Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

DEK1 deletion effect on Physcomitrella patens transcriptome

ABSTRACT: The main phenotype observed in DEK1 deletion (deltadek1) in P. patens is an absence of gametophore (leafy shoot) development. But, bud initiation the first step in gametophore development occurs. This experiments aims to compare transcriptome of wild-type and deltadek1 strains before budding onset, in 6 day old culture, and after budding onset, in 14 days old cultures. At the first time point both culture are morphologically similar as after 14 days wild-type displays young gametophores and deltadek1 has only arrested buds.

INSTRUMENT(S): Illumina HiSeq 2000

ORGANISM(S): Physcomitrella patens

SUBMITTER: Odd-Arne Olsen

PROVIDER: E-MTAB-2588 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Genetic analysis of DEFECTIVE KERNEL1 loop function in three-dimensional body patterning in Physcomitrella patens.

Demko Viktor V Perroud Pierre-François PF Johansen Wenche W Delwiche Charles F CF Cooper Endymion D ED Remme Pål P Ako Ako Eugene AE Kugler Karl G KG Mayer Klaus F X KF Quatrano Ralph R Olsen Odd-Arne OA

Plant physiology 20140902 2

DEFECTIVE KERNEL1 (DEK1) of higher plants plays an essential role in position-dependent signaling and consists of a large transmembrane domain (MEM) linked to a protease catalytic domain and a regulatory domain. Here, we show that the postulated sensory Loop of the MEM domain plays an important role in the developmental regulation of DEK1 activity in the moss Physcomitrella patens. Compared with P. patens lacking DEK1 (∆dek1), the dek1∆loop mutant correctly positions the division plane in the bu ...[more]

PMID: 25185121

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Project description:Endogenous 24nt short interfering RNAs (siRNAs) derived mostly from intergenic and repetitive genomic regions constitute a major class of endogenous small RNAs in Arabidopsis thaliana. Accumulation of A. thaliana 24nt siRNAs requires the Dicer family member DCL3, and clear homologs of DCL3 exist in both flowering and non-flowering plants. However, the absence of a conspicuous 24nt peak in the total RNA populations of several non-flowering plants has raised the question of whether this class of siRNAs might, in contrast to the ancient 21nt microRNAs (miRNAs) and 21-22nt trans-acting siRNAs (tasiRNAs), be an angiosperm-specific innovation. Analysis of non-miRNA, non-tasiRNA hotspots of small RNA production within the genome of the moss Physcomitrella patens revealed multiple loci which consistently produced a mixture of 21-24nt siRNAs with a peak at 23nts. These Pp23SR loci were significantly enriched in transposon content, depleted in overlap with annotated genes, and typified by dense concentrations of the 5-methyl cytosine (5mC) DNA modification. Deep sequencing of small RNAs from two independent Ppdcl3 mutants showed that the P. patens DCL3 homolog is required for the accumulation of 22-24nt siRNAs, but not 21nt siRNAs, at Pp23SR loci. The 21nt component of Pp23SR-derived siRNAs was also unaffected by a mutation in the RNA-dependent RNA polymerase mutant Pprdr6. Transcriptome-wide, Ppdcl3 mutants specifically failed to accumulate 23 and 24nt small RNAs from repetitive regions. We conclude that intergenic/repeat-derived siRNAs are indeed a broadly conserved, distinct class of small regulatory RNAs within land plants. Our results also suggest that Pp DCL3 produces siRNAs of heterogenous size, unlike its A. thaliana homolog which generates exclusively 24nt siRNAs. Small RNAs from Wild-type (two technical replicates), rdr6-19 (two technical replicates), dcl3-5 (one sample), and dcl3-10 (one sample) were sequenced using a Solexa GA. Three different linkers were used for different samples and mixed prior to sequencing. linker 1: CTGTAGGCACCATCAAT (GPL7174 AND GSM313212, GSM313213) linker 2: CACTCGGGCACCAAGGA (GPL7175 AND GSM313214, GSM313215) linker 3: TTTAACCGCGAATTCCAG (GPL7176 AND GSM313216, GSM313217) Raw data: Two FASTQ files represent the raw data underlying the processed sample data in GSE12468. 080616_s_7_seq_GAK-2.fastq corresponds to GSM313212, GSM313214, and GSM313216. 080616_s_8_seq_GAK-3.fastq corresponds to GSM313213, GSM313215, and GSM313217. Experimentally, samples GSM313212, GSM313214, and GSM313216 (i.e., all three genotypes) were sequenced in the same run (hence the same FASTQ file). The sequencing run was a mixture of three different libraries with different linker sequences (as indicated above). These 'barcoded' samples were computationally extracted based on the linker sequences. This is also true for samples GSM313213, GSM313215, and GSM313217. Raw data were filtered to identify small RNAs associated with each linker. Subsequently, small RNAs were filtered to remove rRNA fragments. The remainder were mapped to the Physcomitrella patens draft genome version 1.2 (using oligomap) and filtered to retain only those with one or more perfect match. See Cho et al. for details.

