Browsing by Subject "Interaktion"

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Genetische und physiologische Einflußfaktoren sowie deren Wechselwirkungen auf die Trichothecenbildung bei Roggen, Triticale und Weizen nach Inokulation mit Fusarium culmorum (W. G. Sm.) Sacc.
(2002) Reinbrecht, Carsten; Geiger, Hartwig H.
Fusarium culmorum causes specific hazards of cereal quality by the producion of trichothecenes. Prophylaxis by plant breeding can be highly effective. The aim of this study was to investigate the accumulation of trichothecenes in cereals with regard to host and fungal genotype, to physiological factors and the resulting interactions. To determine the effects of 6 environments (E), 2 inoculation dates (ID), 2 fungal isolates (I), and 5 conidia concentrations (C) and their interactions, field trials with up to 12 rye, 6 triticale, and 8 wheat genotypes (G) were conducted in 1995-1997. Kinetics of trichothecene in the heads were described with 6 harvest dates (H) in 2 host genotypes each. In a growth chamber, 2 levels of temperature (T) and 2 of relative humidity (R) were investigated by using 2 host genotypes each. Average deoxynivalenol (DON) accumulation of rye, triticale, and wheat was 41, 46, and 82 mg kg-1, respectively. Genotypes differed significantly in rye and wheat. In all cereal species, GxE interactions were important. In wheat, DON content was highly correlated to all resistance traits, whereas in rye only a tight correlation existed to the relative specific grain weight. In triticale and wheat, inoculation at full anthesis resulted in higher DON contents than inoculation at heading. In rye, no effect of inoculation date was found. In contrast, GxID interaction was significant in rye. The nivalenol (NIV) producing isolate led to lower trichothecene contents than the DON producing isolate. This effect was found to be significant only in rye and triticale. Significant GxI interactions occurred in wheat only. Even one week after inoculation, considerable DON concentrations could be obtained in harvested heads, especially in wheat. Maximum DON contents were observed between 3 and 6 weeks after inoculation (in wheat: partially above 300 mg kg-1). NIV contents were always lower than DON contents. Until full ripening, DON contents slightly decreased, whereas NIV contents increased continuously. HxE and HxI interactions were most important. Trichothecene content in chaff and spindles was 2-4 fold higher than in the respective kernels at 6 and 8 weeks after inoculation. With higher conidia concentrations, increasing contents of DON+3-Acetyl-DON were measured. GxC interactions were highly significant. Highest heritabilities were found in the upper concentration levels. When the relative humidity was high, trichothecene concentrations of kernels were superior. With the temperature, an inverse effect was obtained. It seems that GxT interaction contributed most to GxE in rye and wheat, in triticale also the GxR interaction. In conclusion, assisting resistance traits may replace an expensive quantification of trichothecenes in early generations. In advanced generations, tests should be conducted in several environments with high conidia concentrations, and a toxin analysis should be carried out directly.
