Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of Arabidopsis Agrobacterium tumefaciens induced tumour and non-tumour tissue

ABSTRACT: Agrobacterium tumefaciens, a bacterial species found in temperate soils world wide, is the causative agent of crown gall disease on many plants. A. tumefaciens-induced tumours are feared in orchards and vineyards because of their pathological interference with nutrient and water supply which results in crop decline. Small wounds at the crown of the plant, usually induced by wind-bending, are potential entry sites for the bacterium. The tumorous growth is initiated by the integration and expression of the T-DNA of the bacterial Ti plasmid within the plant nuclear DNA. The T-DNA encodes enzymes catalysing the synthesis of increased concentrations of auxin and cytokinin, and of opines which stimulate cell division and enlargement. The fast growing tumours have been shown to be a strong nutrient sink on their host plants. As a matter of fact, sugar and K+ content were found to be up to 10- and 5-fold, respectively, higher in this tissue and transpiration was about 15 times increased compared to normal tissue. Whereas the morphological structure as well as some physiological and biochemical parameters of the tumour have been analysed in detail, little is known about the underlying gene expression pattern. Proliferation and growth of the tumour induced by Agrobacterium tumefaciens is obviously due to the extraordinary high concentration of phytohormons, minerals and metabolites. Their influence on regulation of gene transcription will provide information on the mechanisms underlying fast tumour growth. In a project funded by the DFG we recently started to investigate the role of solute transporter for tumour development on the model plant Arabidopsis thaliana. By comparing the expression pattern of RNA preparations from Arabidopsis tumour and non-tumour tissue, we will be able to identify genes which facilitate crown gall development. For the expression analysis with an Affymetrix full genome chip we will induce tumours at the base of an injured Arabidopsis inflorescence stalk (var. Wassilewskija, WS-2). RNA will be extraxted from tumour and injured non-tumor inflorescence stalk tissue using the RNeasy Plant Mini Kit (Qiagen), followed by a DNase treatment to eliminate DNA contamination.

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: unknown unknown

PROVIDER: E-NASC-21 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Agrobacterium tumefaciens, a bacterial species found in temperate soils world wide, is the causative agent of crown gall disease on many plants. A. tumefaciens-induced tumours are feared in orchards and vineyards because of their pathological interference with nutrient and water supply which results in crop decline. Small wounds at the crown of the plant, usually induced by wind-bending, are potential entry sites for the bacterium. The tumorous growth is initiated by the integration and expression of the T-DNA of the bacterial Ti plasmid within the plant nuclear DNA. The T-DNA encodes enzymes catalysing the synthesis of increased concentrations of auxin and cytokinin, and of opines which stimulate cell division and enlargement. The fast growing tumours have been shown to be a strong nutrient sink on their host plants. As a matter of fact, sugar and K+ content were found to be up to 10- and 5-fold, respectively, higher in this tissue and transpiration was about 15 times increased compared to normal tissue. Whereas the morphological structure as well as some physiological and biochemical parameters of the tumour have been analysed in detail, little is known about the underlying gene expression pattern. Proliferation and growth of the tumour induced by Agrobacterium tumefaciens is obviously due to the extraordinary high concentration of phytohormons, minerals and metabolites. Their influence on regulation of gene transcription will provide information on the mechanisms underlying fast tumour growth. In a project funded by the DFG we recently started to investigate the role of solute transporter for tumour development on the model plant Arabidopsis thaliana. By comparing the expression pattern of RNA preparations from Arabidopsis tumour and non-tumour tissue, we will be able to identify genes which facilitate crown gall development. For the expression analysis with an Affymetrix full genome chip we will induce tumours at the base of an injured Arabidopsis inflorescence stalk (var. Wassilewskija, WS-2). RNA will be extraxted from tumour and injured non-tumor inflorescence stalk tissue using the RNeasy Plant Mini Kit (Qiagen), followed by a DNase treatment to eliminate DNA contamination. Experimenter name = Rosalia Deeken; Experimenter phone = +49 931 8886121; Experimenter fax = +49 931 8886158; Experimenter institute = Julius-von-Sachs-Institute of Biosciences Molecular Plant Physiology and Electrophysiology; Experimenter address = Julius-von-Sachs-Platz 2; Experimenter address = Wuerzburg; Experimenter zip/postal_code = D-97082; Experimenter country = Germany Experiment Overall Design: 4 samples were used in this experiment