Project description:Purpose: The plant hormone abscisic acid (ABA) coordinates responses leading to desiccation tolerance in all land plants. RNA-seq was employed to obtain differential expression data in response to ABA, dehydration and mannitol treatments in wild-type tissue. This was compared against the targeted gene knockout line PpanrKO. The ANR gene encodes a novel ABA regulator identified from ABA non-responsive (anr) mutant screens. Methods: RNA-seq on the Illumina HiSeq platform and mapping to the V3.0 genome and transcriptome assemblies using tophat2 enabled gene abundance data to be generated for differential gene expression analysis with edgeR in the Trinity package. Results: We observe a coordinated up-regulation of ca. 900 genes, with extensive overlaps between hormone and stress treatments as well as ca. 900 down-regulated genes. Of the up-regulated genes, those encoding LEA proteins, membrane channel proteins and transporters and oxidative-stress-associated genes comprised a significant fraction while those encoding growth-associated cell wall enzymes (e.g. expansins) and components of secondary metabolism (notably enzymes in the phenylpropanoid pathway) were prominent in the down-regulated genes. The ANR gene, a modular MAP3 kinase comprising an N-terminal PAS domain, a central EDR domain and a C-terminal MAPKKK-like domain unique to basal plant lineages, is found to play a central role in these responses with mutants failing to accumulate the wide spectrum of dehydration tolerance-associated gene products characteristic of the wild-type response and do not acquire ABA-dependent desiccation tolerance. Conclusions: Our study adds considerable data to the stress responses in bryophytes helping understand the mechanisms behind vegetative dehydration tolerance; an important trait for crop species. We also confirmed the vital role that PpANR plays in these responses in the moss Physcomitrella patens likely representing an ancestral mechanism for ABA-mediated dehydration tolerance vital in the conquest of land. RNA from 3 biological replicas was bulked per library. 8 libraries were generated from two genotypes, wild-type and PpanrkO, which included 3 treatments and a control sample for each.

Project description:The Solexa/Illuminaâs digital gene expression (DGE) system was used to gain insight into the broad range of transcriptional responses during the vegetative growth, sclerotial development, myceliogenic germination, carpogenic germination, apothecium formation (stipe) and infection of Sclerotinia sclerotiorum. We obtained a sequencing depth of approximately 3.3 million clean tags per cDNA library. Tag mapping indicated that these six cDNA libraries in total represented more than 66.7% of all of the genes presented in the predicted transcript databases of the Broad Institute. Thouthands of differentially expressed genes were indentified during the various developmental stages compared to the vegetative growth stage. Our results could increase and deepen the understanding of the vegetative and reproductive development as well as the infection of S. sclerotiorum The S. sclerotiorum strain Ep-1PNA367 was grown or treated under different conditions, and samples were collected for RNA extraction for DGE analysis during the following stages: (i) Vegetative stage: activating hyphal agar discs of the Ep-1PNA367 stain were placed on a cellophane membrane overlaid onto PDA medium at 20Â°C; subsequently, the mycelia were collected at 12, 24, 36, 48 and 60 h; (ii) Sclerotial formation stage: the colonies growing on the cellophane membrane overlaid onto PDA medium were further incubated under the same conditions and then the cultures were collected at 84, 96, 108, 120 and 132 h; (iii) Early stages of infection: fresh hyphal fragments of the Ep-1PNA367 strain were overlaid onto sterilized cheese cloth, which was overlaid onto the leaves of the A. thaliana ecotype Columbia-0, followed by inoculation at 20Â°C with 100% relative humility; the cheese cloth with hyphae was then rolled up from the leaves at 9 h and 12 h; (iv) Sclerotial myceliogenic germination stage: sclerotia were surface sterilized with sodium hypochlorite and were sowed on a PDA plate at 20Â°C to induce myceliogenic germination, and when approximately 50% of sclerotia had germinated, the sclerotia were harvested; (v) Sclerotial carpogenic germination stage: sclerotia were dried at room temperature and were pretreated in a freezer (4-6Â°C) for up to one month and then were surface sterilized and sowed on wet sterilized sands in a plate at 15Â°C to induce carpogenic germination; when approximately 50% of the sclerotia germinated (stipes having only emerged from sclerotia), the sclerotia were harvested; and (vi) Early apothecial formation stage: sclerotia were allowed to grow in the same incubator, and the stipes were cut and collected immediately before apothecium formation for RNA extraction. Finally, different time-point samples from the vegetative stage, sclerotial formation stage and infection stage were pooled together respectively according to equal quantities for RNA extraction.

Dataset Information

DEK1 deletion effect on Physcomitrella patens transcriptome

Publications

Genetic analysis of DEFECTIVE KERNEL1 loop function in three-dimensional body patterning in Physcomitrella patens.

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