Genotypic responses of upland rice to an altitudinal gradient
(2012) Shrestha, Suchit Prasad; Asch, Folkard
Adaptation strategies are required for crops to cope with changing climate. The impact of climate change on crop production is not straight forward to predict as extreme events comprise multiple combination of abiotic stresses and their impact differs in crop physiological growth stages. The mechanism on how new abiotic stress combinations translate into phenology and yield, and which cultivars are better adapted is yet unclear. Crop growth models are available that have been parameterized and validated for some aspects of possible climate change scenarios but in view of complex interactions crop responses to climate change are difficult to predict. On the other hand, prediction of the complex ideotype trait combinations may be interesting for breeders but physiological models are required that are well validated for the target environments. In upland rice grown under rainfed conditions without surface water accumulation methane emission is negligible and therefore greenhouse gas emission much lower compared to irrigated paddy rice systems. In addition, growing demand for rice and the increasing pressure on irrigated land leads to development of upland rice areas to supplement irrigated rice. Therefore, this study investigates genetically diverse upland rice genotypes from a wide range of origins across altitudinal gradient locations. The main objective of this study is to investigate genotypic responses of upland rice to different environments in order to calibrate crop growth models, which allow the evaluation of effects of climate change on upland rice systems. Multi-locational field (three locations: 1625, 965 and 25 m asl) trials comprising non-replicated phenological plots with five sowing dates (monthly staggered) in two consecutive years creating thirty different environments, and replicated physiological yield trials with two sowing dates (monthly staggered; early and late sowing) in two consecutive years creating twelve different environments were established in Madagascar. Ten contrasting upland rice genotypes were included in both field trials. Meteorological data were recorded on a daily basis during trial periods. Developmental stages were observed in the phenological plots; in the physiological plots yield and yield components were recorded. In addition, greenhouse trials were conducted with one upland rice genotype subjected to seven N-supply levels in a hydroponic system at the University of Hohenheim in order to understand the relationship between chlorophyll index, photochemical reflectance index and chlorophyll fluorescence parameters. Various statistical tools were applied to analyse field and greenhouse data sets. The phenological trial showed that duration to flowering was 117, 81 and 67 d in high (HA), mid (MA) and low (LA) altitudinal locations respectively. 90% of the total variance was explained by location when pooled over genotype, location, sowing dates and year. In HA, factors such as genotype, sowing date and year equally contributed to the observed variability whereas in MA year was the most determining factor and genotype had no significant contribution. Similarly, in LA sowing date was the main influencing factor and year had no significant effect. Aggregated data over locations, sowing dates and years indicated that each degree Celsius rise in mean air temperature decreased crop duration by 5 to 9 days depending upon genotype. Basic genotypic thermal constants Tbase ranged from 9.8 to 13.9 °C and Tsum from 816 to 1220 °C d within the selected genotypes. Cold tolerant genotypes were less affected by lower Tmin (14 °C) at booting to heading stage regarding spikelet sterility in HA, whereas others were highly affected at 15 °C (cold stress). Similarly, both cold sensitive and tolerant genotypes were affected by Tmax (above 30 °C) at flowering in MA and LA locations (heat stress). Grain yield and yield components were highly affected by location, year, sowing date, and genotypes and the interactions between these yield-determining factors were obvious. In HA, early sown cold tolerant genotypes had more than 5 t ha-1 grain yield and one month delay in sowing led to highly reduced yield whereas other genotypes had very poor yield on both sowing dates due to cold stress. In MA, yield difference between sowing date and genotypes was small (4.3 - 4.9 t ha-1). Grain yield in LA was vulnerable due to frequent tropical storms. Yield stability analysis showed that cold tolerant genotypes had above average stability. AMMI model for grain yield showed that environment and genotype by environment interactions were highly significant. Yield components determined during specific development stages of the genotype such as tillers per hill and percentage of filled spikelets were mainly influenced by environment, spikelets per panicle and thousand grain weight were influenced by genotype, and percentage of productive tillers was equally influenced by both genotype and environment. PCA biplots showed that all HA environments were equally influenced by all weather parameters with minimum air temperature having the strongest positive influence on genotypic performance. In all MA environments genotypic performance in all phenophases was strongly and positively influenced by rainfall, and strongly and negatively influenced by vapour pressure deficit, solar radiation and potential evapotranspiration. In the LA environments, main weather parameters influencing genotypic performance were maximum temperature and high rainfall accompanied by strong winds. The field measured SPAD values of the upper canopy leaves reflected the location specific N-remobilization and leaf senescence levels after flowering. Similarly, PRI values showed the abiotic stress responses among development stages and locations along the altitudinal gradient. These readings showed that genotypes were efficient in radiation use and N-remobilization after flowering in MA. The unsynchronized relationship between source (leaf) and sink (grain) explained the yield penalty. Emphasis on identification of morpho-physiological traits contributing to cold tolerance should be placed for further breeding. We conclude that genotypic responses of upland rice cultivars differed across altitudinal gradients. Genotypes that are well adapted in HA can easily be adapted in MA without yield decrease. But genotypes well adapted in MA may show a huge yield penalty in HA due to lower temperature during reproductive phase and consequently reduced sink formation. Frequent tropical storms and high temperature reduced yield potential in LA. Therefore, HA has a large potential for the future food security considering climate change scenarios. At present, MA is favorable for upland rice production systems, whereas LA is highly vulnerable and is expected to be even more vulnerable in future. Those results on genotype-specific responses to environmental conditions allow further improvement of crop models such as RIDEV and SAMARA (synthesis of SARRAH and EcoMeristem), which can be used to test a number of phenotypic traits x environments combinations to define ideotypes of upland rice varieties adapted to changing climate and cropping calendars. Genotypic responses of phyllochron, biomass production and crop growth rate, and radiation use efficiency across altitudinal gradients will be included to parameterize these models. In this regard, collaborations with AfricaRice, CIRAD and IRRI are ongoing.
