Institut für Biologie

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Adaptations of maize to low phosphate availability : establishing regulatory networks from large-scale quantitative proteomic profiling
(2022) He, Mingjie; Schulze, Waltraud
Maize (Zea mays) is an important crop in global for human food, animal feed and industrial usage. Suboptimal phosphorus (P) availability is one of the primary constraints for maize growth and productivity (Jianbo Shen et al., 2011; L.pez-Arredondo et al., 2014). Over 70% arable land suffers from P-deficiency, and plants can take up small amounts of P from the soil due to P-fixation. However, over-application of P fertilizer has frequently happened in last decades and resulted in environmental pollution (L.pez- Arredondo et al., 2014). Modern agriculture calls for maintaining productivity while reducing synthetic-P fertilizer inputs and losses, thus, requiring breeding of novel cultivars to increase phosphate use efficiency (PUE) (Balemi and Negisho, 2012; X., Li, Mang, et al., 2021; Mardamootoo et al., 2021). Understanding the regulation of maize to low phosphate(LP)-availability at the molecular level will offer unlimited potential for the development of selection markers and engineering targets in breeding programs. Nowadays, “OMIC” approaches and computational science are developing rapidly. They are advanced tools for investigation of molecular adaptations on a large-scale and in a systemic view. Thereby, the major research task within this thesis is to reveal P-deficiency induced responsive components and regulations at protein level based on proteomic profiles, aiming to provide promising candidate genes/proteins for research on the molecular mechanisms of adaptation to LP-stress, and potentially to provide promising candidate gene/proteins for development of selection markers and engineering targets to obtain desired traits, in the long term goal of improving PUE in novel cultivars. In Chapter 1, we focused on six genotypes (EP1, F2, F142, F160, SF1, SM1) with close genetic background but several contrasting traits to LP-stress, such as PUE (X., Li, Mang, et al., 2021). They were cultured in pot with either sufficient or inefficient P-fertilizer in a climate chamber for one month. The young seedlings were sampled by root and shoot for analysis of multiple traits, transcriptome and proteome. Firstly, we constructed the co-expression network of proteins and transcripts separately using WGCNA method (Langfelder and Horvath, 2008), which predicted potential protein-protein interactions or their co-regulations. Secondly, we categorized proteins/transcripts to modules according to their different coexpression patterns, thus, identified potential determining relationships of modules-traits. Thirdly, we compared the responses between transcripts and proteins, presenting their responses being concordant or dis-concordant. Fourthly, we identified common and genotype-specific P-starvation response modules and biological processes. Finally, we focused on protein kinases, which play roles as regulators, to demonstrated protein kinases-centered network and validated protein interactions between mitogenactivated protein kinase-kinase 1 (MEK1, Zm00001d043609) either with sucrose synthase1 (SH1,Zm00001d045042) or translation elongation factor 1-gamma 3 (eEF1B-γ, Zm00001d046352). MEK1 is a potential genotype-specific regulator via sucrose metabolism and translation elongation process. In Chapter 2, we aimed to adapted an experimental workflow for phosphoproteome analysis in maize, addressing the interference to phosphoproteome quantification by fibers, secondary metabolites and low abundant of phosphorylated proteins. In this manuscript, we described a rapid and universal protocol for both proteome and phosphoproteome analysis that is suitable for cereal crops. The results of phosphoproteome in maize root testing samples showed that proteins within kinase-centered network in Chapter 1 can be largely quantified based on this workflow. It provides a possible way to analyze phosphorylation dynamics to P-starvation responses, it allows further investigation for kinase-centered 1 network in Chapter 1 to identify phosphorylation pairs of “protein kinase – protein substrate”, which will largely expand a view on P-starvation regulations through posttranslational modifications.
Analyse von Pathogenresistenzmechanismen in Tomate (Solanum lycopersicum L.)
(2008) Gerhardts, Anja; Pfitzner, Artur J. P.
