Zoltán Bozsó

Novel Extracellular Chitinases Rapidly and Specifically Induced by General Bacterial Elicitors and Suppressed by Virulent Bacteria as a Marker of Early Basal Resistance in Tobacco

Early basal resistance (EBR, formerly known as early induced resistance) is triggered by general bacterial elicitors. EBR has been suggested to inhibit or retard expression of the type III secretion system of pathogenic bacteria and may also prevent nonpathogenic bacteria from colonizing the plant tissue. The quickness of EBR here plays a crucial role, compensating for a low bactericidal efficacy. This inhibitory activity should take place in the cell wall, as bacteria do not enter living plant cells. We found several soluble proteins in the intercellular fluid of tobacco leaf parenchyma that coincided with EBR under different environmental (light and temperature) conditions known to affect EBR. The two most prominent proteins proved to be novel chitinases (EC 3.2.1.14) that were transcriptionally induced before and during EBR development. Their expression in the apoplast was fast and not stress-regulated as opposed to many pathogenesis-related proteins. Nonpathogenic, saprophytic, and avirulent bacteria all induced EBR and the chitinases. Studies using these chitinases as EBR markers revealed that the virulent Pseudomonas syringae pv. tabaci, being sensitive to EBR, must suppress it while suppressing the chitinases. EBR, the chitinases, as well as their suppression are quantitatively related, implying a delicate balance determining the outcome of an infection.

Identification of Virulence-Associated Genes of Pseudomonas viridiflava Activated During Infection by Use of a Novel IVET Promoter Probing Plasmid

Analysis of virulence mechanisms of plant pathogens is often limited by the lack of genetic tools that can be used to identify genes that are preferentially expressed during their interactions with plants. In the present study, we used the newly constructed IVET (in vivoexpression technique) plasmid pIviGK and the corresponding antibiotic resistance–based selection method to identify genes that encode pathogenicity factors of the soft rot-causing bacterium Pseudomonas viridiflava. These included pel, the gene encoding pectate lyase, which is responsible for the development of soft rot symptoms. We have also isolated and characterized the gene mviNpv encoding a putative novel membrane associated virulence factor of P. viridiflava. A mutation in mviNpv was shown to influence motility as well as virulence of P. viridiflava. The mviNpv gene is expressed to a moderate level in LB media and its expression increases under inducing conditions as was shown by measuring in planta expression dynamics of the fused gfp reporter gene.

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.

Hr-Positive Phenotype of the Pseudomonas syringae pv. syringae hrpK Mutant and hrp Gene Superinduction in Tobacco Leaves Treated with Protein Synthesis Inhibitors

Induction period-dependent expression of bacterial hrp (hypersensitive response and pathogenicity) genes is required to cause the hypersensitive reaction (HR) in an incompatible piani. Early induced resistance (EIR) is described as a local and fast (in 3-6 h) developing, light-independent reaction of plants thai can prevent HR. Recently it was found that the same bacterial inoculum can trigger both EIR and HR. A short heai-treatment (50°C, 15 sec) or cycioheximide nullifies EIR’s effect. Here we show thai in heat- or cycloheximide-treated incompatible tobacco leaves, hrpK mutant of Pseudomonas syringae pv. syringae 61 is able to induce the HR, having a significantly longer induction period (3-4 h) than the wild-type parent strain (1 h). Moreover, mutant’s growth is potentiated and hrpK gene is superinduced under these EiR-inhibitory conditions. These data suggest that 1) hrpK mutant triggers EIR beside having the capacity for inducing HR; 2) a mutation in brpK gene extends HR induction period; 3) the faster developing EIR relative to this delayed HR-inducing activity may be responsible for the HR(-)-phenotype of hrpK mutant; 4) the HR-preventive effect of EIR may involve inhibition of hrp gene activity of incompatible bacterial pathogens.

Pattern Triggered Immunity (PTI) in Tobacco: Isolation of Activated Genes Suggests Role of the Phenylpropanoid Pathway in Inhibition of Bacterial Pathogens.

Background Pattern Triggered Immunity (PTI) or Basal Resistance (BR) is a potent, symptomless form of plant resistance. Upon inoculation of a plant with non-pathogens or pathogenicity-mutant bacteria, the induced PTI will prevent bacterial proliferation. Developed PTI is also able to protect the plant from disease or HR (Hypersensitive Response) after a challenging infection with pathogenic bacteria. Our aim was to reveal those PTI-related genes of tobacco (Nicotiana tabacum) that could possibly play a role in the protection of the plant from disease. Methodology/Principal Findings Leaves were infiltrated with Pseudomonas syringae pv. syringae hrcC- mutant bacteria to induce PTI, and samples were taken 6 and 48 hours later. Subtraction Suppressive Hybridization (SSH) resulted in 156 PTI-activated genes. A cDNA microarray was generated from the SSH clone library. Analysis of hybridization data showed that in the early (6 hpi) phase of PTI, among others, genes of peroxidases, signalling elements, heat shock proteins and secondary metabolites were upregulated, while at the late phase (48 hpi) the group of proteolysis genes was newly activated. Microarray data were verified by real time RT-PCR analysis. Almost all members of the phenyl-propanoid pathway (PPP) possibly leading to lignin biosynthesis were activated. Specific inhibition of cinnamic-acid-4-hydroxylase (C4H), rate limiting enzyme of the PPP, decreased the strength of PTI - as shown by the HR-inhibition and electrolyte leakage tests. Quantification of cinnamate and p-coumarate by thin-layer chromatography (TLC)-densitometry supported specific changes in the levels of these metabolites upon elicitation of PTI. Conclusions/Significance We believe to provide first report on PTI-related changes in the levels of these PPP metabolites. Results implicated an actual role of the upregulation of the phenylpropanoid pathway in the inhibition of bacterial pathogenic activity during PTI.

