Disparate Effects of Two Clerodane Diterpenes of Giant Goldenrod (Solidago gigantea Ait.) on Bacillus spizizenii.

New substances with antimicrobial properties are needed to successfully treat emerging human, animal, or plant pathogens. Seven clerodane diterpenes, previously isolated from giant goldenrod (Solidago gigantea) root, were tested against Gram-positive Bacillus subtilis, Bacillus spizizenii and Rhodococcus fascians by measuring minimal bactericidal concentration (MBC), minimal inhibitory concentration (MIC) and half-maximal inhibitory concentration (IC50). Two of them, Sg3a (a dialdehyde) and Sg6 (solidagoic acid B), were proved to be the most effective and were selected for further study. Bacillus spizizenii was incubated with the two diterpenes for shorter (1 h) or longer (5 h) periods and then subjected to genome-wide transcriptional analyses. Only a limited number of common genes (28 genes) were differentially regulated after each treatment, and these were mainly related to the restoration of cell membrane integrity and to membrane-related transports. Changes in gene activity indicated that, among other things, K+ and Na+ homeostasis, pH and membrane electron transport processes may have been affected. Activated export systems can be involved in the removal of harmful molecules from the bacterial cells. Inhibition of bacterial chemotaxis and flagellar assembly, as well as activation of genes for the biosynthesis of secondary metabolites, were observed as a general response. Depending on the diterpenes and the duration of the treatments, down-regulation of the protein synthesis-related, oxidative phosphorylation, signal transduction and transcription factor genes was found. In other cases, up-regulation of the genes of oxidation-reduction processes, sporulation and cell wall modification could be detected. Comparison of the effect of diterpenes with the changes induced by different environmental and nutritional conditions revealed several overlapping processes with stress responses. For example, the Sg6 treatment seems to have caused a starvation-like condition. In summary, there were both common and diterpene-specific changes in the transcriptome, and these changes were also dependent on the length of treatments. The results also indicated that Sg6 exerted its effect more slowly than Sg3a, but ultimately its effect was greater.

A pattern-triggered immunity-related phenolic, acetosyringone, boosts rapid inhibition of a diverse set of plant pathogenic bacteria.

BACKGROUND: Acetosyringone (3,5-dimethoxy-4-hydroxyacetophenone, AS) is a syringyl-type phenolic compound rarely found in plants in free form. It has been shown earlier to inhibit the growth of Pseudomonas bacteria in the presence of hydrogen peroxide and peroxidase (AS mix). RESULTS: We detected elevated levels of free AS in Nicotiana tabacum and N. benthamiana plants after inducing pattern-triggered immunity (PTI) by injecting bacterial elicitor flg22, or pathogenicity-mutant Pseudomonas syringae pv. syringae 61 hrcC- bacteria; but not after inoculations with compatible or incompatible pathogens at the time of PTI onset. In this study, we demonstrate that the antibacterial effect of the AS mix is general, as growth of several Gram-negative and -positive phytopathogenic bacteria was characteristically inhibited. The inhibition of bacterial metabolism by the AS mix was rapid, shown by the immediate drop of luminescence intensity of P. syringae pv. tomato DC3000 lx strain after addition of AS mix. The mechanism of the bacteriostatic effect was investigated using fluorescent reporter dye assays. SYTOX Green experiments supported others' previous findings that the AS mix does not result in membrane permeabilization. Moreover, we observed that the mode of action could be depolarization of the bacterial cell membrane, as shown by assays carried out with the voltage sensitive dye DIBAC4(3). CONCLUSIONS: Level of free acetosyringone is elevated during plant PTI responses in tobacco leaves (N. tabacum and N. benthamiana). When combined with hydrogen peroxide and peroxidase (AS mix), components of the mix act synergistically to inhibit bacterial metabolism and proliferation rapidly in a wide range of plant pathogens. This effect is related to depolarization rather than to permeabilization of the bacterial cell membrane. Similar AS mixture to the in vivo model might form locally at sites of invading bacterial attachment to the plant cells and the presence of acetosyringone might have an important role in the inhibition of bacterial proliferation during PTI.

