Das PR-Protein CBP20: Untersuchungen zur Induktion der Synthese und Akkumulation sowie zum innerzellulären Transport unter Verwendung von in vitro Kulturen des Tabak und der Hefe

Im Mittelpunkt dieser Arbeit stand die Identifizierung und Charakterisierung des wund-induzierbaren Tabakproteins CBP20. Es ist ein basischer Vertreter der 4. Gruppe der sogenannten 'pathogenesis-related' (PR)-Proteine und wird ähnlich reguliert wie die basischen PR-Proteine Beta-1,3-Glukanase und Chitinase, die jeweils zur Klasse I der PR-2- bzw. PR-3-Proteine gehören. Das Protein CBP20 wird konstitutiv in Tabaksuspensionskulturen sowie in der Wurzel und in älteren, 'source' Blättern von Tabakpflanzen akkumuliert. Die Akkumulation des Proteins kann durch Seneszenz, Salicylsäure und Schwermetalle, insbesondere durch Zinkchlorid induziert werden. Wir konnten zeigen, daß die Akkumulation der m-RNA nicht immer in der Akkumulation des entsprechenden Proteins resultiert, so daß CBP20 vermutlich sowohl transkriptional als auch post-transkriptional reguliert wird. Das Auxin 2,4 Dichlorophenoxyessigsäure (2,4 D) stimuliert die extrazelluläre Akkumulation von CBP20 in Suspensionskulturen. Wir konnten zeigen, daß CBP20 generell in die Zellwand von supensionskultivierten Zellen exportiert wird, aber nur bei Anwesenheit von 2,4 D ins Medium entlassen wird. 2,4 D bewirkt eine Erhöhung der Porengröße der Zellwand und vermindert die Bindungsstärke des Proteins an die Zellwand. Weiterhin wurden CBP20-Promotoren isoliert sowie Untersuchungen zum innerzellulären Transport von CBP20 in Hefezellen gezeigt.The aim of this work was the identification and characterisation of the wound-inducible tobacco protein CBP20. It is a basic representative of the pathogenesis-related (PR)-proteins of the group PR-4. It is similarly regulated as the basic proteins class I Beta-1,3-glucanase and class I chitinase of PR-2 and PR-3, respectively. The protein is constitutively accumulated in suspension-cultured cells as well as in roots and lower source leaves of healthy tobacco plants. The accumulation of the protein is induced by senescence, salicylic acid and heavy metals, especially zinc chloride. Accumulation of CBP20-mRNA not always resulted in the accumulation of the respective protein leading to the suggestion that the expression of CBP20 is transcriptionally as well as post-transcriptionally regulated. Depending on the phytohormones present in the culture medium, CBP20 has been also detected in the medium of suspensions. The auxin 2,4 dichlorophenoxyacetic acid (2,4 D) stimulates extracellular accumulation. We have demonstrated that CBP20 is localized in the cell wall of suspension cultured cells but it is only released into the medium in the presence of 2,4 D. 2,4 D increases the pore size of cell walls of suspension-cultered cells and decreases the strengtheness of CBP20-binding to the cell wall. Furthermore, putative CBP20 promoters have been isolated and investigations on innercellular transport of CBP20 in yeast have been shown

Das PR-Protein CBP20: Untersuchungen zur Induktion der Synthese und Akkumulation sowie zum innerzellulären Transport unter Verwendung von in vitro Kulturen des Tabak und der Hefe