For many organisms plants serve as a source of nutrients and energy, but because of their static location they are exposed to various harmful environmental influences. Due to this factor they have developed complex defence mechanisms e. g. for protection against pathogens. An important aspect of these defence mechanisms is the expression of intrinsic resistance genes (R) that detect pathogenic avirulence gene products (Avr) thereby causing a hypersensitive response (HR) in the infected cells and consequently inhibiting the systemic infection of the plant. In this work the resistance genes Tm-2 and Tm-2² of tomato were isolated, cloned and sequenced. The allelic R genes are members of the CC-NBS-LRR group of resistance genes, which is widely spread in plants, and differ only in four amino acids. This is surprising because using resistance breaking ToMV strains Weber et al. (2004) showed that both resistance gene products interact differently with the movement protein (30 kDa MP = Avr) of the virus. To gain further insight into this phenomenon of different pathogen detection, chimeric exchange constructs (A1 and A2) were designed through restriction in the region between the NBS and the LRR domain. These four constructs were used for transformation of MM tomatoes as well as NN and nn tobacco plants. The expression of the resistance gene constructs in MM an nn lines did not confer the expected resistance to ToMV. Nevertheless in older infected nn transformants a formation of spontaneous necrosis was observed, which indicates a delayed development of HR. One possible explanation could be that the presence of only the resistance gene product is not sufficient to detect the viral movement protein and that other host cellular components are involved in this process (as in the guard hypothesis by Dangl and Jones, 2001). This assumption is supported by our yeast two hybrid interaction experiments which showed that a direct interaction of Tm-2 and 30 kDa MP can be excluded. For the NN transformants differences in functionality of the constructs was observed. While NN/Tm-2 and NN/A2 plants showed extreme resistance to ToMV wild type (ToMV0) and the Tm-2² resistance breaking strain ToMV2², the Tm-2² and A1 constructs conferred less resistance to ToMV0 and the Tm-2 resistance breaking strain ToMV1-2. This finding also supports the assumption that there is a difference in pathogen detection between the two alleles. Furthermore it shows that the detection takes place within the LRR region because the exchange construct that behaves in the same way as the endogenous resistance gene carries the C-terminal LRR domain of this allele. The hydroxycinnamoyl-CoA:tyramine N-(Hydroxycinnamoyl)transferase (THT) was found to be another candidate for transmission of pathogen resistance during HR (Gerhardts, 2003). Our in vivo results show that the products of the THT enzymatic reaction induced during HR does not only have an antimicrobiotic effect on the pathogen (von Roepenack-Lahaye et al., 2003; Newman et al., 2001) but also has an apoptotic effect on the plant cell itself.
Changes in the concentration of particular hormones and carbohydrates in apple shoots after "bending" respectively chemical treatments and relationship to the flower induction process
(2005) Boonplod, Nopporn; Bangerth, Fritz
SUMMARY Apples are cultivated commercially throughout the temperate zone. A regular production however does not seem possible because of irregular yields from year to year. Main causes for this are the so called "alternate bearing" behavior which is the result of profuse flowering in one year but few or no flowers in the following year. It is reported that too vigorously growing shoots are part of the reasons for alternate bearing in apple trees. Applications of chemicals or conventional cultural practices, such as bending shoots have been widely used to restrict shoot growth and promote flower induction. However, the physiological mode of action of these methods in FI is still unknown. Phytohormones are thought to be involved in the process of flower induction (FI). In the above experiments, we investigated changes in endogenous hormones, starch and sugar contents after bending upright shoots into a horizontal position and spraying apple trees with the growth regulators Alar plus Ethrel to improve FI. The experiments were carried out during the years 2001 to 2003 at the Experiment Station, of the University of Hohenheim, Germany, whereby the apple cvs. ?Golden Delicious?, ?Boskoop?, ?Elstar? and ?Idared? were used. The apical part of growing shoots and non-growing bourse shoots, beside bark, wood and shoot diffusates were collected. Plant samples were frozen immediately in liquid nitrogen and freeze dried. Phosphate buffer 0.1M, pH 6.2 was used for collecting auxin in the shoot diffusates. All samples were stored at ?20C until extraction and purified, identified and quantified by Radio Immuno Assay (RIA). The results revealed, in general, that shoot bending and spraying with Alar plus Ethrel changed the endogenous hormone concentrations in the apical part of shoots, as well as in wood, bark and shoot exudates of apple trees. The ?Golden Delicious? cultivar and vigorously growing shoots showed clearer tendencies of hormonal changes than the other cvs. and non-growing bourse shoots. Cytokinin concentrations in the apical part of shoots, and in wood and bark increased after both treatments. Contrary to that, GAs and IAA concentrations in the apical part of shoots and in shoot exudates showed the opposite results. Both treatments had no effect on the concentration of ABA. Ethylene production in shoot tips was considerably stimulated by the combined treatment of Ethrel plus Alar probably due to Ethrel being a "synthetic precursor" of ethylene. Considerable variation existed in the mentioned hormonal changes in respect to the year of examination and the cv. under investigation. Time of treatments and in particular climatic conditions were probably the most influential variables. In spite of all this and on the basis of the above results the conclusion can be drawn that higher concentrations of cytokinins and lower concentrations of gibberellins and auxin are favorable for FI. Spraying with Alar plus Ethrel and bending of shoots seemed to decrease the reducing-sugars, as well as sucrose and starch concentrations in growing shoots and their leaves. In non-growing shoots, spraying seemed to reduce starch but to increase reducing-sugars and sucrose concentrations. A correlation between changes in carbohydrate contents (reducing sugar, sucrose and starch) caused by the spraying treatments and FI does not seem to exist. All the observed changes in the carbohydrate concentrations caused by spraying treatments were not particular impressive and did not really support the often published claim that the effect of spraying growth regulators, bending shoots or other cultural practices may mediate their stimulatory effect on FI via a change in carbohydrates. In contrast to that the above observed experimental results rather suggest that hormones are more effectively involved in the flower induction process of fruit trees.
