Zoltán Klement

The role of extracellular polysaccharides as virulence factors for phytopathogenic pseudomonads and xanthomonads

Bacterial exopolysaccharides (EPS) were investigated for their role as virulence factors of leaf spot diseases caused by pseudomonads and xanthomonads. The capacity of these bacteria to induce persistent water-soaking in leaves plays a crucial role during pathogenesis that seems to be accomplished by a synergistic interaction between bacterial EPS and plant polymers. Under conditions of low EPS production (e.g. in continuously darkened plants) the bacteria were not able to cause typical water-soaked disease symptoms. The main EPS components were alginate and levan (Pseudomonas), xanthan (Xanthomonas), as well as lipopolysaccharides (LPS) and a small amount of proteins. It is suggested that alginate which is very similar to plant pectate is required for establishing bacterial infections in later disease stages. This concept was confirmed by evaluating transposon mutants with EPS deficiencies. LPS may be involved in specific interactions with plant polymers leading to agglutination and precipitation (incompatibility) or gel-formation (compatibility). Bacteria which are embedded in a gel-like matrix inplanta are not easily recognized by the plant and are protected against bacteriostatic compounds and desiccation.

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.

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.

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).