Im Mittelpunkt dieser Arbeit stand die Identifizierung und Charakterisierung des wund-induzierbaren Tabakproteins CBP20. Es ist ein basischer Vertreter der 4. Gruppe der sogenannten 'pathogenesis-related' (PR)-Proteine und wird ähnlich reguliert wie die basischen PR-Proteine Beta-1,3-Glukanase und Chitinase, die jeweils zur Klasse I der PR-2- bzw. PR-3-Proteine gehören. Das Protein CBP20 wird konstitutiv in Tabaksuspensionskulturen sowie in der Wurzel und in älteren, 'source' Blättern von Tabakpflanzen akkumuliert. Die Akkumulation des Proteins kann durch Seneszenz, Salicylsäure und Schwermetalle, insbesondere durch Zinkchlorid induziert werden. Wir konnten zeigen, daß die Akkumulation der m-RNA nicht immer in der Akkumulation des entsprechenden Proteins resultiert, so daß CBP20 vermutlich sowohl transkriptional als auch post-transkriptional reguliert wird. Das Auxin 2,4 Dichlorophenoxyessigsäure (2,4 D) stimuliert die extrazelluläre Akkumulation von CBP20 in Suspensionskulturen. Wir konnten zeigen, daß CBP20 generell in die Zellwand von supensionskultivierten Zellen exportiert wird, aber nur bei Anwesenheit von 2,4 D ins Medium entlassen wird. 2,4 D bewirkt eine Erhöhung der Porengröße der Zellwand und vermindert die Bindungsstärke des Proteins an die Zellwand. Weiterhin wurden CBP20-Promotoren isoliert sowie Untersuchungen zum innerzellulären Transport von CBP20 in Hefezellen gezeigt.The aim of this work was the identification and characterisation of the wound-inducible tobacco protein CBP20. It is a basic representative of the pathogenesis-related (PR)-proteins of the group PR-4. It is similarly regulated as the basic proteins class I Beta-1,3-glucanase and class I chitinase of PR-2 and PR-3, respectively. The protein is constitutively accumulated in suspension-cultured cells as well as in roots and lower source leaves of healthy tobacco plants. The accumulation of the protein is induced by senescence, salicylic acid and heavy metals, especially zinc chloride. Accumulation of CBP20-mRNA not always resulted in the accumulation of the respective protein leading to the suggestion that the expression of CBP20 is transcriptionally as well as post-transcriptionally regulated. Depending on the phytohormones present in the culture medium, CBP20 has been also detected in the medium of suspensions. The auxin 2,4 dichlorophenoxyacetic acid (2,4 D) stimulates extracellular accumulation. We have demonstrated that CBP20 is localized in the cell wall of suspension cultured cells but it is only released into the medium in the presence of 2,4 D. 2,4 D increases the pore size of cell walls of suspension-cultered cells and decreases the strengtheness of CBP20-binding to the cell wall. Furthermore, putative CBP20 promoters have been isolated and investigations on innercellular transport of CBP20 in yeast have been shown

A tool to automatically analyze electromagnetic tracking data from high dose rate brachytherapy of breast cancer patients

During High Dose Rate Brachytherapy (HDR-BT) the spatial position of the radiation source inside catheters implanted into a female breast is determined via electromagnetic tracking (EMT). Dwell positions and dwell times of the radiation source are established, relative to the patient's anatomy, from an initial X-ray-CT-image. During the irradiation treatment, catheter displacements can occur due to patient movements. The current study develops an automatic analysis tool of EMT data sets recorded with a solenoid sensor to assure concordance of the source movement with the treatment plan. The tool combines machine learning techniques such as multi-dimensional scaling (MDS), ensemble empirical mode decomposition (EEMD), singular spectrum analysis (SSA) and particle filter (PF) to precisely detect and quantify any mismatch between the treatment plan and actual EMT measurements. We demonstrate that movement artifacts as well as technical signal distortions can be removed automatically and reliably, resulting in artifact-free reconstructed signals. This is a prerequisite for a highly accurate determination of any deviations of dwell positions from the treatment plan

Characterization of the Modular Design of the Autolysin/Adhesin Aaa from Staphylococcus Aureus

BACKGROUND: Staphylococcus aureus is a frequent cause of serious and life-threatening infections, such as endocarditis, osteomyelitis, pneumonia, and sepsis. Its adherence to various host structures is crucial for the establishment of diseases. Adherence may be mediated by a variety of adhesins, among them the autolysin/adhesins Atl and Aaa. Aaa is composed of three N-terminal repeated sequences homologous to a lysin motif (LysM) that can confer cell wall attachment and a C-terminally located cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domain having bacteriolytic activity in many proteins. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show by surface plasmon resonance that the LysM domain binds to fibrinogen, fibronectin, and vitronectin respresenting a novel adhesive function for this domain. Moreover, we demonstrated that the CHAP domain not only mediates the bacteriolytic activity, but also adherence to fibrinogen, fibronectin, and vitronectin, thus demonstrating for the first time an adhesive function for this domain. Adherence of an S. aureus aaa mutant and the complemented aaa mutant is slightly decreased and increased, respectively, to vitronectin, but not to fibrinogen and fibronectin, which might at least in part result from an increased expression of atl in the aaa mutant. Furthermore, an S. aureus atl mutant that showed enhanced adherence to fibrinogen, fibronectin, and endothelial cells also demonstrated increased aaa expression and production of Aaa. Thus, the redundant functions of Aaa and Atl might at least in part be interchangeable. Lastly, RT-PCR and zymographic analysis revealed that aaa is negatively regulated by the global virulence gene regulators agr and SarA. CONCLUSIONS/SIGNIFICANCE: We identified novel functions for two widely distributed protein domains, LysM and CHAP, i.e. the adherence to the extracellular matrix proteins fibrinogen, fibronectin, and vitronectin. The adhesive properties of Aaa might promote S. aureus colonization of host extracellular matrix and tissue, suggesting a role for Aaa in the pathogenesis of S. aureus infections

Multiple roles of Pseudomonas aeruginosa TBCF10839 PilY1 in motility, transport and infection