Molekulare Interaktionen von Milchsäurebakterien mit enterohämorrhagischen Escherichia coli und humanen Darmepithelzellen
(2011) Stöber, Helen; Schmidt, Herbert
The interactions of 19 benign strains of lactic acid bacteria, bifidobacteria and staphylococci with five enterohemorrhagic Escherichia coli (EHEC) strains of different serotypes and virulence gene spectrum were investigated using a HT29 cell culture infection model. As a parameter for the infection the secretion of Interleukin 8 (IL-8) of the infected cells was analyzed by ELISA. None of the used benign strains induced an IL-8 secretion, whereas the infection with the EHEC strains leads ? independent of their virulence profile - to high amounts of IL-8. In coinfection assays with the pathogen EDL933 (O157:H7) and different test strains the secretion of IL-8 of the cultured cells was decreased by a few strains. With 12 of 19 tested strains, a weak reduction < 30 % of IL-8 secretion of HT29 cells after coinfection with EHEC O157:H7 strain EDL933 was observed. Six strains reduced the IL-8 secretion up to 60 % and the strain B. breve DSMZ 20083 decreased the IL-8 production about 73 %. Coinfection assays with different strains of one species (B. adolescentis DSMZ 20083 and DSMZ 20086 as well as L. johnsonii BFE 633 and DSMZ 10533) showed the strain specificity of the observed anti-inflammatory effect, due to different capabilities of IL-8 reduction. In further coinfection assays with different EHEC strains of the serotypes O103:H2, O26:H-, 0157:H- and O113:H21 different abilities of the benign strains to influence the infection with the different pathogen strains were noted. Therefore the protective anti-inflammatory effect is strain specific for the tested benign bacteria and also depends on the application of EHEC strains with different sero- and virulence types. Further investigations indicated the imperative of living bacteria for the observed protective effect; neither culture supernatant nor inactivated bacteria showed an effect on the IL-8 secretion of the EDL933 infected HT29 cells. The analysis of the cell culture supernatants 6 h after infection with different bacteria detected the production of lactic and acetic acid. The application of these acids in infection assays with EDL933 did not lead to an reduced IL-8 secretion of the infected cells. Therefore the production of organic acids did not explain the protective effect. The induction of IL-8 could not be traced back to the influence of a single virulence factor. Four PMK5 strains with deletions in different virulence genes induced similar IL-8 secretions in comparison to cells infected with the wild-type strain. Coinfection assays with the mutants and S. pasteuri LTH 5211 showed also similar IL-8 reductions than coinfection assays with the wild-type strain. It is to suppose that the anti-inflammatory effects of the benign bacteria do not influence a single virulence factor of the tested EHEC strains. As a second parameter the activation of the transcription factor ?Nuclear Factor kappa B? (NF-κB) of coinfected HT29 cells was monitored using a reporter-genassay. In comparison to the single EHEC-infection, the NF-κB activation was reduced by all tested lactic acid bacteria, bifidobacteria and S. pasteuri LTH 5211 in coinfection trials significantly. No strain-specificity and no pathogen-specificity could be observed. Interestingly, stimulation of the HT29 cells with benign bacteria led to inhibition of NF-κB activity, the measured values were less than the values of the negative control PBS. A gene expression analysis of toll-like receptors (TLRs), recognizing bacteria on cell surfaces and initiating the immune response, showed no regulation for TLR2. Infection with EDL933 led to down regulation of TLR4 and to up regulation of TLR9. Stimulation with L. rhamnosus GG, L. johnsonii DSMZ 10533 or L. fermentum DSMZ 20052 led neither to regulation of TLR4 nor TLR9. The benign bacteria did not influence the EHEC-induced TLR4 regulation in coinfection trials; in contrast the regulation of TLR9 was reduced significantly. The model described here is useful for screening basic effects of protective bacteria that are able to counteract EHEC-mediated effects on human cells and to study the molecular interaction between bacteria as well as between bacteria and human cultured cells.