Project description:Agrobacterium tumefaciens, a bacterial species found in temperate soils world wide, is the causative agent of crown gall disease on many plants. A. tumefaciens-induced tumours are feared in orchards and vineyards because of their pathological interference with nutrient and water supply which results in crop decline. Small wounds at the crown of the plant, usually induced by wind-bending, are potential entry sites for the bacterium. The tumorous growth is initiated by the integration and expression of the T-DNA of the bacterial Ti plasmid within the plant nuclear DNA. The T-DNA encodes enzymes catalysing the synthesis of increased concentrations of auxin and cytokinin, and of opines which stimulate cell division and enlargement. The fast growing tumours have been shown to be a strong nutrient sink on their host plants. As a matter of fact, sugar and K+ content were found to be up to 10- and 5-fold, respectively, higher in this tissue and transpiration was about 15 times increased compared to normal tissue. Whereas the morphological structure as well as some physiological and biochemical parameters of the tumour have been analysed in detail, little is known about the underlying gene expression pattern. Proliferation and growth of the tumour induced by Agrobacterium tumefaciens is obviously due to the extraordinary high concentration of phytohormons, minerals and metabolites. Their influence on regulation of gene transcription will provide information on the mechanisms underlying fast tumour growth. In a project funded by the DFG we recently started to investigate the role of solute transporter for tumour development on the model plant Arabidopsis thaliana. By comparing the expression pattern of RNA preparations from Arabidopsis tumour and non-tumour tissue, we will be able to identify genes which facilitate crown gall development. For the expression analysis with an Affymetrix full genome chip we will induce tumours at the base of an injured Arabidopsis inflorescence stalk (var. Wassilewskija, WS-2). RNA will be extraxted from tumour and injured non-tumor inflorescence stalk tissue using the RNeasy Plant Mini Kit (Qiagen), followed by a DNase treatment to eliminate DNA contamination. This Series record represents a partial dataset. The complete dataset was submitted under GEO accession number GSE13927. Experimenter name = Rosalia Deeken Experimenter phone = +49 931 8886121 Experimenter fax = +49 931 8886158 Experimenter institute = Julius-von-Sachs-Institute of Biosciences Molecular Plant Physiology and Electrophysiology Experimenter address = Julius-von-Sachs-Platz 2 Experimenter address = Wuerzburg Experimenter zip/postal_code = D-97082 Experimenter country = Germany Keywords: pathogenicity_design

Project description:Purpose: There is a quest for novel non-invasive diagnostic markers for the detection of breast cancer. The goal of this study is to identify circulating microRNA signatures using a cohort of Asian Chinese breast cancer patients, and to compare microRNA profiles between tumour and serum samples. Experimental design: MicroRNAs from paired breast cancer tumours, normal tissue and serum samples derived from 32 patients were comprehensively profiled using microarrays (1300 microRNAs against tumour and normal tissues) or LNA RT-PCR panels (742 microRNAs against serum samples). Serum samples from healthy individuals (n=22) were also employed as normal controls. Significant serum microRNAs, identified by logistic regression, were validated in an independent set of serum samples from patients (n=82) and healthy controls (n=53). Results: The 20 most significant microRNAs differentially expressed in breast cancer tumours included miR-21, miR-10b, and miR-145, previously shown to be dysregulated in breast cancer. Interestingly, 16 of the 20 most significant microRNAs differentially expressed in serum samples were novel. MiR-1, miR-92a, miR-133a and miR-133b were identified as the most important diagnostic markers, and were successfully validated; receiver operating characteristic curves derived from combinations of these microRNAs exhibited areas under the curves of 0.944-0.946. Only seven microRNAs were overexpressed in both tumours and serum, suggesting that microRNAs may be released into the serum selectively. Conclusion: The clinical employment of microRNA signatures as a non-invasive diagnostic strategy is promising, but should be further validated for different subtypes of breast cancers. "_A" and "_B" are two tissue sections of the same sample; "_1" and "_2" represents 2 runs of the same sample; na = not available All tissue samples were histologically confirmed by a pathologist using hematoxylin and eosin staining of cryosectioned specimens. One tumour sample was rejected due to failure to detect any tumour cells. Except for two samples (with 30% and 40% tumour cells), all tumour tissues employed had a minimum of 60% tumour cells, as estimated microscopically. Overall, the breast cancer tumour samples had an average of 71% tumour cells. The criteria for adjacent normal tissue were absence of tumour cells and presence of epithelial cells. Hence, after histological confirmation, 31 breast cancer tumours and 23 matched normal tissues were employed for microRNA extraction and profiling using microarray.