Changes in apoplast protein pattern suggest an early role of cell wall structure remodelling in flagellin-triggered basal immunity

The leaf apoplast is a dynamic compartment in contact with plant pathogenic bacteria after infection. Among the very first interaction events is the receptor-mediated perception of bacterial surface molecules such as flagellin or other conserved microbe-associated molecular patterns (MAMPs). Apoplast proteins likely play a role in basal resistance (BR) or pattern-triggered immunity (PTI). Here, a proteomic approach was carried out on water soluble — potentially the most mobile — apoplast proteins from flagellin-treated tobacco (Nicotiana tabacum) leaves. As the quickness of BR/PTI seems crucial for its efficacy, samples were taken as early as 2.5 and 7 h post inoculation. Proteins were separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and identified by liquid chromatography tandem mass spectrometry (LC-MS/MS). Forty-nine different proteins from 28 protein spots changed in their density compared to the water-inoculated control. Eleven protein spots appeared de novo in response to EBR induction. There are glycohydrolases and redox-active proteins besides pathogenesis-related proteins among them, predicting plant cell wall structural modifications and more direct antimicrobial effectors as earliest changes related to BR/PTI.

The Mechanism of Symptomless Reaction of Plants Induced by Pathogenic Pseudomonads

Leaf spot-inducing pseudomonads usually cause a hypersensitive reaction (HR) in non-host plants. In certain cases, however, the necrotic response does not occur. The mechanism of this symptomless “immune” response was not known before. We have shown, that a single bacterial inoculation induces two iocai host responses at an early stage of pathogenesis. One of these is the HR and the other is the early induced resistance (ElR) of plants 2. It was found that the symptomless response is a consequence of the development of the EIR prior to the induction of HR. This situation exists either a) when the EIR develops very quickly (e.g. at high temperatures) or b) when the HR induction time of pathogens is delayed (e.g. in some pathovars or mutants). We have results supporting both cases: a) the EIR developed in tobacco leaves at 20 °C by 3-6 h, but at 30 °C by 1-2 h. Therefore, Pseudomonas syringae pv. phaseolicola, the HR induction time of which is rather long (2.5-3 h) at both temperatures, causes HR at 20 °C but not at 30 °C; b) the induction time of some Tn5-mutants of pv. phaseolicola is delayed. During this longer induction time of HR (3-6 h) the EIR can develop also at normal temperature (20 °C). Therefore, the hypersensitive necrosis does not appear. These findings were verified in experiments in which the length of the HR induction time was extended by chloramphenicol treatment of wild strains of pv, phaseolicola. When the development of EIR was inhibited in tobacco leaves by inhibition of plant protein synthesis (cycioheximide or heat shock at 50 °C for 13 sec), the HR appeared.

Early Induced Resistance, a General, Symptomless Plant Response to Bacteria

Many micro-organisms including pathogenic and saprophytic bacteria react with plant cells in the intercellular spaces inducing different defence responses. The local Early Induced Resistance (EIR) is a first line defence mechanism against bacteria. Here an overview will be given of this local, nonspecific, symptomless defence mechanism as a separate entity from the incompatible-specific Hypersensitive Response (HR). The EIR operates 1–6 h after inoculation (hpi) for about one day depending on temperature and leaf age. The EIR can be inhibited by a short heat shock (50°C for 15 sec) of leaves or by a plant protein synthesis inhibitor, cycloheximide (5 µg m1−1). In a compatible host-pathogen relationship (Pseudomonas syringae pv. tabaci/tobacco) the effect of EIR does not eventuate. However, the EIR develops simultaneously with the HR and sometimes is able to prevent it when the induction time of HR is longer than the time required for the development of the EIR (e.g. P. s. pv. phaseolicola does not induce HR in tobacco above 28°C). It seems that the EIR inhibits the metabolism of bacteria and the activity of hrp genes. Moreover, EIR activates the accumulation of H2O2 at the bacterial attachment site expressing new peroxidase isoenzymes in the initiated plant tissue. Further investigations, hopefully, will clarify the relationship of other complementary defence mechanisms like local late induced resistance (LIR) examined by Sequeira (1983) and Mazzucchi and co-workers (1979), Minardi (1995), Newman et al., (2001).

Non-specific, Peroxidase and H2O2 Associated Reactions of Tobacco Leaves after Infiltration with hrp/hrmA Mutants of P. syringae pv. syringae 61

Plants recognize the infection of both pathogenic and non-pathogenic bacteria and respond with specific and non-specific defense reactions after bacterial infections. One of the specific defense responses of plants is the hypersensitive reaction (HR). The HR is accompanied by an accumulation of active oxygen species including H2O2 and other resistance associated responses. The hrp/hrc genes are indispensable for phytopathogenic bacteria to induce HR or disease in plants. The plant cells sense not only the plant pathogenic bacteria and their elicitors (harpin or avr proteins) but also the non-pathogenic and the HR-negative bacteria (non-specific reactions). During these non-specific reactions plants respond with different resistance related reactions such as induction of mRNA accumulation of several defense associated genes, and large papilla formation at the site of attachment of bacteria. The plants probably recognize the bacterial common surface molecules e.g. flagellin protein (1) or bacterial lipopolysaccharides (2, Kecskes et al., unpublished).