Novel serine phosphorylation sites of IRS2 mediate 14-3-3 binding and regulate insulin signal transduction
(2012) Neukamm, Sabine Sarah; Schleicher, Erwin D.
Insulin and insulin like growth factor (IGF)-1 mediate their metabolic and mitogenic effects on target tissues through activation of the insulin and IGF-1 receptor. Insulin receptor substrate (IRS) proteins function as intermediate docking platforms to transduce the insulin/IGF-1 signal to intracellular effector molecules that regulate glucose homoeostasis, lipid metabolism, cell proliferation and ß-cell survival. The activated receptors function as tyrosine kinases that phosphorylate IRS proteins on tyrosine residues, thereby generating interaction motifs for Src homology (SH) 2 domain containing proteins. Signal transduction via IRS proteins is furthermore regulated by their serine/threonine phosphorylation by several kinases and this can result in inhibitory or stimulatory consequences for downstream signalling. Hyperphosphorylation of IRS1 has been shown to be involved in the desensitization process that can result in insulin resistance. Both IRS1 and IRS2 undergo proteasomal degradation while particularly IRS2 levels are additionally regulated by cAMP-dependent gene activation. 14-3-3 proteins are versatile regulators of a variety of intracellular processes like control of cell cycle, cell growth, gene transcription and apoptosis. Serine/threonine phosphorylation within distinct motifs on the interaction partner is a prerequisite for 14-3-3 binding. IRS1 and IRS2 have been described as 14-3-3 interaction proteins and interaction of IRS2 with 14-3-3 proteins was specifically characterized in this thesis. Insulin/IGF-1-dependent PI 3-kinase stimulation as well as elevated cAMP levels were identified to modulate 14-3-3 binding to IRS2. IGF-1 stimulation led to increased binding of 14-3-3 to IRS2 in transfected HEK293 cells and this bind- ing was prevented by inhibition of the PI 3-kinase pathway and an Akt/PKB inhibitor. Insulin-stimulated interaction between endogenous IRS2 and 14-3-3 was observed in rat hepatoma cells and in mice liver after an acute insulin stimulus or refeeding. Application of different IRS2 fragments enabled localization of the IGF-1-dependent 14-3-3 binding region spanning amino acids 300-600. Mass spectrometric analysis produced a total of 24 phosphorylated serine/threonine residues on IRS2 after IGF-1 stimulation with 12 sites unique for IRS2 while the other residues are conserved in IRS1 and IRS2. The 24 identified phosphorylated residues on IRS2 included several 14-3-3 binding candidates in the region 300-600 and single alanine mutants of these candidates led to the identification of Ser573 as 14-3-3 binding site by overlay assays. A phosphosite specific antibody was generated to further characterize Ser573. IGF-1-dependent phosphorylation of Ser573 was reduced by inhibition of PI 3-kinase and Akt/PKB. The alanine mutant of Ser573 showed enhanced phosphorylation of Akt/PKB in an IGF-1 time course experiment. In summary, the data presented in this thesis indicate a negative impact of Ser573 phosphorylation on downstream signalling. Binding of 14-3-3 to IRS2 upon stimulation with forskolin and the cAMP analogue CPT-cAMP (8-(4-chlorophenylthio) adenosine 3?,5?-cyclic monophosphate) was demonstrated in HEK293 cells, that was prevented with the PKA inhibitor H89. The amino acid region behind position 952 on IRS2 was identified as cAMP/PKA-dependent 14-3-3 binding region by GST-14-3-3 pulldown as- says. Mass spectrometric analyses revealed Ser1137/Ser1138 as cAMP-dependent, potential PKA phosphorylation sites. Inhibition of Akt/PKB or ERK did not prevent the cAMP-dependent phosphorylation of IRS2 on PKA consensus motifs. Mutation of Ser1137/Ser1138 to alanine strongly reduced the cAMP-dependent 14-3-3 binding as shown by GST-14-3-3 pulldown experiments and co-immunoprecipitation assays. IRS2 protein degradation was demonstrated by the application of cycloheximide and an increased IRS2 protein stability was observed when HEK293 cells stably expressing IRS2 or primary hepatocytes were incubated with forskolin. This reduced IRS2 protein degradation was dependent on the presence of Ser1137/Ser1138, since stimulation with forskolin did not increase protein stability of the double Ala1137/Ala1138 mutant. To conclude, Ser1137/Ser1138 are presented as novel cAMP-dependent phosphorylation sites on IRS2 and their importance in 14-3-3 binding and IRS2 protein stability is demonstrated. This represents a novel mechanism for the cAMP- dependent upregulation of IRS2 protein levels that can be of importance for hepatic metabolism and ß-cell survival.