Charakterisierung der Matrixmetalloproteinasen (SlMMP1 & SlMMP2) aus Tomate (Solanum lycopersicum) und ihre Rolle in der pflanzlichen Entwicklung und Pathogeninteraktionen
(2010) Pasule, Christian; Schaller, Andreas
Matrixmetalloproteinases belong to the family of metzincins and are widely distributed in prokaryotic as well as eukaryotic organisms. Mammalian matrixmetalloproteinases function in the formation of peptide hormones, growth factors and receptor proteins and regulate important physiological processes. Little is known about the function of the related plant matrixmetalloproteinases, except for a few enzymes in plants. Their function in tomato plants is still unresolved. However, preliminary data suggest that matrixmetalloproteinases may play essential roles in the regulation of development and pathogen defense in tomato plants. The present study aimed at the elucidation of the function of the tomato matrixmetalloproteinases SlMMP1 and SlMMP2 by purification and biochemical characterization of the recombinant proteins, detailed expression analysis in tomato plants, as well as the phenotypical and molecular analysis of transgenic plants with reduced expression levels for SlMMP1/2 (SlMMP1/2-RNAi). SlMMP1/2 were expressed in E. coli and purified by affinity chromatography. Activity of recombinant MMPs was stimulated by Ca2+-ions. The highest activity was measured for SlMMP1 at pH 6,5 and for SlMMP2 at pH 7. The apparent Km-values for a fluorigenic peptide substrate were 19,5 µM for SlMMP1 and 19,9 µM for SlMMP2, with a catalytic efficiency (kcat/KM) of 0,010 and 0,024 s-1*µM-1 respectively. These data suggest redundant functions for SlMMP1 and SlMMP2. SlMMP1 was found to be expressed in all tested organs and developmental stages with highest expression levels in eight days old seedlings and stems from six weeks old plants. A constitutive expression in leaves from six weeks old plants was not observed for SlMMP2. In eight days old seedlings, SlMMP2 showed higher expression in roots and hypocotyls than in cotyledons. The tissue-specific expression of SlMMP1 and SlMMP2 suggests specific functions and substrates for the two enzymes in different tissues, inspite of their similar biochemical properties. SlMMP1/2 were localised to cell walls from hypocotyls and roots using immunohistochemistry. This confirms the assumed extracellular localisation of the enzymes. Transgenic plants with reduced SlMMP1/2 expression levels exhibit alterations in surface structure leading to necrotic lesions two weeks after germination. The lesions spread over the entire hypocotyl during later stages of development. Cellular organisation was altered in cortical tissues accompanied by cell death events in the epidermis and subepidermal cell layer. These effects spread over leaves during later stages of development. SlMMP1/2-RNAi-plants accumulate massive amounts of phenolic compounds, and exhibit impaired root development with a reduction in overall root length and reduced numbers of primary and secondary lateral roots. Many of the observed effects suggest an involvment of ethylene and a higher ethylene production was in fact confirmed in SlMMP1/2-RNAi-plants. The effects of the loss of SlMMP1/2 on gene expression were analyzd with microarrays in hypocotyls from SlMMP1/2-RNAi-plants. 522 genes were found to be differentially regulated in SlMMP1/2-RNAi-hypocotyls, with 332 up- and 190 downregulated as compared with widtype plants. Genes with roles in plant development, stress, reaction during abiotic and biotic stimuli, and genes with unknown roles in biological processes appeared to be overrepresented among the differentially regulated genes, however, statistically not significantly. Genes with functions in transport were found to be underrepresented in RNAi-plants. Based on the observation that infection with Sclerotinia sclerotiorum led to induction of SlMMP1/2 gene expression, a possible function for the enzymes in pathogen defense was analyzed. A direct involvment of the enzymes in plant defense against the fungus can be excluded, however, because no differences in fungal growth were detected between wildtype and SlMMP1/2-RNAi-plants. Furthermore, it was analyzed if there are differences in resistance against the biotrophic pathogen Xanthomonas campestris pv. vesicatoria (Xcv) between wildtype and SlMMP1/2-RNAi-plants. Bacterial growth was found to be reduced for virulent, avirulent and non-pathogenic bacteria, accompanied by strong necrosis in SlMMP1/2-RNAi-plants. These data suggest a function for SlMMP1/2 in the regulation of non-specific rections against biotic stresses. The results of the present study provide a basis for a more complete understanding of the function of SlMMP1/2 in development and pathogene defense of tomato plants.
