Unknown,Transcriptomics,Genomics,Proteomics

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Expression profiles of MicroRNA and SiRNA of Arabidopsis thaliana Col-0 and transgenic plants

ABSTRACT: Expression profiles of MicroRNA and SiRNA of Arabidopsis thaliana Col-0 and transgenic plants with constitutive expression of the chimeric receptors NRG1 grown at different temperature To reveal the underlying molecular mechanism of de-cosuppression with memory by high temperature in Arabidopsis, we performed the expression profiles of microRNA and SiRNA in transgenic plants with constitutive expression of the chimeric receptors NRG1 and wide type Col-0 grown at different temperature using the Custom LC Sciences Arabidopsis microRNA and SiRNA array. Keywords: high temperature, de-cosuppression, MicroRNA, SiRNA Total RNA was prepared from wild type and NRG1 plants grown at 22ºC and 30ºC for 4 weeks with TRIzol reagent according to the manufacturer’s protocol (Invitrogen). Small RNAs (<300nt) were isolated using a YM-100 Microcon centrifugal filter (Millipore), and 3’-extended with a poly(A) tail for later fluorescent dye staining. Two different tags were used for the two RNA samples labeled with Cy3 or Cy5 fluorescent dyes in dual-sample experiments. Also dye-swap experiments were carried out to control for labeling bias. Hybridization was performed overnight on a µParafloTM microfluidic chip. Each miRNA and siRNA had 6 and 3 repeat prints, respectively. Six replicate arrays were performed with six independent pairs of RNA samples. After hybridization, array images were obtained using an Axon GenePix 4000B laser scanner and digitized using the Array-Pro image software (Media Cybernetics). The data were corrected by subtracting the background and normalized using the LOWESS (locally weighted regression) method. All data calculations were performed by LC Science (Houston, TX).

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: Li Qun

PROVIDER: E-GEOD-11535 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Background: Xylella fastidiosa, a Gram-negative fastidious bacterium, grows exclusively in the xylem of several plants, causing diseases such as citrus variegated chlorosis. As the xylem sap contains low concentrations of amino acids and other compounds, X. fastidiosa needs to cope with nitrogen limitation in its natural habitat. Results: In this work, we performed a whole-genome microarray analysis of the X. fastidiosa nitrogen starvation response. A time-course experiment (2, 8 and 12 hours) revealed many differentially expressed genes under nitrogen starvation, such as genes related to transport, nitrogen assimilation, amino acid biosynthesis, transcriptional regulation, and many genes encoding hypothetical proteins. In addition, a decrease in the expression levels of many genes involved in carbon metabolism and energy generation pathways was also observed. Comparison of gene expression profiles between the wild type strain and the rpoN null mutant allowed the identification of genes induced by nitrogen starvation in a ?54-dependent manner. A more complete picture of the ?54 regulon was achieved by combining the transcriptome data with an in silico search for potential ?54-dependent promoters, using a position weight matrix approach. One of these ?54-predicted binding sites, located upstream of the glnA gene (encoding a glutamine synthetase), was validated by primer extension assays, confirming that this gene has a ?54-dependent promoter and contains a predicted NtrC binding site. Conclusions: Together, these results show that nitrogen starvation causes intense changes in the X. fastidiosa transcriptome and some of these differentially expressed genes belong to the ?54 regulon. For time-course studies, cells cultivated at late-exponential phase in PWG medium were used to inoculate a culture in 100 ml XDM2 medium to an optical density at 600 nm (OD600 nm) of 0.1. Cells were grown during 12 days in the XDM2 medium (mid-log phase) and harvested by centrifugation. Then, the culture was divided into two 6 portions and cells were washed with XDM2 medium (zero time) or XDM2 medium lacking all nitrogen sources (XDM0), respectively. The cultivation was continued for 2h, 8h and 12h in XDM0 to establish nitrogen starvation conditions. For each time point, cells in a 25-ml culture were collected by centrifugation and rapidly frozen in dry ice, until RNA isolation. Three RNA samples isolated from independently grown cultures of the cells at each starvation period (2h, 8h and 12h) were examined, and each preparation was subjected to microarray analysis. As the genes were spotted at least in duplicate, we obtained six replicates for each gene from three independent data sets per gene per starvation period. Comparison of gene expression profiles between the wild type strain and the rpoN null mutant allowed the identification of genes induced by nitrogen starvation in a ?54-dependent manner. To determine the effect of rpoN inactivation on gene expression after nitrogen starvation, the transcriptomes of the wild type and the rpoN strains were compared using DNA microarrays, with both strains grown on XDM2 medium and submitted to nitrogen starvation (XDM0) during 2 hours. Three RNA samples isolated from independently grown cultures of the cells were examined. Due to the platform design, each microarray slide was divided into "LEFT" and "RIGHT", allowing the probing of two technical replicates per slide.

Project description:Xylella fastidiosa is the etiologic agent of a wide range of plant diseases including citrus variegated chlorosis (CVC), a major threat to the Brazilian citrus industry. Genome sequences of several strains of this phytopathogen are accessible, enabling large-scale functional studies. Transcript levels in different iron availabilities were assessed with DNA microarrays representing 2608 (91.6%) coding sequences (CDS) of X. fastidiosa CVC strain 9a5c. When treated with the iron chelator 2,2-dipyridyl, 193 CDS were considered as up-regulated and 216 as down-regulated. In the presence of 100uM of ferric pyrophosphate, 218 and 256 CDS were considered as up- and down-regulated, respectively. Differential expression for a subset of 44 CDS was further evaluated by reverse transcription - quantitative PCR that showed a Pearson correlation of 0.77 with array results. The CDS differentially expressed upon the iron concentration shift participate in diverse cellular functions. Many CDS involved with regulatory functions, pathogenicity and cell structure, were modulated in both conditions tested suggesting that major changes in cell architecture and metabolism occur when X. fastidiosa cells are exposed to extreme variations in iron concentration. Interestingly, the modulated CDS include those related to colicin V-like bacteriocin synthesis and secretion and to pili/fimbriae functions. We also investigated the contribution of the ferric uptake regulator Fur to the iron regulon of X. fastidiosa. The promoter regions of strain 9a5c genome were screened for putative Fur boxes and candidates were analyzed by electrophoretic mobility shift assays. Taken together, our data support the hypothesis that Fur is not solely responsible for the modulation of the iron regulon of X. fastidiosa and present novel evidence for iron regulation of pathogenicity determinants. Keywords: stress response; response to iron-depleted condition Direct comparison between low iron content (200uM 2,2-dipyridyl) and control condition. Hybridizations are dye-swaped. There are 2 biological replicates (independent harvest) and 2 technical replicates of each array (L - left and R - right).

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Expression profiles of MicroRNA and SiRNA of Arabidopsis thaliana Col-0 and transgenic plants

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