Project description:Coilin is a marker protein for subnuclear organelles known as Cajal bodies, which are sites of various RNA metabolic processes including the biogenesis of spliceosomal small nuclear ribonucleoprotein particles. Through self-associations and interactions with other proteins and RNA, coilin provides a structural scaffold for Cajal body formation. However, despite a conspicuous presence in Cajal bodies, most coilin is dispersed in the nucleoplasm and expressed in cell types that lack these organelles. The molecular function of coilin, particularly of the substantial nucleoplasmic fraction, remains uncertain. We identified coilin loss-of-function mutations in a genetic screen for mutants showing either reduced or enhanced expression of an alternatively spliced GFP reporter gene in Arabidopsis thaliana The coilin mutants feature enhanced GFP fluorescence and diminished Cajal bodies compared with wild-type plants. The amount of GFP protein is several-fold higher in the coilin mutants owing to elevated GFP transcript levels and more efficient splicing to produce a translatable GFP mRNA. Genome-wide RNA-sequencing data from two distinct coilin mutants revealed a small, shared subset of differentially expressed genes, many encoding stress-related proteins, and, unexpectedly, a trend toward increased splicing efficiency. These results suggest that coilin attenuates splicing and modulates transcription of a select group of genes. The transcriptional and splicing changes observed in coilin mutants are not accompanied by gross phenotypic abnormalities or dramatically altered stress responses, supporting a role for coilin in fine tuning gene expression. Our GFP reporter gene provides a sensitive monitor of coilin activity that will facilitate further investigations into the functions of this enigmatic protein.
Project description:DNA methylation directed by 24-nucleotide small RNAs involves the small RNA-binding protein ARGONAUTE4 (AGO4), and it was previously shown that AGO4 localizes to nucleolus-adjacent Cajal bodies, sites of snRNP complex maturation. Here we demonstrate that AGO4 also localizes to a second class of nuclear bodies, called AB-bodies, which are found immediately adjacent to condensed 45S ribosomal DNA (rDNA) sequences. AB-bodies also contain other proteins involved in RNA-directed DNA methylation including NRPD1b (a subunit of the RNA Polymerase IV complex, RNA PolIV), NRPD2 (a second subunit of this complex), and the DNA methyltransferase DRM2. These two classes of AGO4 bodies are structurally independent--disruption of one class does not affect the other--suggesting a dynamic regulation of AGO4 within two distinct nuclear compartments in Arabidopsis. Abolishing Cajal body formation in a coilin mutant reduced overall AGO4 protein levels, and coilin dicer-like3 double mutants showed a small decrease in DNA methylation beyond that seen in dicer-like3 single mutants, suggesting that Cajal bodies are required for a fully functioning DNA methylation system in Arabidopsis.
Project description:Arabidopsis thaliana and Arabidopsis lyrata are two closely related Brassicaceae species, which are used as models for plant comparative biology. They differ by lifestyle, predominant mating strategy, ecological niches and genome organization. In order to explore molecular basis of specific traits, we performed RNA-sequencing of vegetative rosettes from both species. Additionally, we sequenced apical meristems and inflorescences of A. lyrata that allow for intra-specific transcriptome comparison in several major developmental stages. Arabidopsis lyrata and Arabidopsis thaliana aerial tissues were collected from mock treated plants, total RNA isolated and poly-A RNA populations sequenced
Project description:We produced RNA-seq reads from messenger RNA isolated from aerial seedling tissue for Arabidopsis thaliana mutants in the HULK gene family. The read data were generated with biological replication (two replicates). The resulting RNA-seq data provide a resource to assess the function of HULK genes in the control of downstream gene expression in A. thaliana. Examination of RNA transcript levels in HULK T-DNA mutants (and mutant combinations therein) in Arabidopsis thaliana.
