Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

EXPRSS: an Illumina based high-throughput expression-profiling method to reveal transcriptional dynamics - II

ABSTRACT: Background Next Generation Sequencing technologies have facilitated differential gene expression analysis through RNA-seq and Tag-seq methods. RNA-seq has biases associated with transcript lengths, lacks uniform coverage of regions in mRNA and requires 10–20 times more reads than a typical Tag-seq. Most existing Tag-seq methods either have biases or not high throughput due to use of restriction enzymes or enzymatic manipulation of 5’ ends of mRNA or use of RNA ligations. Results We have developed EXpression Profiling through Randomly Sheared cDNA tag Sequencing (EXPRSS) that employs acoustic waves to randomly shear cDNA and generate sequence tags at a relatively defined position (~150-200 bp) from the 3? end of each mRNA. Implementation of the method was verified through comparative analysis of expression data generated from EXPRSS, NlaIII-DGE and Affymetrix microarray and through qPCR quantification of selected genes. EXPRSS is a strand specific and restriction enzyme independent tag sequencing method that does not require cDNA length-based data transformations. EXPRSS is highly reproducible, is high-throughput and it also reveals alternative polyadenylation and polyadenylated antisense transcripts. It is cost-effective using barcoded multiplexing, avoids the biases of existing SAGE and derivative methods and can reveal polyadenylation position from paired-end sequencing. Conclusions EXPRSS Tag-seq provides sensitive and reliable gene expression data and enables high-throughput expression profiling with relatively simple downstream analysis. Five weeks old Arabidopsis (Col-0, ein2-5, jar1-1 and npr1-1) leaf discs treated with water or flg22 and a time course of samples were collected; mRNA profiles were generated by deep sequencing on Illumina GAIIx using EXPRSS protocols.

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: Ghanasyam Rallapalli

PROVIDER: E-GEOD-51720 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

EXPRSS: an Illumina based high-throughput expression-profiling method to reveal transcriptional dynamics.

Rallapalli Ghanasyam G Kemen Eric M EM Robert-Seilaniantz Alexandre A Segonzac Cécile C Etherington Graham J GJ Sohn Kee Hoon KH MacLean Daniel D Jones Jonathan D G JD

BMC genomics 20140506

<h4>Background</h4>Next Generation Sequencing technologies have facilitated differential gene expression analysis through RNA-seq and Tag-seq methods. RNA-seq has biases associated with transcript lengths, lacks uniform coverage of regions in mRNA and requires 10-20 times more reads than a typical Tag-seq. Most existing Tag-seq methods either have biases or not high throughput due to use of restriction enzymes or enzymatic manipulation of 5' ends of mRNA or use of RNA ligations.<h4>Results</h4>W ...[more]

PMID: 24884414

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Project description:Plant nucleotide-binding leucine-rich repeat (NB-LRR) disease resistance (R) proteins recognize specific “avirulent” pathogen effectors and activate immune responses. NB-LRR proteins structurally and functionally resemble mammalian Nod-like receptors (NLRs). How NB-LRR and NLR proteins activate defense is poorly understood. The divergently transcribed Arabidopsis R genes, RPS4 (resistance to Pseudomonas syringae 4) and RRS1 (resistance to Ralstonia solanacearum 1), function together to confer recognition of Pseudomonas AvrRps4 and Ralstonia PopP2. RRS1 is the only known recessive NB-LRR R gene and encodes a WRKY DNA binding domain, prompting suggestions that it acts downstream of RPS4 for transcriptional activation of defense genes. We define here the early RRS1-dependent transcriptional changes upon delivery of PopP2 via Pseudomonas type III secretion. The Arabidopsis slh1 (sensitive to low humidity 1) mutant encodes an RRS1 allele (RRS1SLH1) with a single amino acid (leucine) insertion in the WRKY DNA-binding domain. Its poor growth due to constitutive defense activation is rescued at higher temperature. Transcription profiling data indicate that RRS1SLH1-mediated defense activation overlaps substantially with AvrRps4- and PopP2-regulated responses. To better understand the genetic basis of RPS4/RRS1-dependent immunity, we performed a genetic screen to identify suppressor of slh1 immunity (sushi) mutants. We show that many sushi mutants carry mutations in RPS4, suggesting that RPS4 acts downstream or in a complex with RRS1. Interestingly, several mutations were identified in a domain C-terminal to the RPS4 LRR domain. Using an Agrobacterium-mediated transient assay system, we demonstrate that the P-loop motif of RPS4 but not of RRS1SLH1 is required for RRS1SLH1 function. We also recapitulate the dominant suppression of RRS1SLH1 defense activation by wild type RRS1 and show this suppression requires an intact RRS1 P-loop. These analyses of RRS1SLH1 shed new light on mechanisms by which NB-LRR protein pairs activate defense signaling, or are held inactive in the absence of a pathogen effector. Five weeks old Arabidopsis (Ws-2 and rrs1-1) leaves were infiltrated with Pf0-1 carrying PopP2 or PopP2C321A in pEDV6 and samples were collected at 2, 4, 6 and 8 hours post infiltration (hpi); mRNA profiles were generated in triplet, by deep sequencing on Illumina GAIIx using EXPRSS tag-seq protocol.