Oszillation und Musterbildung in reinen und Polypeptid-dotierten Bilayern
(2017) Gaugele, Sebastian; Hanke, Wolfgang
The question of how lipids and proteins interact in bilayers and membranes and why cells and organelles have different lipid compositions in their membranes has been in focus of research for decades. Which role these different lipid compositions play for interaction between integrated and associated proteins has not yet been clarified until today. This thesis with the title "Oszillation und Musterbildung in reinen und Polypeptid-dotierten Bilayern" focuses on forces that influence the interaction of lipids and proteins. In both, living cells as well as in bilayer experiments, the protein-lipid interaction is a complex process involving a huge number of molecules. Different test series with temperature and amperage have been carried out using electrophysiological experiments with bilayers and fluorescence in vesicles. The results were used to determine similarities and differences that indicate certain characteristics of the setup and the interaction among the molecules. Since the question of the interaction between proteins and lipids is a very complex process, the already well-known pore former alamethicin has been used for this work. Alamethicin was used in the both types of experiments, bilayer and vesicles, with different lipid compositions consisting of DOPC, DOPE, DOPS and DMPC. The lipids were solved in ethanol first and then added with certain concentrations of alamethicin in two test chambers which were connected to each other by a small hole. In this hole the bilayer was formed by the addition of aqueous KCl solution. After that the current-clamp-method with different currents was used and subjected to the bilayer. The resulting voltage profiles were observed for oscillation patterns. Subsequently, the obtained data were subjected to the method of Fourier-Transformation to separate the results from noise and interference as well as overlays which resulted from the multi-channel system of alamethicin. The Fourier-Transformation has shown a large number of heterogeneous oscillation patterns. Due to the great heterogeneity of the results that did not allow direct comparisons of individual data with each other the average time-period of oscillation was calculated. This data were used to form data clouds where regression lines were generated. In addition the slopes of the lines were calculated for comparison. The results show that the orientation and slope angle of the slopes directly dependent on the concentrations of alamethicin and KCl. It was found that the addition of a small amount of negatively charged lipid like DOPS to a mixture of DOPC and DOPE has a big impact on the results and can change the orientation of the slopes of the average time-period of oscillation. Similar results were found while using asolectin bilayer containing inositol that is negatively charged, too. As a result, it was concluded that contrary to previous assumptions of some earlier work groups, bilayers are not pure hydrophobic barrier layers between two aqueous phases but highly complex structures that affect the gating of its integrated pores in different ways. The insert and operation of alamethicin strongly depends on the used lipids. Moreover a major dependency from the used salt concentration of the alamethicin pores and bilayer stability was found. The experiments with DMPC and temperature showed that the chain length of the lipids play an important role for integrated proteins on the one hand. On the other hand it could be confirmed that the phase transition of lipids can comprise a wide range of temperature of several degree Celsius depending on the experimental conditions. It could also be confirmed that alamethicin remains active in the liquid-crystal domains during phase transition of a bilayer. In addition it was found that temperature dependent changes appear in voltage oscillation patterns that affect both the average time-period and its resulting slopes in angle and orientation.