Detection and molecular characterization of canine babesiosis causative agent Babesia canis in naturally infected dogs in the Dobrogea area (Southeastern Romania)
(2023) Ionita, Mariana; Leica, Laurentiu; Wassermann, Marion; Mitrea, Emanuel; Nicorescu, Isabela Madalina; Mitrea, Ioan Liviu
Canine babesiosis is an emerging tick-borne disease of major veterinary concern in Europe. Its prevalence has increased in the last two decades and is spreading rapidly toward the north. The aim of this study was to investigate the genetic diversity of Babesia spp. strains isolated from naturally infected dogs in a tick-endemic area (Dobrogea) in southeastern Romania. For this purpose, a total of twenty-three samples from dogs diagnosed with various clinical forms of babesiosis, evaluated by means of clinical history, physical examination, and hematological tests, were subjected to a molecular investigation using PCR, sequencing analysis, and genetic characterization. A microscopic examination of thin Diff-quick-stained blood smears revealed large intra-erythrocytic Babesia piroplasms in all dogs. The PCR and sequencing analysis results indicated the presence of Babesia canis in 22 dogs (95.7%) and Babesia vogeli in 1 dog (4.3%). Among the B. canis isolates, two genotypes were distinguished based on two nucleotide substitutions (GA→AG) observed in the 18S rRNA gene sequences (at positions 609 and 610), with the AG genotype predominating (54.5% of samples), while the GA variant was identified in 9.1% of samples. In the remaining isolates (36.4%), both variants were identified. The B. vogeli-positive dog also tested positive for antibodies against Ehrlichia canis and displayed severe disease. This study reports, for the first time, the presence of genetically heterogenic B. canis strains in dogs with clinical babesiosis in Romania. These findings provide a basis for future studies on the relationship between the genetic structure of the causative agents of canine babesiosis in Romania and the course of the disease.
Entwicklung und Testung neuer DNA- und Protein-basierter Multikomponentenvakzinen sowie regulatorischer Adjuvanzien gegen eine Infektion mit B. anthracis in Auszucht-Mäusen und Ziegen
(2015) Köhler, Susanne Melanie; Beyer, Wolfgang
The discovery of the Sterne spore live vaccine (SSLV) and subsequently its application in a veterinary context contributed to the global reduction of Anthrax related outbreaks since 1930. Nonetheless the causative agent Bacillus anthracis is still prevalent in some mediterranean countries, South and Central America, Africa and Central Asia, as well as the USA and Canada. Reasons for this are the persistence of the pathogen in the soil, as well as still undefined factors for an ongoing cycle of outbreak and spread of the disease and the limited applicability of the SSLV. This includes the necessity to revaccinate annually, the residual virulence in certain sensitive species (e. g. goats and llamas) and the incompatibility to treat and vaccinate simultaneously. To participate in the ongoing search for alternative vaccines this work was dedicated to evaluate protein- and DNA-based components as potential ingredients for a multi-component non-living vaccine formulation (NLV). For the protein-based NLV these included rPA83 as part of the Anthrax toxin, rBclA and Formalin inactivated spores (FIS) as spore specific antigens, a Capsule-Lipopeptide conjugate as part of the vegetative form of the pathogen and a Lipopeptide-adjuvant. The DNA-vaccines consisted of vector-backbones comprising signal sequences able to direct the integrated antigens (rPA83, PAD4LFD1 and BclAD1D3) to the MHCI, MHCII and the secretory pathway. A sperate vector encoding for a positive MHCII-regulator (CIITA) and a vector internal sequence for the Interferon-ß promotor stimulator (mIPS1) served as adjuvants for the DNA-vaccines. The majority of the groups showed detectable antibody titres against their respective antigens, with protein vaccines generally eliciting higher titres against rPA83 than the DNA-vaccines. Regarding rBclA equivalent high titres were measured for protein- and DNA-vaccines alike, which also corresponded to the anti-FIS titres for groups immunized with rBclA, FIS or both. The Capsule-Lipopeptide conjugate did not elicit high titres against the capsule, possibly due to an immune suppressing