Project description:Small RNAs (sRNAs) play important roles in plant development and host-pathogen interactions. Several studies have highlighted the relationship between viral infections, endogenous sRNA accumulation and transcriptional changes associated with symptoms. However, few studies have described a global analysis of endogenous sRNAs by comparing related viruses at early stages of infection, especially before viral accumulation reaches systemic tissues. An sRNA high-throughput sequencing of Arabidopsis thaliana leaf samples infected either with Oilseed rape mosaic virus (ORMV) or crucifer-infecting Tobacco mosaic virus (TMV-Cg) with slightly different symptomatology at two early stages of infection (2 and 4 dpi) was performed. At early stages, both viral infections strongly alter the patterns of several types of endogenous sRNA species in distal tissues with no virus accumulation suggesting a systemic signaling process foregoing to virus spread. A correlation between sRNAs derived from protein coding genes and the associated mRNA transcripts was also detected, indicating that an unknown recursive mechanism is involved in a regulatory circuit encompassing this sRNA/mRNA equilibrium. This work represents the initial step in uncovering how differential accumulation of endogenous sRNAs contributes to explain the massive alteration of the transcriptome associated with plant-virus interactions.
Project description:We performed transcriptome and ribosome immunoprecipitation studies of transgenic Arabidopsis (Arabidopsis thaliana) Columbia seeds expressing a His6FLAG-tagged version of the ribosomal large subunit protein L18B (35S:HF-RPL18B)
Project description:microRNAs (miRNAs) are small, endogenous RNAs of 20 approximately 25 nucleotides, processed from stem-loop regions of longer RNA precursors. Plant miRNAs act as negative regulators of target mRNAs predominately by slicing target transcripts, and a number of miRNAs play important roles in development. We analyzed a number of published datasets from Arabidopsis thaliana to characterize novel miRNAs, novel miRNA targets, and miRNA-regulated developmental changes in gene expression. These data include microarray profiling data and small RNA (sRNA) deep sequencing data derived from miRNA biogenesis/transport mutants, microarray profiling data of mRNAs in a developmental series, and computational predictions of conserved genomic stem-loop structures. Our conservative analyses identified five novel mature miRNAs and seven miRNA targets, including one novel target gene. Two complementary miRNAs that target distinct mRNAs were encoded by one gene. We found that genes targeted by known miRNAs, and genes up-regulated or down-regulated in miRNA mutant inflorescences, are highly expressed in the wild type inflorescence. In addition, transcripts upregulated within the mutant inflorescences were abundant in wild type leaves and shoot meristems and low in pollen and seed. Downregulated transcripts were abundant in wild type pollen and seed and low in shoot meristems, roots and leaves. Thus, disrupting miRNA function causes the inflorescence transcriptome to resemble the leaf and meristem and to differ from pollen and seed. Applications of our computational approach to other species and the use of more liberal criteria than reported here will further expand the number of identified miRNAs and miRNA targets. Our findings suggest that miRNAs have a global role in promoting vegetative to reproductive transitions in A. thaliana.
Project description:Plants can re-programme their transcriptome, proteome and metabolome to deal with environmental and biotic stress. It has been shown that the rhizosphere microbiome has influence on the plant metabolome and on herbivore behaviour. In the present study, Trichoderma gamsii was isolated from Arabidopsis thaliana rhizosphere soil. The inoculation of roots of Arabidopsis thaliana with T. gamsii significantly inhibited the feeding behaviour of Trichoplusia ni and affected the metabolome as well as the content of phytohormones in Arabidopsis leaves. T. gamsii-treated plant leaves had higher levels of amino acids and lower concentrations of sugars. In addition, T. gamsii-treated plant leaves had more abscisic acid (ABA) and lower levels of salicylic acid (SA) and indole-3-acetic acid (IAA) in comparison with the untreated plants. Furthermore, the inoculation with T. gamsii on different signalling mutants showed that the induction of defences were SA-dependent. These findings indicate that T. gamsii has potential as a new type of biocontrol agent to promote plant repellence to insect attacks.