Browsing by Subject "Tomate"

Now showing 1 - 4 of 4

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.
Analysis of the structure of tomato mosaic virus movement protein based on virus host interactions
(2011) Tanwir, Fariha; Pfitzner, Artur J. P.
Viruses are obligatory plant pathogens causing sever diseases, and ultimately great losses in crop yield. Plant viruses, once entered in the cell, make use of host machinery for its own replication and moves from one cell to the other. Natural resistance against virus attack is achieved by the presence of resistance genes (R genes). R genes recognize viral avirulence (Avr) factors in elicitor-receptor manner to initiate resistance cascade. In tomato, the resistance genes Tm-I, Tm-2 and Tm-22 are used to protect the plants against infection by tomato mosaic virus.Tm-2 and Tm-22 require recognition of the viral 30kDa movement protein (MP) for triggering resistance response. Sequence analysis of Tm-2 and Tm-22 resistance breaking viruses have shown an amino-acid exchange at position 133 (E>K) is found in all Tm-2 resistance breaking virus strains, whereas, amino-acid exchange at position 130 (K>E) is associated with Tm-22 resistance breaking phenotype (Calder and Palukaitis, 1992). This suggests a physical interaction between resistance genes and 30kDa MP. In the present study, a unique Split GFP approach is used to analyse the structure and localization of different domains of 30kDa MP in S. cerviceae and N. benthamiana. Different deletion mutants were fused between two non-overlapping halves of GFP and expressed. Results showed that both N and C terminus as well as the middle part of 30kDa MP (aa 80-150) is present in the cytoplasm with two integral membrane loops. These findings are in contrast with previous in-vitro results, which suggest that middle part of 30kDa MP is present in ER lumen, whereas N and C terminus in cytoplasm (Brill et al., 2000). Fluorescence microscopy revealed that GFP fused 30kDa MP deletion mutants were localized on the cytoplasmic side of plasmamembrane and near plasmodesmata. Membrane association of fusion protein confirmed the proper folding and functionality of deletion mutants. Therefore, the structural model of ToMV 30kDa MP has to be revised. Secondly, to identify the host factors involved in resistance mechanism, initiated by Tm-2 and Tm-22 resistance genes, tomato mosaic virus based vectors were constructed. Two different types of in-vivo transcription vectors were constructed, one containing both right and left border of the T-DNA (pBinSLN) and one without the right border (pBinSLN-RB). Self replication of these vectors were analysed in N. benthamiana, N. tabacum and S. lycopersicum. It was found that the deletion of RB does not affect virus replication, when agro-infiltrated in N. benthamiana. pBinSLN-RB was used further for the isolation of a stable and vigorous Tm-2 and Tm-22 resistance breaking ToMV strain through a novel selection scheme. ToMV2-22 contains two amino-acid exchanges at position 54(N>D) and 133(E>K). ToMV2-22 is the first mutant strain of ToMV, which can escape both Tm-2 and Tm-22 resistance simultaneously.
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.
The functional role of phosphorus-mobilizing bacteria in the rhizosphere of tomato and maize
(2017) Nassal, Dinah; Kandeler, Ellen
Phosphorus (P) is an essential plant nutrient. However, global P reserves are being increasingly exploited and surplus P applied by P fertilization is steadily accumulating in the form of plant-unavailable P compounds in arable soils. Future plant production will therefore require a more effective and sustainable P fertilization regime. One promising approach is the use of phosphorus-mobilizing bacteria (PMB), which are able to mobilize P in soil through mineralization or solubilization so effectively that plant P supply is improved. Increases in plant growth and P uptake by the addition of PMB have been reported several times, but PMB’s functional mechanisms in soils and plants are still poorly understood. However, an understanding of PMB’s functional mechanisms is necessary to evaluate both the potential and limitations of their use as well as to develop practical application recommendations. This thesis aimed to provide a better understanding of PMB’s functional mechanisms in soil; the foci here were mechanisms and interactions of P mineralization with indigenous soil microorganisms. We aimed to identify P mineralization-dependent and -independent as well as direct and indirect mechanisms of PMB on soil and plants. To this end, three rhizobox experiments were performed in the greenhouse using tomato and maize as the test plants and Pseudomonas sp. RU47 (RU47) as the PMB. To identify effective P mineralization beyond the level of endogenous microbial activity, a treatment using unselectively cultivated soil bacteria for inoculation was included. Furthermore, the addition of devitalized RU47 cells provided the opportunity to identify indirect mechanisms. In all three rhizobox experiments the activities of acid and alkaline phosphomonoesterases in rhizosphere and bulk soil were determined, as the latter could be clearly identified as being of microbial origin. Effects on microbial community structure in soil were estimated by denaturing gradient gel electrophoresis (DGGE) and/or phospholipid fatty acid analysis. For deeper investigations of potential effects on microbial population composition and possible dependencies on soil conditions, a fourth experiment was performed using maize, three different Pseudomonas strains possessing PMB abilities, and three different soils varying in parameters which included organic C, pH, and P content. Microbiome shifts in soil were quantitatively determined via quantitative PCR using domain- (bacteria, archaea, fungi) and six bacterial phylum-specific primers. Our experiments showed that tomato plants grown under low P availability soil conditions improved in both growth and P uptake when viable RU47 cells were added. This effect was accompanied by increased alkaline phosphatase activity (PA) in the rhizosphere. We also observed plant growth-promotion effects and a trend of increased PA by the addition of dead RU47 cells. Based on DGGE results, which indicated the promotion of indigenous rhizobacteria, we assume a priming effect induced by the addition of C sources in the form of bacterial residues (dead RU47), which resulted in increased indigenous microbial activity in the rhizosphere. In each rhizobox experiment viable RU47 cells were able to colonize the rhizosphere at high abundances, persisting up to 50 days after sowing. We found indications of phytohormonal influences with the addition of both viable and dead RU47 cells, but this was more pronounced in dead than in viable RU47 treatments. Increasing P availability in soil by mineral P fertilization seemed to improve RU47’s ability to colonize and persist, which was shown by an increased RU47 abundance in both rhizosphere and bulk soils. However, despite an observable slight tendency, strengthened plant growth-promotion that positively correlated with improved RU47 abundance in the rhizosphere could not be detected. In general, colonization by viable RU47 cells did not significantly affect microbial community structure, either in the rhizosphere or in bulk soil. Using three different PMB strains, including RU47, in three contrasting soils, inoculation effects on the microbial community occurred heterogeneously, differing between the strains, soils, and time. Changes at the domain level were due primarily to nutrient availability in the soil, which differed between the soils and over time. Individual shifts in microbial community structure occurred more frequently in the rhizosphere than in bulk soil, but colonizing PMB neither increased bacterial abundance in rhizosphere bacteria, nor displaced copiotrophic rhizobacteria (indicative of C competition).