A combination of bioinformatic tools, high-throughput gene expression profiles, and the use of synthetic promoters is a powerful approach to discover and evaluate novel cis-sequences in response to specific stimuli. With Arabidopsis (Arabidopsis thaliana) microarray data annotated to the PathoPlant database, 732 different queries with a focus on fungal and oomycete pathogens were performed, leading to 510 up-regulated gene groups. Using the binding site estimation suite of tools, BEST, 407 conserved sequence motifs were identified in promoter regions of these coregulated gene sets. Motif similarities were determined with STAMP, classifying the 407 sequence motifs into 37 families. A comparative analysis of these 37 families with the AthaMap, PLACE, and AGRIS databases revealed similarities to known cis-elements but also led to the discovery of cis-sequences not yet implicated in pathogen response. Using a parsley (Petroselinum crispum) protoplast system and a modified reporter gene vector with an internal transformation control, 25 elicitor-responsive cis-sequences from 10 different motif families were identified. Many of the elicitor-responsive cis-sequences also drive reporter gene expression in an Agrobacterium tumefaciens infection assay in Nicotiana benthamiana. This work significantly increases the number of known elicitor-responsive cis-sequences and demonstrates the successful integration of a diverse set of bioinformatic resources combined with synthetic promoter analysis for data mining and functional screening in plant-pathogen interaction.
The dominant tobacco mosaic virus (TMV) resistance gene N induces a hypersensitive response upon TMV infection and protects tobacco against systemic spread of the virus. It has been proposed to change disease resistance specificity by reprogramming the expression of resistance genes or their corresponding avirulence genes. To reprogramme the resistance response of N towards bacterial pathogens, the helicase domain (p50) of the TMV replicase, the avirulence gene of N, was linked to synthetic promoters 4D and 2S2D harbouring elicitor-responsive cis-elements. These promoter::p50 constructs induce local necrotic lesions on NN tobacco plants in an Agrobacterium tumefaciens infiltration assay. A tobacco genotype void of N (nn) was transformed with the promoter::p50 constructs and subsequently crossed to NN plants. Nn F1 offspring selected for the T-DNA develop normally under sterile conditions. After transfer to soil, some of the F1 plants expressing the 2S2D::p50 constructs develop spontaneous necrosis. Transgenic Nn F1 plants with 4D::p50 and 2S2D::p50 expressing constructs upregulate p50 transcription and induce local necrotic lesions in an A. tumefaciens infiltration assay. When leaves and stems of Nn F1 offspring harbouring promoter::p50 constructs are infected with oncogenic A. tumefaciens C58, transgenic lines harbouring the 2S2D::p50 construct induce necrosis and completely lack tumor development. These results demonstrate a successful reprogramming of the viral N gene response against bacterial crown gall disease and highlight the importance of achieving tight regulation of avirulence gene expression and the control of necrosis in the presence of the corresponding resistance gene.
Flooding, a natural cause of anaerobiosis, is often accompanied by high CO2 concentrations in the flood water. Plants need to respond to these environmental conditions. Strong anaerobic reporter gene activity in tobacco (Nicotiana tabacum) controlled by the glycolytic glyceraldehyde-3-phosphate dehydrogenase (GapC4) promoter from maize (Zea mays) depends on the presence of CO2 and light. To identify factors required for CO2 regulated gene expression, promoter deletions fused to the bglucuronidase reporter gene were studied in transgenic tobacco. Deletion of a 40 bp fragment directly upstream of the TATA box leads to increased anaerobic reporter gene activity both, in the presence and absence of CO2. This deletion does not affect light specific anaerobic expression. A positive correlation between increasing CO2 concentrations and gene activity is observed. Electrophoretic mobility shift experiments indicate that tobacco nuclear extracts harbour proteins that bind to part of the 40 bp fragment. Database assisted as well as experimental analysis reveal a role for AP2/EREBP transcription factors for conferring the high CO2 specificity to the GapC4 promoter in tobacco leaves. This work highlights the importance for plants to respond to high environmental CO2 concentrations under anaerobic conditions.
The dominant tobacco mosaic virus (TMV) resistance gene N confers a hypersensitive response (HR) at the site of TMV infection and protects tobacco against systemic spread of the virus. To study N gene activity in seeds and early seedling development, the avirulence gene of N, the helicase domain (p50) of the TMV replicase, was constitutively expressed in a tobacco genotype without N (nn). Transgenic F1 expressing N and p50 were generated by crossing with an NN genotype. Surprisingly, Nn F1 seeds expressing p50 are viable and germinate. Only about 5 days after sowing, seedlings started to show an HR. This paralleled the upregulation of several pathogenesis-related and HR genes. The timing of the HR is consistent with the upregulation of N gene transcript 4-6 days after sowing. The expression of p50 has a stimulating effect on the N gene transcript level during germination. These results show that tobacco seeds and very young seedlings do not express a functional N gene product.
The main and closure domain structures of cylindrical and prismatic 2.5% silicon-iron single crystals with (110) axes are investigated using powder pattern technique and Kerr magnetooptic effect. The main domains are leaf-shaped only in small effective fields. AB closure domains we find on (110) faces the chess-board structure at zero effective field and &-domains combined with the complicated zigzag pattern in the field range of magnetization rotation. The zigzag pattern ia found to be independent of side face orientation in a wide range of orientation. Models of these closure domain structures are presented. Aspecta of the calculation of the domain period and its field dependence are discussed.Die Grund-und AbschluDstrukturen zylindrischer und prismatischer 2,5%-Silizium-Eisen-Einkristalle mit (110) als Stabachee werden mit Hilfe der Bitter-Technik und des magnetooptischen Kerr-Effekta untersucht. Die Grundbereiche sind nur in kleinen inneren Feldern plattenformig. Als Abschlu6struktur finden wir auf (1 IO)-Flachen das Schachbrettmuster im Bereich He = 0 und Q-Bereiche zusammen mit dem komplizierten Zickzackmueter im Bereich der Drehprozesse. Das Zickzackmueter ist in einem weitsn Winkelbereich unabhiingig von der Seitenfliichenorientierung. Es werden Modelle der Abschlullstrukturen angegeben. Gesichtapunkte fiir die Berechnung der Bereichsbreite und ihrer Feldabhiingigkeit werden diskutiert.
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