Prediction of protein-protein complexes by combining size exclusion chromatography and mass spectrometric analysis
(2021) Gilbert, Max; Schulze, Waltraud
Two major objectives were pursued and met in this study. First, the goal was to add to the scientific toolbox a diligent method for uncovering PPi dynamics on a proteomic scale, with a focus on plant membranes. There are large-scale or high-throughput approaches, but they rely on genetically modified proteins or heterologous expression systems to describe PPi outside of their natural context. Similarly, those methods are incapable of describing the dynamics of protein interactions. In course of this study, a co-elution based approach was combined with modern mass spectrometric label free quantification in order to investigate PPi and interaction dynamics on a proteomic scale. A rigorous data processing pipeline was developed to not only address known fallacies of using co-elution based methods (such as for example random co elution), but also to access and utilize meta-information in form of protein abundance and protein network connectivity to draw conclusions not only on proteomic scale, but also for individual proteins. In total, 6.928 individual proteins extracted from Arabidopsis thaliana root membranes were detected under different nutritional conditions (full nutrition, nitrogen starvation and nitrogen resupply). The data processing pipeline described in this study was used to predict and discover connectivity information for at least 2.058 of these proteins. Each step in data processing was validated by comparison to database confirmed interactions to improve filtering criteria. Protein abundance was evaluated through a unique ranking system, allowing a seamless integration as network attributes for each condition. From the suggested interaction data, an interactome network of the various nutritional conditions was reconstructed. Using different network parameters from graph theory, protein significance and dynamic conditional changes were described. Second, this study applied the aforementioned approach to identify relevant proteins involved in nitrogen signaling in Arabidopsis thaliana root membranes. Through correlation analysis and network reconstruction, receptor kinase AT5G49770 was identified as a component of the nitrogen signaling network that collaborates with co-receptor QSK1, BAK1, the nitrogen transporter NRT2.1 and proton pump AHA2. In response to nitrogen deficiency, the network parameters of AT5G49770 reacted strongly and its involvement was demonstrated by a phenotypic similarity to knock-out lines of NRT2.1, NRT1.1 and AHA2 during a root growth assay of Arabidopsis seedlings. The interaction between QSK1 and BAK1 was further confirmed using FRET/FLIM microscopy and pulldown assays. These findings show that combining a co-elution based approach with a rigorous data processing pipeline and network analysis is suitable to study the protein interaction environment and signal response dynamics in plant root membranes. The modular experimental design allows for a simple adaptation to study different stimuli and the unbiased proteomic approach yields results for proteins regardless of the individual scientific focus. Meta-information such as protein abundance and network connectivity parameters can be used to prospect and identify important proteins involved in stress response dynamics. The author of this study is confident that the proteomic data produced can be utilized in further research and contributes to the understanding of nitrogen signaling in plant root membranes. Through integration of the data processing pipeline and adaptation to different scientific scenarios, valuable information beyond protein interaction is gained. Thus, this work makes an important contribution to the advancement of proteomic analysis and data interpretation methodology.