epitope. Survival rates ranged between 10 and 100 %, with full protection only achieved in a combination of all antigens including FIS. All DNA-vectors induced 30 – 50 % protectiveness when given alone. Notably DNA-vectors including BclAD1D3 elicited 50 % survival and sterile immunity. A combination of the most promising vectors encoding for toxin and spore specific antigens achieved 90 % protectiveness in mice. According to the results from the mice trials, the auspicious protein- and DNA-vaccine combinations were tested in goats in comparison to the SSLV in cooperation with our project partners in South Africa and Turkey. The efficacy of the SSLV was assessed in 3 groups which were challenged shortly after the first immunisation, one year after the first immunisation or after the revaccination. Apart from the comparison of immunogenicity and protectiveness between SSLV and NLV in goats, assessment of data concerning the titre development of SSLV-immunized goats during the course of a year as well as detailed diagnostic data during the infection (behavior, temperature, bacterial loads, correlations and minimal infective dose) were integral part of this study. Compared to one another the SSLV-immunized animals showed equal or higher antibody titres against the measured antigens, with FIS and rPA83 being the most immunogen antigens. Utilizing a higher dose (75 µg) the protein-based NLV protected equivalently to the SSLV (60 – 100 %) yielding 50 % protectiveness without FIS and 80 % if FIS was included. The DNA-vaccines showed little to no immunogenicity in goats, thus no challenge was performed on these animals. The humoral reaction against BclA was generally poor in goats, which has not been noted before and could be a basis for further improvements concerning the SSLV and NLV alike. The different immunizations with the SSLV revealed a broad range for the efficacy of the first vaccination as well as a notable difference in the antibody spectrum between first vaccination and revaccination. Together with the recorded data of the antibody titre development throughout a year a more optimal protocol for immunisation with the SSLV, possibly in combination with an NLV was postulated.
External nutrition stimuli induced proteome and phosphoproteome responses of maize root hairs and arabidopsis root microsomal fraction
(2021) Li, Zhi; Schulze, Waltraud
This work studied how the proteome from young maize root hair cells responds to different nutrition deprivation, and gives perspectives to the possible involvement of NRT1.1 and NRT2.1 in regulating root membrane phosphoproteome responses. This work also proposes a phospho-switch model that may explain how the NRT2.1 activity was regulated.
Helminths in invasive raccoons (Procyon lotor) from Southwest Germany
(2023) Reinhardt, Nico P.; Wassermann, Marion; Härle, Jessica; Romig, Thomas; Kurzrock, Lina; Arnold, Janosch; Großmann, Ernst; Mackenstedt, Ute; Straubinger, Reinhard K.
As hosts of numerous zoonotic pathogens, the role of raccoons needs to be considered in the One Health context. Raccoons progressively expand their range as invasive alien species in Europe. This study aimed to investigate the intestinal helminth fauna of raccoons in Baden-Wuerttemberg, Germany, as no such screening had ever been conducted there. In total, we obtained 102 animals from hunters in 2019 and 2020. Intestinal helminths were retrieved using the SSCT (segmented sedimentation and counting technique) and identified morphologically and by PCR-based Sanger sequencing. Fecal samples were assessed using the ELISA PetChekTM IP assay (IDEXX, Germany) and flotation technique. The artificial digestion method was employed for analyzing muscle tissue. We detected species of four nematode genera (Baylisascaris procyonis, Toxocara canis, Capillaria spp., and Trichuris spp.), three cestode genera (Atriotaenia cf. incisa/procyonis, Taenia martis, and Mesocestoides spp.), and three trematode genera (Isthmiophora hortensis/melis, Plagiorchis muris, and Brachylaima spp.). Echinococcus spp. and Trichinella spp. were not found. The invasive behavior and synanthropic habits of raccoons may increase the infection risk with these helminths in wildlife, domestic and zoo animals, and humans by serving as a connecting link. Therefore, it is crucial to initiate additional studies assessing these risks.
