Parallel analysis of RNA ends enhances global investigation of microRNAs and target RNAs of Brachypodium distachyon
ABSTRACT: The wild grass Brachypodium distachyon has emerged as a model system for temperate grasses and biofuel plants. However, the global analysis of miRNAs, molecules known to be key for eukaryotic gene regulation, has been limited in B. distachyon to studies examining a few samples or that rely on computational predictions. Similarly an in-depth global analysis of miRNA-mediated target cleavage using Parallel Analysis of RNA Ends (PARE) data is lacking in B. distachyon. B. distachyon small RNAs were cloned and deeply sequenced from 17 libraries that represent different tissues and stresses. Using a computational pipeline, we identified 116 miRNAs including not only conserved miRNAs that have not been reported in B. distachyon, but also non-conserved miRNAs that were not found in other plants. To investigate miRNA-mediated cleavage function, four PARE libraries were constructed from key tissues and sequenced to a total depth of approximately 70 million sequences. The roughly 5 million distinct genome-matched sequences that resulted represent an extensive dataset to analyze small RNA-guided cleavage events. Analysis of the PARE and miRNA data provided experimental evidence for miRNA-mediated cleavage of 264 sites in predicted miRNA targets. In addition, PARE analysis revealed that differentially expressed miRNAs in the same family guide specific target RNA cleavage in a correspondingly tissue-preferential manner. B. distachyon miRNAs and target RNAs were experimentally identified and analyzed. Knowledge gained from this study should provide insights into the roles of miRNAs and the regulation of their targets in B. distachyon and related plants. Examination of various tissues and stresses in Brachypodium by high throughput sequencing for small RNA profiling and PARE (Parallel Analysis of RNA Ends)
Project description:MicroRNAs (miRNAs) are a class of small RNAs which typically function by guiding cleavage of target messenger RNAs. They have been shown to play major roles in a variety of plant processes including development, and responses to pathogens and environmental stresses. To identify new miRNAs and regulation in Arabidopsis thaliana, 27 small RNA libraries were constructed and sequenced from various tissues, stresses and small RNA biogenesis mutants, resulting in 95 million genome-matched sequences. The use of rdr2 to enrich the miRNA population greatly enhanced this analysis and led to the discovery of 44 new miRNAs arising from both known and new precursors. Parallel Analysis of RNA Ends (PARE) data provide evidence that the majority guide target cleavage. The inclusion of novel stress/tissue conditions, such as submergence-stressed flowers, enabled identification of new stress regulation of both miRNAs and their targets, all of which were validated in wild type plants. By combining small RNA expression analysis with ARGONAUTE (AGO) immunoprecipitation data and global target cleavage data from PARE, a much more complete picture of Arabidopsis miRNAs was obtained. This combinatorial approach led to the discovery of AGO loading and target cleavage biases, which gave important insights into tissue-specific expression patterns, pathogen responses and the role of sequence variation among closely related miRNA family members. Examination of various tissues, stresses and small RNA biogenesis mutants in Arabidopsis by high-throughput sequencing for small RNA profiling. We have used AGO-IP and PARE data from the published data, which were downloaded from NCBI GEO with the following accession number. AGO-IP from GSM253622, GSM707682, GSM642335, GSM642336, GSM512703, GSM512702, GSM707683, GSM707684, GSM707685, GSM149080, GSM253623, GSM304283, GSM642337, GSM642338, GSM253624, GSM415788, GSM707686, GSM707687, GSM707688, GSM707689, GSM415787, GSM149081, GSM253625, GSM415789, GSM415790, GSM304285, GSM415791, GSM415792 PARE sequencing data from xrn4 flowers were obtained from Gene Expression Omnibus with accession number GSM280227.
Project description:Small RNAs have variety of important roles in plant development, stress responses, and other processes. They exert their influence by guiding mRNA cleavage, translational repression, and chromatin modification. To identify novel and regulated rice miRNAs, 62 small RNA libraries were constructed from rice plants and deeply sequenced with Illumina technology. The libraries represent several tissues from control plants and plants subjected to different environmental stress treatments. More than 94 million genome-matched reads were obtained resulting in more than 16 million distinct small RNA sequences. This allowed an evaluation with current criteria of about 400 annotated miRNAs and the finding that among these, about 150 had siRNA-like characteristics. Seventy nine new miRNAs were identified and miRNAs were distinguished that are regulated in response to water stress, nutrient stress, or temperature stress. Among the new examples of miRNA regulation were members of the same miRNA family that were differentially regulated in different organs and had distinct sequences Some of these distinct family members result in differential target cleavage and provide new insight about how an agriculturally important rice phenotype could be controlled in the panicle. This high resolution analysis of rice miRNAs should be relevant to plant miRNAs in general, particularly in the Poaceae. Examination of different tissue types and tissues treated with various environmental and nutrient stresses in rice by high throughput sequencing for small RNA profiling
Project description:We used Brachypodium distachyon (BD21) as a model grass to gain insight into the affected host molecular pathways upon infection of Panicum Mosaic Virus (PMV) together with its satellite virus, Satellite Panicum Mosaic Virus (SPMV). Brachypodium plants at 2-3 leaf stage were either mock inoculated or inoculated with PMV and PMV+SPMV. Total RNA was isolated from shoot tissues of control and treated plants and was subjected to microarray analysis.