Polymorphonuclear neutrophils are the most important mammalian host defence cells against infections with Pseudomonas aeruginosa. Screening of a signature tagged mutagenesis library of the non-piliated P. aeruginosa strain TBCF10839 uncovered that transposon inactivation of its pilY1 gene rendered the bacterium more resistant against killing by neutrophils than the wild type and any other of the more than 3000 tested mutants. Inactivation of pilY1 led to the loss of twitching motility in twitching-proficient wild-type PA14 and PAO1 strains, predisposed to autolysis and impaired the secretion of quinolones and pyocyanin, but on the other hand promoted growth in stationary phase and bacterial survival in murine airway infection models. The PilY1 population consisted of a major full-length and a minor shorter PilY1* isoform. PilY1* was detectable in small extracellular quinolone-positive aggregates, but not in the pilus. P. aeruginosa PilY1 is not an adhesin on the pilus tip, but assists in pilus biogenesis, twitching motility, secretion of secondary metabolites and in the control of cell density in the bacterial population

Fitness of Isogenic Colony Morphology Variants of Pseudomonas aeruginosa in Murine Airway Infection

Chronic lung infections with Pseudomonas aeruginosa are associated with the diversification of the persisting clone into niche specialists and morphotypes, a phenomenon called ‘dissociative behaviour’. To explore the potential of P. aeruginosa to change its morphotype by single step loss-of–function mutagenesis, a signature-tagged mini-Tn5 plasposon library of the cystic fibrosis airway isolate TBCF10839 was screened for colony morphology variants under nine different conditions in vitro. Transposon insertion into 1% of the genome changed colony morphology into eight discernable morphotypes. Half of the 55 targets encode features of primary or secondary metabolism whereby quinolone production was frequently affected. In the other half the transposon had inserted into genes of the functional categories transport, regulation or motility/chemotaxis. To mimic dissociative behaviour of isogenic strains in lungs, pools of 25 colony morphology variants were tested for competitive fitness in an acute murine airway infection model. Six of the 55 mutants either grew better or worse in vivo than in vitro, respectively. Metabolic proficiency of the colony morphology variant was a key determinant for survival in murine airways. The most common morphotype of self-destructive autolysis did unexpectedly not impair fitness. Transposon insertions into homologous genes of strain PAO1 did not reproduce the TBCF10839 mutant morphotypes for 16 of 19 examined loci pointing to an important role of the genetic background on colony morphology. Depending on the chosen P. aeruginosa strain, functional genome scans will explore other areas of the evolutionary landscape. Based on our discordant findings of mutant phenotypes in P. aeruginosa strains PAO1, PA14 and TBCF10839, we conclude that the current focus on few reference strains may miss modes of niche adaptation and dissociative behaviour that are relevant for the microevolution of complex traits in the wild

New breeding technologies – what can be achieved with which methods:

Um eine hocheffziente und gleichzeitig umweltschonende Landwirtschaft zu verwirklichen, ist die Züchtung neuer Pfanzensorten unerlässlich. Dies gewinnt zusätzlich an Bedeutung angesichts der klimatischen Veränderungen und der Notwendigkeit, den Einsatz von chemischen Pfanzenschutzmitteln und mineralischem Dünger zu reduzieren. Die neuen Züchtungsmethoden der GenomEditierung liefern hierfür wertvolle Werkzeuge, die auf unterschiedliche Weise eingesetzt werden können. Bereits heute wurden mehr als 60 Arten von Kulturpfanzen mit diesen Werkzeugen züchterisch bearbeitet und landwirtschaftlich relevante Merkmale realisiert. Pfanzenzüchter sehen ein großes Potenzial vor allem für Kulturpfanzen, für die schon umfangreiche genetische Informationen vorliegen. Nach langen kontroversen Diskussionen liegt nun ein Entwurf der Europäischen Kommission für eine Neuregulierung von Pfanzen, die mittels „Neuer Genomischer Techniken“ (NGTs) entwickelt wurden, vor.To achieve a highly effcient und at the same time environmentally friendly agriculture the breeding of new varieties of plants is imperative. This has become even more vital in the face of climate change und the necessity to reduce the application of chemical plant protection products and mineral fertilizers. The new breeding methods of genome editing provide valuable tools which can be used in a number of ways. Already today, more than 60 crop species have been bred using these tools, and agriculturally relevant traits have been optimized. Plant breeders see a great potential especially for crops for which comprehensive genetic information is already available. After long controversial discussions, a draft of the European Commission for a new regulation of plants that have been bred by using “New Genomic Techniques“ (NGTs) is now available

A Barley ROP GTPase ACTIVATING PROTEIN Associates with Microtubules and Regulates Entry of the Barley Powdery Mildew Fungus into Leaf Epidermal Cells[C][W]

Little is known about the role the cytoskeleton plays in signaling with regard to the outcome of plant–microbe interactions. This work describes a regulator of cytoskeleton organization and of disease susceptibility shuttling between microtubules and the monomeric G-protein RACB at the cell periphery