Industrial chicory genome gives insights into the molecular timetable of anther development and male sterility
(2023) Waegneer, Evelien; Rombauts, Stephane; Baert, Joost; Dauchot, Nicolas; De Keyser, Annick; Eeckhaut, Tom; Haegeman, Annelies; Liu, Chang; Maudoux, Olivier; Notté, Christine; Staelens, Ariane; van der Veken, Jeroen; van Laere, Katrijn; Ruttink, Tom
Industrial chicory (Cichorium intybus var. sativum) is a biannual crop mostly cultivated for extraction of inulin, a fructose polymer used as a dietary fiber. F1 hybrid breeding is a promising breeding strategy in chicory but relies on stable male sterile lines to prevent self-pollination. Here, we report the assembly and annotation of a new industrial chicory reference genome. Additionally, we performed RNA-Seq on subsequent stages of flower bud development of a fertile line and two cytoplasmic male sterile (CMS) clones. Comparison of fertile and CMS flower bud transcriptomes combined with morphological microscopic analysis of anthers, provided a molecular understanding of anther development and identified key genes in a range of underlying processes, including tapetum development, sink establishment, pollen wall development and anther dehiscence. We also described the role of phytohormones in the regulation of these processes under normal fertile flower bud development. In parallel, we evaluated which processes are disturbed in CMS clones and could contribute to the male sterile phenotype. Taken together, this study provides a state-of-the-art industrial chicory reference genome, an annotated and curated candidate gene set related to anther development and male sterility as well as a detailed molecular timetable of flower bud development in fertile and CMS lines.
Limitations of soil-applied non-microbial and microbial biostimulants in enhancing soil P turnover and recycled P fertilizer utilization: A study with and without plants
(2024) Herrmann, Michelle Natalie; Griffin, Lydia Grace; John, Rebecca; Mosquera-Rodríguez, Sergio F.; Nkebiwe, Peteh Mehdi; Chen, Xinping; Yang, Huaiyu; Müller, Torsten
Introduction: Phosphorus recovery from waste streams is a global concern due to open nutrient cycles. However, the reliability and efficiency of recycled P fertilizers are often low. Biostimulants (BS), as a potential enhancer of P availability in soil, could help to overcome current barriers using recycled P fertilizers. For this, a deeper understanding of the influence of BSs on soil P turnover and the interaction of BSs with plants is needed. Methods: We conducted an incubation and a pot trial with maize in which we testednon-microbial (humic acids and plant extracts) and microbial BSs (microbial consortia) in combination with two recycled fertilizers for their impact on soil P turnover, plant available P, and plant growth. Results and discussion: BSs could not stimulate P turnover processes (phosphatase activity, microbial biomass P) and had a minor impact on calcium acetate-lactate extractable P (CAL-P) in the incubation trial. Even though stimulation of microbial P turnover by the microbial consortium and humic acids in combination with the sewage sludge ash could be identified in the plant trial with maize, this was not reflected in the plant performance and soil P turnover processes. Concerning the recycled P fertilizers, the CAL-P content in soil was not a reliable predictor of plant performance with both products resulting in competitive plant growth and P uptake. While this study questions the reliability of BSs, it also highlights the necessity toimprove our understanding and distinguish the mechanisms of P mobilization in soil and the stimulation of plant P acquisition to optimize future usage.