Project description:Background Heterodera schachtiia is an economically important plant parasitic nematode that forms a syncytium from a cell superficial to the formed vascular bundle by progressive recruitment of other cells into the structure. The pattern of plant gene expression changes dramatically inside the syncytium. The pathogen probably plays a major role in defining the plant response by choice of initial plant cell during precise behaviour in planta and/or by the secretions it releases. The modified plant cells enable a high feeding rate by the female nematode so enhancing its rate of development and subsequent daily egg production. Arabidopsis is widely used as a model plant to characterise molecular responses to nematodes (e.g. Sijmons et al., 1991 Plant J. 1:245-254.). A complete overview of the changes in plant gene expression when sedentary nematodes establish has not yet been gained using Arabidopsis or any other host plant. Experimental Approaches Our initial studies will focus on the H. schachtii/Arabidopsis interaction. To assure reliable microarray screening care has been taken to minimise extraneous differences between samples (see "Growth conditions" section). At 21 days (Growth stage 3.2-3.5 Boyes et al., 2001 Plant Cell 13:1499-1510) Arabidopsis plants were challenged with rigorously sterilised, infective nematodes of H. schachtii as before (Urwin et al., (1997) Plant Journal 12: 455-461.). 35 sterile J2s were pipetted onto small ~0.5mm2 squares of sterile GF/A filter paper. The GF/A paper was left in direct contact with the zone of elongation on 3 lateral roots per plant for 48 hours. Control plants were mock inoculated with sterile water. Sections of root containing syncytia have been excised from the thin and transparent roots of Arabidopsis and collected into RNAlater solution (Ambion) at 21 days post infection (Growth Stage 6.1 Boyes et al. 2001). The female nematode has been removed with watch-maker's forceps. Equivalent sections of root have been harvested from non-infected plants. Material has been collected from c. 1000 plants for each of the two samples and the uninfected material serves as an internal control. Total RNA has been prepared from the reference and test root material using an RNeasy plant RNA preparation kit (Qiagen) according to methods required by GARNET.

Project description:Aim: To determine the effect of an AtrbohC mutation on the gene expression pattern in primary root tissue, to identify candidate genes acting downstream of AtrbohC, particularly any encoding antioxidant-related proteins, signal transduction components or proteins known to be required for normal root-hair development. Background: Root-hairs are a model system for investigating plant cell polarity. The root-hair mutant rhd2 (Schiefelbein and Somerville, 1990. Plant Cell, 2:235) has short hairs that burst at their tips, (Jones and Smirnoff, unpublished). RHD2 has been cloned and is identical to AtrbohC (L. Dolan, pers. comm.), which encodes a homologue of the superoxide-generating neutrophil respiratory burst oxidase catalytic subunit gp91phox (Torres et al., 1998. Plant J., 14:365). Superoxide rapidly dismutates to hydrogen peroxide (H2O2), suggesting that the rhd2 phenotype may result from reduced H2O2 levels in root-hair cells. Low doses of exogenous antioxidants phenocopy the rhd2 root-hair phenotype in wild-type plants (Jones and Smirnoff, unpublished) further supporting a role for H2O2 in root-hair growth. Fluorescent dyes that detect H2O2 show distinct localisation patterns in growing root-hair cells, (Jones and Smirnoff, unpublished). H2O2 may be an important second messenger in plant cell signalling with proposed roles in the development of cotton fibres (Potikha et al., 1999. Plant Physiol., 119: 849) and in ABA-induced stomatal closure (Zhang et al., 2001. Plant Physiol., 126: 1438). In cultured Arabidopsis cells H2O2 induces gene expression, including that of a gp91phox homologue, (Desikan et al., 1998. J. Exp. Bot., 49: 1767; Desikan, et al., 2000. Free Rad. Biol. Med., 28: 773; Baxter-Burrell et al., 2002. Science, 296: 2026) and activates a MAP kinase cascade (Desikan et al., 1999. J. Exp Bot., 50: 1863). cDNA microarray technology has been used previously to examine the effects of H2O2 on gene expression during oxidative stress (Desikan et al., 2001. Plant Physiol., 127: 159). We wish to investigate the effects of H2O2 on gene expression during root development using the rhd2 mutant. We are currently determining the expression pattern of RHD2. By extracting RNA from the small region of the primary root (for wild-type and rhd2 plants grown in sterile conditions) where root hairs are growing we hope to enrich for root-hair RNAs. This may reveal candidate genes that could be examined more closely at the single-cell level. This approach will provide new insights into the role of H2O2 in root-hair development.

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Transcription profiling of Arabidopsis Agrobacterium tumefaciens induced tumour and non-tumour tissue

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