Browsing by Subject "Tabak"

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Die Bedeutung von Aquaporinen und ihren Interaktionspartnern für die Zelltodregulation in Pflanzen
(2011) Hoch, Tanja; Pfitzner, Artur J. P.
Programmed cell death (PCD, apoptosis) is an induced cell suicide process that plays an important role during the differentiation and pathogen defense responses of plants and animals. BHRF1 (?BamHI fragment H rightward open reading frame no. 1?) is a cell-death modulating protein of the Epstein-Barr virus (EBV), a human lymphotrophic herpes virus. The expression of BHRF1 in transgenic plants led to the formation of necrotic lesions. Further experiments showed that BHRF1 associated necrotic lesions are caused due to stress, senescence and pathogen defense responses. Yeast-two-hybrid-screening of a tobacco cDNA library identified two different aquaporins as partners for interacting with BHRF1. Aquaporins were identified as water channels/carriers within red blood cells, but are also present in all other organisms. Over the last years, more information was gathered indicating that, apart from transporting water, aquaporins had other functional activities. E. g. Henzler and Steudle (2000) demonstrated that aquaporins can act as hydrogen peroxide channels in the algae Chara corallina. Furthermore, publications by Bienert et al. (2006), indicating that aquaporins in plants as well as in animals are also able to transport H2O2. Hydrogen peroxide and other reactive oxygen species (ROS) have long been recognized as important signal molecules during the pathogen defense response in plants, therefore establishing a logical connection between cell death and aquaporins for the first time. It was assumed that the aquaporins NtPIP2.2a, NtPIP2.2b und NtTIP1.1a identified during the yeast-two-hybrid-screen can act as H2O2 channels. In further experiments it could indeed be established that these aquaporins have the ability to transport H2O2 in yeast cells. Yeasts expressing aquaporins could be influenced in their H2O2 sensitivity by the expression of BHRF1. BHRF1 without transmembrane domain (BHRF1deltaTMwt) led to an enhanced H2O2 sensitivity and also to an increase in cell death. In addition, the transient expression of aquaporin could induce necrotic lesions and cell death in Nicotiana benthamiana. Deletion experiments identified a common binding domain for interacting with BHRF1 in these aquaporins. This binding domain consists of the conserved region containing the first NPA motive (?loop? B) that is also half of one pore. Further studies showed that BHRF1 interacts with all kinds of different aquaporins from plants, animals (rAQP8) and humans (hAQP1). BHRF1 most likely binds with the alpha3 helix to the highly conserved NPA region of aquaporins. A cellular protein showing sequence homology to M20 proteases and aminoacylases was isolated when looking for interaction partners of aquaporins in plants. Like BHRF1, this protein binds to the conserved NPA region of the aquaporins. Although the cellular substrate for this protein has to be found yet, an interesting observation was made. Co-expression of the isolated aminoacylase with NtTIP1.1a or NtPIP2.2b in Nicotiana benthamiana led to the inhibition of cell death induced by these aquaporins.
Pathogenitätsfaktoren und induzierte Resistenz in der Interaktion von Orobanche ramosa L. und Nicotiana tabacum L.
(2005) Gonsior, Guido; Sauerborn, Joachim
Parasitic weeds of the genus Orobanche, cause severe damage to vegetables, legumes and sunflower in Europe, the Middle East and North Africa. Recently there is evidence for increasing occurrence of branched broomrape (Orobanche ramosa L.) in rape and tobacco cultivars in France and Germany. This research concerns on biology and pathogenicity of O. ramosa with special focus on the interactions with the host plant. In addition mechanisms of induced resistance of the host plant against the parasite were considered. The results showed genetic differences between the populations of O. ramosa in Germany, which partially parasitize different hosts. As possible factors of pathogenicity the accumulation of reactive oxygen species and a high peroxidase activity together with a high antioxidative capacity caused by the phenylpropanoids Verbascosid and Oraposid were identified. The control of branched broomrape was possible by application of agents, which are described to induce disease resistance. Control of branched broomrape could be based on systemic acquired resistance (SAR) as well as on induced systemic resistance (ISR). As defense mechanisms in tobacco roots against O. ramosa the biosynthesis of the hydroxycoumarin scopoletin and a significant increase of soluble and bound phenolics was discussed. This is underlined by a high activity and an increased expression of phenylalanine ammonia-lyase (PAL) the key enzyme of the phenylpropanoid pathway. Beside phytoalexin production we recognized an increased activity of peroxidases in Orobanche ramosa infected tobacco roots. Further it could be shown that the plant hormone Ethylene as well as the regulation of the sugar metabolism have importance for the resistance of the host plant.