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.

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.

Two Non-Necrotic Disease Resistance Types Distinctly Affect the Expression of Key Pathogenic Determinants of Xanthomonas euvesicatoria in Pepper.

Pepper (Capsicum annuum L.) carrying the gds (corresponding to bs5) gene can prevent the development of bacterial leaf spot disease without HR. However, little is known regarding the development of the resistance mechanism encoded by gds, especially its influence on the bacterium. Here, the effect of gds was compared with pattern-triggered immunity (PTI), another form of asymptomatic resistance, to reveal the interactions and differences between these two defense mechanisms. The level of resistance was examined by its effect on the bacterial growth and in planta expression of the stress and pathogenicity genes of Xanthomonas euvesicatoria. PTI, which was activated with a Pseudomonas syringae hrcC mutant pretreatment, inhibited the growth of Xanthomonas euvesicatoria to a greater extent than gds, and the effect was additive when PTI was activated in gds plants. The stronger influence of PTI was further supported by the expression pattern of the dpsA bacterial stress gene, which reached its highest expression level in PTI-induced plants. PTI inhibited the hrp/hrc expression, but unexpectedly, in gds plant leaves, the hrp/hrc genes were generally expressed at a higher level than in the susceptible one. These results imply that different mechanisms underlie the gds and PTI to perform the symptomless defense reaction.

Global transcriptome and targeted metabolite analyses of roots reveal different defence mechanisms against Ralstonia solanacearum infection in two resistant potato cultivars.

Ralstonia solanacearum (Rs), the causal agent of bacterial wilt disease in an unusually wide range of host plants, including potato (Solanum tuberosum), is one of the most destructive phytopathogens that seriously reduces crop yields worldwide. Identification of defence mechanisms underlying bacterial wilt resistance is a prerequisite for biotechnological approaches to resistance breeding. Resistance to Rs has been reported only in a few potato landraces and cultivars. Our in vitro inoculation bioassays confirmed that the cultivars 'Calalo Gaspar' (CG) and 'Cruza 148' (CR) are resistant to Rs infection. Comparative transcriptome analyses of CG and CR roots, as well as of the roots of an Rs-susceptible cultivar, 'Désirée' (DES), were carried out two days after Rs infection, in parallel with their respective noninfected controls. In CR and DES, the upregulation of chitin interactions and cell wall-related genes was detected. The phenylpropanoid biosynthesis and glutathione metabolism pathways were induced only in CR, as confirmed by high levels of lignification over the whole stele in CR roots six days after Rs infection. At the same time, Rs infection greatly increased the concentrations of chlorogenic acid and quercetin derivatives in CG roots as it was detected using ultra-performance liquid chromatography - tandem mass spectrometry. Characteristic increases in the expression of MAP kinase signalling pathway genes and in the concentrations of jasmonic, salicylic, abscisic and indoleacetic acid were measured in DES roots. These results indicate different Rs defence mechanisms in the two resistant potato cultivars and a different response to Rs infection in the susceptible cultivar.

Diverse Effect of Two Cytokinins, Kinetin and Benzyladenine, on Plant Development, Biotic Stress Tolerance, and Gene Expression.

