Browsing by Subject "Parasit"

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Identifizierung und Charakterisierung der Succinsemialdehyd-Dehydrogenase aus parasitischen und nichtparasitischen Arthropoden
(2008) Rothacker, Boris; Ilg, Thomas
The objective of the PhD project was the molecular and enzymological characterization of SSADH in parasitic and nonparasitic insects and acarids, organism groups where this enzyme was virtually unexplored. In this project, the list of investigated non-arthropod organisms included the bacterium Escherichia coli (Ec) and the mammal Mus musculus (mouse). Their SSADH served as reference enzymes or were used for biophysical experiments. Amongst the arthropods, Drosophila melanogaster (Dm) (fruit fly) was chosen as nonparasitic model insect. Lucilia cuprina (Lc) (sheep blowfly) and Ctenocephalides felis (Cf) (cat flea) were included as important parasitic insects, as was the acarid Rhipicephalus microplus (Rm) as the economically most important tick species. At the start of this thesis, expressed sequence tag studies and whole genome sequencing on Dm suggested the existence of a single copy gene candidate that, based on amino acid sequence homology, was considered to be a candidate for a SSADH ortholog in this species (DmSSADH). The Dm SSADH gene candidate was cloned and expressed in Ec as a soluble protein. To compare the enzymological properties of DmSSADH, another so far uncharacterized Dm gene candidate for an acetaldehyde dehydrogenase (DmALDH) was cloned and expressed in Ec as a soluble protein. Both expression products showed the expected enzymological properties: a NAD+-dependent ssa-oxidizing activity for DmSSADH and a NAD+-dependent acetaldehyde-oxidizing activity for Dm-ALDH. Site-directed mutageneses on DmSSADH performed in this study demonstrated that two residues essential for catalysis are glutamate 277 and cysteine 311. The second part of the thesis encompassed the gene identification, full length gene cloning of SSADH in Lc (LcSSADH) and Cf (CfSSADH), as well as functional expression of one gene version, respectively. Substrate/cosubstrate specificity determinations combined with enzyme kinetics studies showed that both enzymes are predominantly NAD+-dependent SSADHs. Bioinformatics analyses detected N-terminal mitochondrial import sequences in both Lc and Cf SSADH suggesting that these enzymes are localized in vivo in the mitochondrial matrix. The investigation of the genomic structure of the LcSSADH and CfSSADH genes revealed significant differences to the previously known gene organizations: firstly, different to the single copy SSADH gene situation in Ec, Dm, Mus musculus and Homo sapiens, Lc appears to possess 2-3 SSADH genes, while in Cf up to 8 gene copies may be present. Secondly, compared to Dm (2 exons, 1 intron), the exon/intron structure of the SSADH genes in Lc and Cf is not conserved: the one SSADH genomic gene version investigated in detail in Lc contained 3 exons and 2 introns, while the genomic gene version of Cf analysed in this study was devoid of any introns. The central topic of the third part of this thesis was the gene identification and biochemical characterization of SSADH from the tick Rm. By a combination of different PCR methods a tick gene orthologous to insect and mammalian SSADH (RmSSADH) could be identified and isolated. The results of Southern blot analyses of Rm DNA are incompatible with a single copy situation and suggest the presence of 2-3 RmSSADH genes. To compare RmSSADH with a mammalian SSADH, the respective gene was isolated from mouse. Both the tick and the mouse SSADH genes were then expressed as soluble functional proteins in Ec. The initial comparison showed that both proteins are potent NAD+-dependent ssa-oxidizing enzymes with very similar enzyme kinetics. A more detailed comparison of both enzymes suggests that in general, the mouse enzyme appears to be more specific for succinic semialdehyde than the tick enzyme. In the fourth section of the experimental part of this thesis, saturation transfer difference nuclear magnetic resonance spectroscopy (STD-NMR) experiments were performed on three of the above SSADH preparations, to answer questions on the aldehyde substrate and cosubstrate binding to these enzymes. Importantly, the long-standing question whether the free aldehyde of succinic semialdehyde or its hydrated gem-diol form (present in aqueous solution in equimolar amounts) is the binding substrates was answered conclusively in favour of the aldehyde form for both the Ec and the Dm enzyme. Most remarkably, STD-NMR experimental investigation of the ssa interaction with the Dm SSADH cysteine311alanine mutant enzyme demonstrated binding of both the aldehyde and the gem-diol form. This experiment strongly suggests that cysteine311 is acting as an aldehyde versus gem-diol selectivity filter in the active site of the enzyme. Furthermore, STD-NMR epitope mapping of the NAD+/NADP+ binding to Ec SSADH and Dm SSADH was performed. In both cases, the data suggested that the dominant protein-ligand interactions are via the adenine and the nicotinamide ring systems, while the ribose moieties interact much less intensely with the enzymes.