Project description:Purpose: The goal of this study is to compare the transcriptomes expressed during submergence stress of two Brachypodium distachyon ecotypes with contrasting survival under this stress. Bd21 is a submergence sensitive ecotype with EC50 of 2.5 days and Bd2-3 is a tolerant ecotype with EC50 of 4 days. Methods (Stress): Brachypodium Bd21 and Bd2-3 plants (14-day-old, 6 leaves stage) were submerged in a water column of 30 cm inside opaque-wall plastic tanks. Light still reached the plants at 40 uE m-2 s-1. Ecotypes were submerged side-by-side in a randomized manner; only plants submerged in the same tank were compared. Controls were grown in plastic tanks without a water column. Submergence stress started at ZT14 (2h before night, long-day regime 16h light, 8h dark). Above ground tissue was collected after 48 h submergence stress in liquid nitrogen and stored at -80C in an ultra freezer until further processing. Tissue was ground to powder with mortar, pestle and liquid nitrogen avoiding thawing. Control and submerged total RNA was extracted with TRIzol reagent (Invitrogen, 15596018), purified with Direct-zol RNA mini prep columns (Zymo Research, R2050) and digested in-column with DNAse I (ThermoScientific, EN0521). RNA integrity and concentration was verified in denaturing 1.0% agarose gels, Nanodrop 2000 (ThermoScientific) and in a Bioanalyzer 2100 (Agilent) with the integrated software 2100 Expert, samples had a RNA Integrity Number (RIN) between 6.4-7.2 characteristic of aerial plant tissue (Babu and Gassman, 2011). Total RNA extracted from control and submerged tissue from three independent experiments consisting each of four individuals were used to construct cDNA indexed libraries and sequenced in a HiSeq2500 (Illumina) at 1x50 format, making a total of 12 sequenced libraries (tolerant and intolerant ecotype, control and submerged, all experimental triplicates) in a 2-lane format. RNA integrity, library construction and sequencing was performed as a service at the Unidad Universitaria de Secuenciacion Masiva, Instituto de Biotecnologia, Universidad Nacional Autonoma de Mexico (IBT-UNAM). Differential Gene Expression (DGE) analysis was performed with edgeR using a generalized linear model and false discovery rate <0.05 (FDR). To group differentially expressed transcripts a logFC value of 1.5 (up-regulated) or -1.5 (down-regulated) and a FDR <0.05x10-5 were selected. GO analysis of differential transcripts was performed at phytozome.org Results: We identified commonly up-regulated genes (317) and exclusively up-regulated in Bd2-3 (466) or Bd21 (706). Regarding down-regulation, 330 transcripts were common, an exclusively 851 and 1026 for Bd2-3 and Bd21, respectively. GO analysis indicated that oxidative stress, pathogen responses and nitric oxide homeostasis were the most differential characteristics of tolerant ecotype Bd2-3. Conclusions: The use of triplicate RNAseq data of transcriptomes expressed in ecotypes with contrasting tolerance to submergence under long-day light regime, allowed us to identify common responsive routes such as SUSY, glycolysis, anaerobic routes (alanine, ethanol, lactate, GABA) and glyoxylate cycle. It also enabled us to discover integrated oxidative stress and NO homeostasis pathways that are differentially expressed in the tolerant ecotype. We expect that this information can be translated to agricultural relevant plants to increase our knowledge and biotechnological possibilities on plant submergence stress. Sequenced libraries (triplicates, HiSeq2500 Illumina, 1x50 format) of aerial tissue (control and 48h submergence stress) of Brachypodium distachyon Bd21 (sensitive) and Bd2-3 (tolerant).
Project description:We have applied whole transcriptome profiling to infer genetic determinants of pathogenicity and host specialization in Z. tritici. Our data includes RNAseq data from early infection stages of a compatible (wheat) and a non-compatible host (Brachypodium distachyon). Overall transcription of AC genes is remarkably lower than genes on core chromosomes (CC) and only 40% of the genes are transcribed. We identify 31 AC and 1069 CC genes showing plant specific expression. In addition 21 CC genes are only upregulated in wheat supporting functional relevance in host specificity. We further explore the genomic composition and distribution of unique and paralogous genes in Z. tritici focusing on the evolutionary origin of AC genes. In contrast to previous studies we show that ACs mainly encode unique genes. Phylogenetic analyses suggest that rare duplication events in the Z. tritici genome precede diversification of Zymoseptoria species and demonstrate that ACs have been maintained in the genome of Zymoseptoria over long evolutionary times. Examination of gene expression at 3 different growth condition of the wheat pathogen Z. tritici.