Morphological and molecular studies on cerebral and non-cerebral coenurosis in sheep and goats
(2017) Christodoulopoulos, Georgios; Mackenstedt, Ute
In dieser Forschungsarbeit wurden Isolate von Taenia multiceps als Erreger der zerebralen und nicht-zerebralen Coenurose bei Schafen und Ziegen vergleichend untersucht. Erreger von nicht-zerebraler Coenurose aus einem breiten geographischen Bereich in Afrika und Westasien wurden verglichen mit Erregern zerebraler Coenuruszysten von gesammelten Drehkrankheitsfällen aus Griechenland, wo zerebrale Coenurose häufig vorkommt, wo aber nicht-zerebrale Coenurose noch nie beschrieben wurde. Diese Forschungsarbeit enthält eine feld- und eine laborexperimentelle Komponente und beschäftigt sich mit Aspekten wie: (i) das Vorhandensein oder Fehlen von nicht-zerebraler Coenurose bei Schafen; (ii) die Beschreibung von nicht-zerebraler Coenurose im Zwischenwirt (Schafe und Ziegen); und (iii) die phylogenetische Auflösung des T. multiceps-Clusters sowie eine mögliche Erklärung, warum nicht-zerebrale Coenurose in bestimmten geographischen Gebieten unbekannt ist. Im Rahmen der Felduntersuchung wurde eine Gesamtzahl von 90,415 geschlachtete Schafe und 2,284 geschlachtete Ziegen aus Schlachthöfen der Vereinigten Arabischen Emirate (UAE) und Ägypten untersucht, die aus verschiedenen tropischen und subtropischen Ländern wie Indien, Pakistan, Iran, Oman, Sudan, Somalia und Äthiopien stammten. Die Feldarbeit umfaßte außerdem die Sammlung von zerebralen Coenurosezysten von 20 Schafen und sechs Ziegen, die vom griechischen Festland und Umgebung stammten. Im Labor wurden vier Teilstudien durchgeführt: (1) eine morphologische Untersuchung der Merkmale von nicht-zerebralen Coenurosezysten, deren Cluster und der Protoskolizes, (2) ein morphologischer Vergleich der Rostellarhaken der zerebralen und nicht-zerebralen Coenurosezysten, (3) ein morphologischer Vergleich von erwachsenen Würmern aus Hunden, die experimentell mit Protoskolizes von zerebralen und nicht-zerebralen Zysten von Schafen und Ziegen infiziert worden waren, und (4) eine molekulare Analyse von Teilsequenzen dreier mitochondrialer Gene (nad1, cox1 und 12S rRNA) der oben genannten Isolate von zerebralen und nicht-zerebralen Zysten. Die Prävalenz von nicht-zerebraler Coenurose betrug bei Ziegen 1,75% und nur 0,008% bei Schafen. Die Zysten wurden in verschiedenen Muskeln sowie an den Nieren, am Mesenterium und am Herz gefunden. Sechsundsiebzig nicht-zerebrale Coenuruszysten von Ziegen wurden gesammelt und statistisch ausgewertet. Die Anzahl der Protoskolizes korrelierte signifikant positiv mit dem Volumen von Zysten und die Anzahl von Clustern korrelierte signifikant positiv mit der Anzahl der Protoskolizes, was auf positives allometrisches Wachstum hinweist. Die Anzahl von Clustern korrelierte mit der Anzahl von Zysten, was auf ein negatives allometrisches Wachstum hinweist. Die biologische Bedeutung dieser Allometrie ist nicht bekannt, aber der Parasit investiert offenbar seine Ressourcen mehr in das Wachstum von Protoskolizes und weniger in das Wachstum von Zysten und Clustern. Die morphologischen Untersuchungen der Rostellarhaken von Protoskolizes aus zerebralen und nicht-zerebralen Zysten und die in Hunden experimentell erzeugten adulten Würmer zeigten keine Unterschiede in Bezug auf die Wirtstierart (Schafe oder Ziegen). Im Gegensatz dazu wiesen adulte Würmer, die aus der Inokulation zerebraler und nicht-zerebraler Zysten hervorgingen, deutliche morphologische Unterschiede auf. Die meisten Meßwerte der Haken und Proglottiden unterschieden sich signifikant, aber die Form der kleinen Haken, die Verteilung der Hoden in den reifen Proglottiden und das Aussehen der Spiralen des Vas deferens waren die Merkmale mit den auffälligsten Unterschieden. Diese morphologischen Unterschiede fallen allerdings in den Bereich der Variationen von T. multiceps. Die phylogenetische Analyse der mitochondrialen Haplotypen ergab drei distinkte Cluster: eines, das sowohl zerebrale Isolate aus Griechenland als auch nicht-zerebrale Isolate aus tropischen und subtropischen Ländern umfaßte, und zwei Cluster, die ausschließlich aus zerebralen Isolaten aus Griechenland bestanden. Die meisten der nicht-zerebralen Proben gruppierten zusammen, bildeten aber keine monophyletische Gruppe. Dasselbe gilt für geographische Aspekte, obwohl Proben aus derselben Region zu Clustern tendierten. Die Daten zeigten eine hohe intraspezifische Diversität. Die Ergebnisse dieser Studie unterstützen den Zusammenhang zwischen genetischer Identität der T. multiceps-Isolate und der geographischen Herkunft, und führten zum Vorschlag einer neuen Hypothese zum Vorkommen von zerebralen und nicht-zerebralen Formen. Unsere phylogenetische Analyse legt nahe, daß die Entwicklung der zerebralen Coenuri bei Schafen eine ursprüngliche Eigenschaft von T. multiceps sein könnte. Alle Varianten wären damit in der Lage, zerebrale Coenurose bei Schafen zu verursachen, wogegen nur einige Varianten, vor allem aus einem genetischen Cluster, sekundär die Fähigkeit erworben haben, das Gehirn von anderen Arten (Ziegen und Rindern) zu befallen, sowie nicht-zerebrale Formen (vor allem in Ziegen) hervorzubringen. Unsere phylogenetische Analyse zeigt somit eindeutig eine molekulare Basis für nicht-zerebrale Pathogenität innerhalb der Art T. multiceps. Spezifische Biosicherheitsmaßnahmen sollten daher erwogen werden, um die Einführung von T. multiceps Varianten mit einer solchen Pathogenität zu verhindern.