The plant hormones cytokinins affect a various array of plant growth and development processes as well as responses to biotic and abiotic stresses. In this study, the opposite effect of two different cytokinins kinetin (N6-furfuryladenine) and benzyladenine (BA) on development and on the tolerance of Arabidopsis and tobacco plants to virus, bacteria, and fungi infection was reported. Treatments of Arabidopsis and tobacco seedlings with saturated solutions of BA inhibited plant progress, while treatments with saturated water solution of kinetin promoted plant development. Furthermore, BA pre-treatments strongly reduced the number of TMV (Tobacco mosaic virus) lesions on tobacco and the tissue damage caused by the incompatible Pseudomonas bacteria on Arabidopsis and tobacco leaves. Similarly, BA pre-treatment significantly reduced the necrotic disease symptoms of Botrytis cinerea infection. Kinetin pre-treatments had a much weaker or no protective effect on the damage caused by the above pathogens. Accordingly, Arabidopsis gene expression profiles after treatments also showed that the two cytokinins have different effects on several plant processes. The gene expression results supported the more robust effect of BA, which up and downregulated more than 2000 genes, while only 436 genes were influenced by kinetin treatment. It is noteworthy that BA and kinetin treatment changed gene expressions in the same direction only in a relatively few cases (73 upregulated and 70 downregulated genes), and even 28 genes were regulated into the opposite directions by BA and kinetin. Both treatments had a strong effect on auxin and gibberellin-related genes, but only BA had a significant effect on cytokinin-induced processes. While kinetin exclusively activated the flavonoid synthesis genes, BA affected more significantly protein synthesis, photosynthesis, and plant defence-related genes. In conclusion, BA solution had sometimes the opposite and generally a much stronger effect than kinetin solution not only on the development and on biotic stress tolerance of tobacco and Arabidopsis plants but also on the gene expressions. The stronger protective effect of BA to necrotic stresses is probably due to its stronger senescence inhibitory effect on plant tissues, as supported by the stronger chlorophyll retardation of the BA-treated leaves.

Transcriptome analysis of a bacterially induced basal and hypersensitive response of Medicago truncatula.

Research using the well-studied model legume Medicago truncatula has largely focused on rhizobium symbiosis, while little information is currently available for this species on pathogen-induced transcriptome changes. We have performed a transcriptome analysis of this species with the objective of studying the basal (BR, no visible symptoms) and hypersensitive response (HR, plant cell death) in its leaves at 6 and at 24 h after infection by HR-negative (hrcC mutant) and HR-inducing Pseudomonas syringae pv. syringae strains, respectively. Although there were no visible symptoms at the BR, the alterations in gene expression were comparable to those found with the HR. Both responses resulted in the transcriptional alteration of hundreds of plant genes; however, the responses in the HR were usually more intense. The reactions to HR-inducing and HR-negative bacterial strains were significantly overlapping. Parallel up- or down-regulation of genes with the same function occurred frequently. However, some plant processes were regulated in one direction; for example, most of the protein synthesis-related genes were activated and all of the photosynthetic/chloroplast genes were suppressed during BR. The possible roles of several functional classes (e.g., cell rescue, signaling, defense, cell death, etc.) of transcriptionally altered genes are discussed. The results of the comparison with available mycorrhizal and nodule expression data show that there is a significant overlap between nodulation and the leaf defense response and that during the early stage of the nodulation in roots, Sinorhizobium meliloti induces a fluctuation in the transcription of BR- and HR-responsive genes.

Contribution of cell wall peroxidase- and NADPH oxidase-derived reactive oxygen species to Alternaria brassicicola-induced oxidative burst in Arabidopsis.

Cell wall peroxidases and plasma membrane-localized NADPH oxidases are considered to be the main sources of the apoplastic oxidative burst in plants attacked by microbial pathogens. In spite of this established doctrine, approaches attempting a comparative, side-by-side analysis of the functions of extracellular reactive oxygen species (ROS) generated by the two enzymatic sources are scarce. Previously, we have reported the role of Arabidopsis NADPH oxidase RBOHD (respiratory burst oxidase homologue D) in plants challenged with the necrotrophic fungus Alternaria brassicicola. Here, we present results on the activity of apoplastic class III peroxidases PRX33 (At3g49110) and PRX34 (At3g49120) investigated in the same Arabidopsis-Alternaria pathosystem. ROS generated by Arabidopsis peroxidases PRX33 and PRX34 increase the necrotic symptoms and colonization success of A. brassicicola. In addition, the knockdown of PRX33 and PRX34 transcript levels leads to a reduced number of host cells showing an extracellular burst of ROS after inoculation with A. brassicicola. Our results also reveal an age-dependent transcript distribution of ROS-producing peroxidase and NADPH oxidase enzymes, and some potential new components of the RBOHD, PRX33 and PRX34 signalling networks.

