two isolates belonging to the Podoviridae and ten to the Myoviridae families. Examining plaque morphology, host range and molecular characterization by PCR established that these phages are not identical neither to the three North American strains used as references nor the earlier isolated Hungarian Siphoviridae strains. Studying the efficacy of selected phages in apple blossoms and green pear fruit slices it was found that a combination of three phage isolates (ΦEaH2A, ΦEaH5K and ΦEaH7B) significantly reduced bacterial multiplication and fire blight symptoms as compared to untreated controls. Combined application of these new E. amylovora-specific phages as biocontrol agents may contribute to a better control of E. amylovora under field conditions.

Cold hardening protects cereals from oxidative stress and necrotrophic fungal pathogenesis

Abstract The effects of cold hardening of cereals on their cross-tolerance to treatments leading to oxidative stress were investigated. Long-term exposure to low non-freezing temperatures provided partial protection to wheat and barley plants from the damage caused by paraquat and hydrogen peroxide treatments. It also conferred resistance in two barley cultivars to the necrotic symptoms and growth of the fungal phytopathogen Pyrenophora teres f. teres. Pathogen-induced oxidative burst was also reduced in cold hardened plants. The possible roles of host-derived redox factors and other signaling components in the observed forms of cereal cross-tolerance are discussed.

Description of the Nicotiana benthamiana−Cercospora nicotianae Pathosystem

Nicotiana benthamiana is a valuable model organism in plant biology research. This report describes its extended applicability in the field of molecular plant pathology by introducing a nonbiotrophic fungal pathogen Cercospora nicotianae that can be conveniently used under laboratory conditions, consistently induces a necrotic leaf spot disease on Nicotiana benthamiana, and is specialized on solanaceous plants. Our inoculation studies showed that C. nicotianae more effectively colonizes N. benthamiana than its conventional host, N. tabacum. The functions of two critical regulators of host immunity, coronatine-insensitive 1 (COI1) and ethylene-insensitive 2 (EIN2), were studied in N. benthamiana using Tobacco rattle virus-based virus-induced gene silencing (VIGS). Perturbation of jasmonic acid or ethylene signaling by VIGS of either COI1 or EIN2, respectively, resulted in markedly increased Cercospora leaf spot symptoms on N. benthamiana plants. These results suggest that the N. benthamiana-C. nicotianae host-pathogen interaction is a prospective but hitherto unutilized pathosystem for studying gene functions in diseased plants.