Regulation von NIMIN- und PR1-Genen aus Arabidopsis thaliana (L.) Heynh. und Nicotiana tabacum (L.) in der Salicylat-abhängigen Pathogenabwehr
(2009) Hermann, Meike; Pfitzner, Artur J. P.
Systemic acquired resistance (SAR) is an important defense mechanism of plants against a broad range of pathogens. NPR1 acts as a central regulator controlling the salicylic acid (SA)-dependent formation of SAR through interaction with TGA transcription factors leading to the induction of ?pathogenesis-related? (PR) proteins. The SA-activated expression of the PR1 genes in Arabidopsis thaliana (At) and Nicotiana tabacum (Nt) depends on cis-acting as-1-like elements with a TGACG sequence. This dissertation studies the functional relevance of NIMIN proteins and SA-dependent PR gene induction using the analysis of gene regulation. Arabidopsis has four NIMIN-genes ? N1, N2, N3 and N1b which interact independently of TGA transcription factors with NPR1. N1 and N2 have a common interaction motif and bind to the C-terminus of AtNPR1, whereas N3 binds to the N-terminus of AtNPR1. The binding site for the TGA transcription factors is located relatively central in the AtNPR1 protein. The analysis of the NIMIN gene expression in the SA-dependent signaling pathway of SAR as well as their possible involvement in the Jasmonic (JA) signaling network ought to offer new aspects for understanding the regulation of plant pathogen defense. The relevance of different as-1-like elements was studied by establishing a yeast one-hybrid system. N1b is likely to be an inactive pseudogene. Neither could transcripts be detected in untreated, SA- or JA-treated Arabidopsis plants nor was the construct N1b[GUS] with the 1135 bp 5?-region able to induce reporter gene expression in transgenic tobacco plants. Expression of N3 occurs constitutively at low levels and independently of NPR1. Treatment with SA or JA does not lead to induction of N3. Likewise, the N3 promoter is not affected by treatment with SA, JA, TMV and phytohormones. Reporter gene expression of the N3 promoter occurs constitutively in transgenic tobacco seedlings. In contrast, N1 and N2 are clearly SA-induced. After SA induction, the expression of N2 is immediate and long-lasting and regulated independently of NPR1. N1 is expressed transiently at a later point in time and is NPR1-dependent. The expression of both, N1 and N2, clearly occurs before PR1 gene expression. The analysis of GUS-reporter gene constructs confirms the early SA-dependent induction of the N1 and N2 promoters before activation of the NtPR1a promoter. Similar to the PR1a promoter, both NIMIN promoters can be induced by thiamine-HCl and show an inhibitory effect of the JA signaling network on the strength of reporter gene expression during simultaneous treatment with SA and JA. At the histological level, the N1 and N2 promoters display SA-dependent activation in leaf and root tissue of young tobacco seedlings. This activity clearly differs from the N3 promoter. The N2 promoter ? just like the AtPR1 and NtPR1a promoters ? contains an as-1-like element with a tandem repeat of TGACG, responsible for the SA sensitivity of the promoter. However the N2 as-1-like element structurally differs from the as-1-like elements in the PR1 promoters. In the N1 promoter a SA-responsive element was located in the region of -436 to -402 with respect to the translation starting point of the N1 gene. However, mutation of a TGATG repeat within this region did not result in a loss of promoter activity. Analysis of chimeric promoter constructs with foreign as-1-like elements showed that the different expression kinetics of PR1 and NIMIN genes are not encoded by the genetic information of the respective as-1-like elements. On the contrary, the as-1-like cis-acting elements affect the promoters? tissue specificity. Due to the integration of the N2-as-1-like element, the NtPR1a promoter, which is solely active in leaf tissue, adopts the typical NIMIN activity in root tissue. The presence of the 35S-as-1 element in the NtPR1a promoter leads to constitutional activation in root tissue. However, the activation in leaf tissue is still SA-dependent. In the yeast one-hybrid system, the interaction of TGA factors with as-1 and the as-1-like elements of the AtPR1, NtPR1a and N2 promoters shows only small differences in