A simplified and efficient Agrobacterium tumefaciens electroporation method.

Agrobacterium tumefaciens is a widely used microbial tool in plant molecular biology to transfer DNA into plant cells and produce, e.g., stable or transient transformants or induce gene silencing. In our study, we present a simplified version of electrocompetent cell preparation that is not only time and cost efficient, but it requires minimal handling of bacterial cells. Liquid cultures are normally used to prepare competent Agrobacterium cells. To overcome the difficulties of working with liquid cultures, we propose suspending bacterial cells directly from overnight agar plate cultures. In addition, we optimized several parameters to simplify the procedure and maximize the number of transformants (e.g., Agrobacterium strains, number of washing steps, amount of required plasmid DNA, electroporation parameters, type of incubation media, or incubation time). This optimized, simple, and fast protocol has proved to be efficient enough to obtain transformed colonies with low amounts (as little as 1 ng) of plasmid DNA. In addition, it also enabled us to introduce ligated plasmids directly into Agrobacterium omitting the E. coli transformation step and accelerating the cloning procedure further.

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.

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 (Ca(2+) 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.

Local early induced resistance of plants as the first line of defence against bacteria.

This paper is an overview of a non-specific local early induced resistance (EIR) mechanism, distinct from the incompatible-specific hypersensitive reaction (HR). We have shown that the local induced resistance (LIR) described earlier is not a single and uniform response to pathogen infection, because an early (EIR) and a late form can be distinguished. EIR operates from 3-6 h post-inoculation (hpi) until about 20 hpi, and is inhibited by a short heat-shock or the eukaryotic protein synthesis inhibitor, cycloheximide. In contrast, LIR, which corresponds to the induced resistance forms discovered earlier, requires more time (about 24 h) and intensive illumination to develop, and is effective for a longer period. EIR develops parallel with HR and is sometimes able to prevent it when the induction time of HR is longer than the time required for the development of EIR. It seems that EIR inhibits the metabolism of bacteria and the activity of hrp genes which otherwise are required for the induction of HR. In a compatible host-pathogen relationship the effect of EIR fails to take place. The rapid development of EIR is greatly influenced by temperature and the physiological state of the plant. EIR activates the accumulation of hydrogen peroxide at the bacterial attachment, expressing new peroxidase isoenzymes in the initiated plant tissue. It seems that this is a native general local defence mechanism which can localise foreign organisms even at the penetration site.

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.

Characterisation of basal resistance (BR) by expression patterns of newly isolated representative genes in tobacco.

Increasing evidence indicates that plants, like animals, use basal resistance (BR), a component of the innate immune system, to defend themselves against foreign organisms. Contrary to the hypersensitive reaction (HR)-type cell death, recognition in the case of BR is unspecific, as intruders are recognised based on their common molecular patterns. Induction of BR is not associated with visible symptoms, in contrast to the HR-type cell death. To analyse the early events of BR in tobacco plants we have carried out a subtractive hybridisation between leaves treated with the HR-negative mutant strain Pseudomonas syringae pv. syringae 61 hrcC and non-treated control leaves. Random sequencing from the 304 EBR clones yielded 20 unique EST-s. Real-time PCR has proved that 8 out of 10 clones are activated during BR. Six of these EST-s were further analyzed. Gene expression patterns in a time course showed early peaks of most selected genes at 3-12 h after inoculation (hpi), which coincided with the development-time of BR. Upon treatments with different types of bacteria we found that incompatible pathogens, their hrp mutants, as well as non-pathogens induce high levels of expression while virulent pathogens induce only a limited gene-expression. Plant signal molecules like salicylic acid, methyl jasmonate, ethylene and spermine, known to be involved in plant defense were not able to induce the investigated genes, therefore, an unknown signalling mechanism is expected to operate in BR. In summary, we have identified representative genes associated with BR and have established important features of BR by analysing gene-expression patterns.