Na+-coupled respiration and reshaping of extracellular polysaccharide layer counteract monensin-induced cation permeability in Prevotella bryantii B14
(2021) Trautmann, Andrej; Schleicher, Lena; Pfirrmann, Jana; Boldt, Christin; Steuber, Julia; Seifert, Jana
Monensin is an ionophore for monovalent cations, which is frequently used to prevent ketosis and to enhance performance in dairy cows. Studies have shown the rumen bacteria Prevotella bryantii B14 being less affected by monensin. The present study aimed to reveal more information about the respective molecular mechanisms in P. bryantii, as there is still a lack of knowledge about defense mechanisms against monensin. Cell growth experiments applying increasing concentrations of monensin and incubations up to 72 h were done. Harvested cells were used for label-free quantitative proteomics, enzyme activity measurements, quantification of intracellular sodium and extracellular glucose concentrations and fluorescence microscopy. Our findings confirmed an active cell growth and fermentation activity of P. bryantii B14 despite monensin concentrations up to 60 µM. An elevated abundance and activity of the Na+-translocating NADH:quinone oxidoreductase counteracted sodium influx caused by monensin. Cell membranes and extracellular polysaccharides were highly influenced by monensin indicated by a reduced number of outer membrane proteins, an increased number of certain glucoside hydrolases and an elevated concentration of extracellular glucose. Thus, a reconstruction of extracellular polysaccharides in P. bryantii in response to monensin is proposed, which is expected to have a negative impact on the substrate binding capacities of this rumen bacterium.
New AMS 14C dates track the arrival and spread of broomcorn millet cultivation and agricultural change in prehistoric Europe
(2020) Filipović, Dragana; Meadows, John; Corso, Marta Dal; Kirleis, Wiebke; Alsleben, Almuth; Akeret, Örni; Bittmann, Felix; Bosi, Giovanna; Ciută, Beatrice; Dreslerová, Dagmar; Effenberger, Henrike; Gyulai, Ferenc; Heiss, Andreas G.; Hellmund, Monika; Jahns, Susanne; Jakobitsch, Thorsten; Kapcia, Magda; Klooß, Stefanie; Kohler-Schneider, Marianne; Kroll, Helmut; Makarowicz, Przemysław; Marinova, Elena; Märkle, Tanja; Medović, Aleksandar; Mercuri, Anna Maria; Mueller-Bieniek, Aldona; Nisbet, Renato; Pashkevich, Galina; Perego, Renata; Pokorný, Petr; Pospieszny, Łukasz; Przybyła, Marcin; Reed, Kelly; Rennwanz, Joanna; Stika, Hans-Peter; Stobbe, Astrid; Tolar, Tjaša; Wasylikowa, Krystyna; Wiethold, Julian; Zerl, Tanja
Broomcorn millet (Panicum miliaceum L.) is not one of the founder crops domesticated in Southwest Asia in the early Holocene, but was domesticated in northeast China by 6000 bc. In Europe, millet was reported in Early Neolithic contexts formed by 6000 bc, but recent radiocarbon dating of a dozen 'early' grains cast doubt on these claims. Archaeobotanical evidence reveals that millet was common in Europe from the 2nd millennium bc, when major societal and economic transformations took place in the Bronze Age. We conducted an extensive programme of AMS-dating of charred broomcorn millet grains from 75 prehistoric sites in Europe. Our Bayesian model reveals that millet cultivation began in Europe at the earliest during the sixteenth century bc, and spread rapidly during the fifteenth/fourteenth centuries bc. Broomcorn millet succeeds in exceptionally wide range of growing conditions and completes its lifecycle in less than three summer months. Offering an additional harvest and thus surplus food/fodder, it likely was a transformative innovation in European prehistoric agriculture previously based mainly on (winter) cropping of wheat and barley. We provide a new, high-resolution chronological framework for this key agricultural development that likely contributed to far-reaching changes in lifestyle in late 2nd millennium bc Europe.

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