binding quality, whereas considerable differences can be detected in quantitative binding strength. Mutation of the as-1-like elements in the NtPR1a and N2 promoters results in the loss of TGA factor binding. The N1 promoter region from -436 to -399 contains a TGA binding site. Mutation of the contained TGATG repeat leads to a total loss of binding of TGA transcription factors. The neighbouring promoter context can exert both positive and negative influence on TGA factor binding. In case of the N1 promoter, the presence of adjacent promoter regions results in increased binding affinity of TGA factors. In contrast, additional NtPR1a promoter context shows a considerable reduction of TGA factor binding to as-1-like elements. Despite independent binding sites, NIMIN proteins and TGA transcription factors compete for binding of AtNPR1 in the yeast three-hybrid system. The presence of N1 and N3 thereby impedes interaction of TGA factors with NPR1, whereas simultaneous binding of N2 and TGA factor is possible without any restrictions. Binding of N1 or N2 simultaneously with N3 at the C- and N-termini of NPR1 also results in reciprocal interference suggesting a spatial folding of NPR1 where the N- and C-termini lie closely together.
Untersuchungen zum asexuellen Gentransfer bei biotrophen Oomyceten anhand der Fallbeispiele Plasmopara halstedii und Peronospora tabacina
(2009) Hammer, Timo; Spring, Otmar
Evidence for gene transfer during the asexual life cycle of certain biotrophic oomycetes was searched for in this study in order to evaluate the potential impact of such parasexual recombination on the variability of these important plant pathogens. Therefore, two case studies with Plasmopara halstedii, the causal agent of sunflower downy mildew, and with the tobacco blue mould pathogen, Peronospora tabacina, were conducted. Although the life cycles of both pathogens lack the possibility of genetic recombination, the organisms differ significantly in their variability. Using molecular methods, several indications for interspecific parasexual recombination between the near relatives Plasmopara halstedii and Plasmopara angustiterminalis were found, giving a possible explanation for the unexpected variability of these pathogens. Asexually formed offspring from dual infection experiments with the two Plasmopara species showed pheno- and genotypic parental traits in new combination and could be cultivated under double selective pressure. The recombinant strain ?R? was studied over 30 generations. Up to the 9th generation and after single sporangium infection, nuclear and mitochondrial traits of both parents were detected in ?R?, indicating a heterokaryon with nuclei and mitochondria of both parental strains. Starting with the 10th generation only fungicide-resistance remained as Pl. halstedii-specific trait, whereas all other detected signals were Pl. angustiterminalis-specific, which indicated true genetic recombination. As one possible mechanism for the genetic exchange it was shown that nuclei can be exchanged between neighboured hyphae via anastomoses and that more than one nucleus can be distributed into one developing sporangium. To prove anastomoses between the different Plasmopara species, specific optical labelling of the participating hyphae is a prerequisit. However, only transient expression of an optical marker gene was achieved, yet. Additional experiments for establishing a stable transformation system were conducted, but were not yet successful in selecting transgenic strains. In contrast to the study on Plasmopara, there was no evidence for recombination in Peronospora tabacina. Neither anastomoses nor heterokaryotic sporangia were found throughout the study. The results are concordant with the findings that tobacco blue mould shows very low variability and that only two phenotypes are known so far from studies in Europe. As the two types of the pathogen, which differ in fungicide sensitivity, did not interact during parallel infection of the same host tissue, they were characterized in detail. Several new pheno- and genotypic differences could be revealed, showing the genetic distance between the two types of the pathogen. A simple PCR detection system to differentiate the two genotypes was created and more than 50 European isolates of tobacco blue mould were monitored.