Project description:PARE (parallel analysis of RNA ends) was performed to study the change of uncapped mRNAs before and after cold treatment in Brachypodium. Different change patterns were identified. We have provided a complete view of uncapped transcriptome under cold stress condition, which will deepen our understanding of gene expression regulation in cold stress response as well as cold stress response mechanism for monocot plants. The uncapped mRNA profiles of 12-day-old Brachypodium seedlings with and without cold treatment (4 °C for 24 h) were generated by deep sequencing using Illumina GAIIx
Project description:The small RNA transcriptomes of bread wheat (Triticum aestivum L.) and its emerging model (Brachypodium distachyon (L.) Beauv) were obtained by using deep sequencing technology. Small RNA compositions were analyzed in these two species. In addition to 70 conserved microRNAs (miRNA) from 25 families, 23 novel wheat miRNAs were identified. For Brachypodium, 12 putative miRNAs were predicted from a limited number of ESTs, of which one was a potential novel miRNA. Also, 94 conserved miRNAs from 28 families were identified in this species. Expression validation was performed for several novel wheat miRNAs. RNA ligase-mediated 5' RACE experiments demonstrated their capability to cleave predicted target genes including three disease resistant gene analogs. Differential expression of miRNAs was observed between Brachypodium vegetative and reproductive tissues, suggesting their different roles at the two growth stages. Our work significantly increases the novel miRNA numbers in wheat and provides the first set of small RNAs in Brachypodium distachyon. Keywords: Small RNA One wheat small RNA library (Tae) and two Brachypodium small RNA libraries (BdR and BdV) were sequenced.
Project description:In the current study we did microarray of upland rice cultivar Nagina22 for drought stress at reproductive stage (panicle initiation) and analyzed drought stress responsive genes. We have taken flag leaf for our study as it is most essential organ for photosynthesis in rice. Normal watering Vs Drought Stress Flag leaf of Control (Three biological replicates) plant of Nagina22: C1, C2, C3 Flag leaf of drought stressed (Three biological replicates) plant of Nagina 22: S1, S2, S3
Project description:ABSTRACT: Exposure to abiotic stresses triggers global changes in the expression of thousands of eukaryotic genes at the transcriptional 70 and post-transcriptional levels. Small RNA (smRNA) pathways and splicing both function as crucial mechanisms regulating stress-responsive gene expression. However, examples of smRNAs regulating gene expression remain largely limited to effects on mRNA stability, translation, and epigenetic regulation. Also, our understanding of the networks controlling plant gene expression in response to environmental changes, and examples of these regulatory pathways intersecting, remains limited. Here, to investigate the role of smRNAs in stress responses we examined smRNA transcriptomes of Brachypodium distachyon plants subjected to various abiotic stresses. We found that exposure to different abiotic stresses specifically induced a group 75 of novel, endogenous small interfering RNAs (stress-induced, UTR-derived siRNAs, or sutr-siRNAs) that originate from the 3′ UTRs of a subset of coding genes. Our bioinformatics analyses predicted that sutr-siRNAs have potential regulatory functions and that over 90% of sutr-siRNAs target intronic regions of many mRNAs in trans. Importantly, a subgroup of these sutr- siRNAs target the important intron regulatory regions, such as branch point sequences, that could affect splicing. Our study indicates that in Brachypodium, sutr-siRNAs may affect splicing by masking or changing accessibility of specific cis-elements 80 through base-pairing interactions to mediate gene expression in response to stresses. We hypothesize that this mode of regulation of gene expression may also serve as a general mechanism for regulation of gene expression in plants and potentially in other eukaryotes. Analysis of smRNA populations in Brachypodium plants challenged by abiotic stresses: To profile the populations of smRNAs in the model monocot Brachypodium distachyon and examine their regulation in response to abiotic stresses, we conducted high-throughput sequencing of small RNAs from plants exposed to four different abiotic stress conditions, cold, heat (air), heat (water immersion), and salt, in the wild type Brachypodium cultivar Bd21. For our experiments we used information from the literature to select two time-points for stress durations, short and long, which differed for each stress: cold (6 and 24 hours), heat-air (1 and 3 hours), heat-water (1 and 3 hours), and salt (48 hours). We generated small RNA libraries for Illumina sequencing (GAII) from the leaves of Brachypodium plants subjected to stresses and selected smRNAs between 15 and 40 nt in length, which we mapped to the Brachypodium genome.
Project description:The functions of miRNAs and their target mRNAs in Arabidopsis development have been widely documented, however, roles of stress responsive miRNAs and their targets are not as well understood. Using small RNA deep sequencing and ATH1 microarrays to profile mRNAs, we identified IAR3 (IAA-Ala Resistant 3) as a novel target of miR167a. 2 organs X 3 treatments X 3 replicates This submission represnts ATH1 microarrays component of study.