Strategies and mechanisms of cellular interaction between the parasitic weed Orobanche cumana WALLR. and its host Helianthus annuus L.
(2020) Krupp, Anna Clarissa; Spring, Otmar
Sunflower broomrape, Orobanche cumana WALLR., is a root parasitic plant causing considerable yield losses in sunflower cultivation in Europe, North Africa and Asia. Comprehensive knowledge about early interaction stages between host and parasite is necessary to find new ways of controlling this weed. In this thesis, three aspects regarding the biology of O. cumana were studied: 1) the chemotropism of O. cumana germtubes which bend towards the host root, 2) the development of O. cumana on resistant and susceptible sunflower lines and 3) the development of the phloem connection between the O. cumana haustorium and the sunflower host root. Sesquiterpene lactones in sunflower root exudates act as germination stimulants for O. cumana. As sesquiterpene lactones are known inhibitors of plant elongation growth and seem to play a role in the phototropic curvature of sunflower hypocotyls, a chemotropism bioassay on water agar was established to test if they also serve as chemotropic signals for the host-finding of O. cumana germtubes. When sesquiterpene lactone containing sunflower root exudate, sunflower seed oil extract or the sesquiterpene lactone reference costunolide were applied on filter discs, 70 % of the germtubes showed orientation towards them. The artificial strigolactone GR24, however, did not induce chemotropism. A concentration gradient of sesquiterpene lactones exudated from the host root is likely to be responsible for a stronger inhibition of elongation growth on the host-facing flank of the germtube. This would confer a double role of sesquiterpene lactones from root exudates in the sunflower-broomrape-interaction, namely as germination stimulants and as chemotropic signals. One way of controlling O. cumana is the cultivation of resistant sunflower lines. However, this resistance is rapidly overcome by more aggressive pathotypes of the parasite. Therefore, the resistance or tolerance reaction of the sunflower genotype T35001 was investigated in comparison to six other sunflower genotypes with different resistance characteristics. The development of O. cumana was monitored in a root chamber system which allowed permanent assessment of germination, attachment and tubercle formation in the different host-parasite-combinations. All seven tested sunflower lines induced germination and attachment of O. cumana, independent of the expected resistance or susceptibility of the host. A difference between compatibility or incompatibility of the interactions was only observed at the tubercle stage. On T35001, tubercles never occurred, neither in root chamber nor in pot experiments. To find out why the development stopped before the tubercle stage, samples of sunflower roots with attached O. cumana seedlings were analysed by bright field-, fluorescence- and transmission electron microscopy. Histological studies revealed that O. cumana penetrated the host root, but never reached the host’s vascular bundle. The root cortex cells surrounding the Orobanche haustorium showed no ultrastructural changes such as cell wall thickening. Fluorescence microscopy revealed no callose depositions or signs of phytoalexin release. However, ultrastructural examination of the host-parasite-interface showed degeneration processes in both cortex and haustorial cells. Cortex cells were flooded with bacteria, haustorium cells showed degeneration of cytoplasm and nuclei. The resistance mechanism that prevented further development of the O. cumana haustorium did not express itself in a histologically visible way. As holoparasite, O. cumana acquires its entire demand for water, minerals and organic nutrients from the host’s vascular system. The development of the xylem connection between O. cumana and sunflower had previously been reported, but the phloem connection is far more relevant for the parasite in terms of organic nutrients. Accordingly, the ultrastructure of the phloem connection between the haustorium of young O. cumana tubercles and the sunflower root was examined. Parasite and host tissues were intermingled at the contact site and difficult to distinguish, but sieve-tube elements of O. cumana and sunflower could be differentiated according to their plastid ultrastructure. While sieve-element plastids of O. cumana were larger, often irregular in shape and contained few, small starch inclusions, sieve-element plastids of the host were significantly smaller, always round with more and larger starch inclusions. This made it possible to trace the exact contact site of host and parasite sieve elements to show a direct symplastic phloem connection between the two species. The interspecific sieve plate showed more callose on the host side. This allowed detection of newly formed plasmodesmata between host sieve-tube elements and parenchymatic parasite cells, thus showing that undifferentiated cells of the parasite can connect to fully differentiated sieve elements of sunflower.