E. Besenyei

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.

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.

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).