Project description:Plant nucleotide-binding leucine-rich repeat (NB-LRR) disease resistance (R) proteins recognize specific M-bM-^@M-^\avirulentM-bM-^@M-^] pathogen effectors and activate immune responses. NB-LRR proteins structurally and functionally resemble mammalian Nod-like receptors (NLRs). How NB-LRR and NLR proteins activate defense is poorly understood. The divergently transcribed Arabidopsis R genes, RPS4 (resistance to Pseudomonas syringae 4) and RRS1 (resistance to Ralstonia solanacearum 1), function together to confer recognition of Pseudomonas AvrRps4 and Ralstonia PopP2. RRS1 is the only known recessive NB-LRR R gene and encodes a WRKY DNA binding domain, prompting suggestions that it acts downstream of RPS4 for transcriptional activation of defense genes. We define here the early RRS1-dependent transcriptional changes upon delivery of PopP2 via Pseudomonas type III secretion. The Arabidopsis slh1 (sensitive to low humidity 1) mutant encodes an RRS1 allele (RRS1SLH1) with a single amino acid (leucine) insertion in the WRKY DNA-binding domain. Its poor growth due to constitutive defense activation is rescued at higher temperature. Transcription profiling data indicate that RRS1SLH1-mediated defense activation overlaps substantially with AvrRps4- and PopP2-regulated responses. To better understand the genetic basis of RPS4/RRS1-dependent immunity, we performed a genetic screen to identify suppressor of slh1 immunity (sushi) mutants. We show that many sushi mutants carry mutations in RPS4, suggesting that RPS4 acts downstream or in a complex with RRS1. Interestingly, several mutations were identified in a domain C-terminal to the RPS4 LRR domain. Using an Agrobacterium-mediated transient assay system, we demonstrate that the P-loop motif of RPS4 but not of RRS1SLH1 is required for RRS1SLH1 function. We also recapitulate the dominant suppression of RRS1SLH1 defense activation by wild type RRS1 and show this suppression requires an intact RRS1 P-loop. These analyses of RRS1SLH1 shed new light on mechanisms by which NB-LRR protein pairs activate defense signaling, or are held inactive in the absence of a pathogen effector. Arabidopsis No-0 and slh1 plants were grown for 4 weeks at 28M-BM-0C after germination on MS plate. Plants were transferred to 19M-BM-0C growth chamber at the beginning of the light cycle and samples were harvested at 0 h, 9 h, 12 h, 16 h and 24 h after transfer for total RNA extraction. mRNA profiles were generated by deep sequencing on Illumina GAIIx using EXPRSS tag-seq protocol. Please note that all 16 samples submitted for this study were sequenced in one lane of Illumina GAIIx and the "No-0_slh1_tempshift_nobarcode.fq" (linked to 'unassigned reads' sample) contains unassigned sequence reads, once sample de-multiplexing has been carried out based on barcode.

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Dataset Information

EXPRSS: an Illumina based high-throughput expression-profiling method to reveal transcriptional dynamics - II

Publications

EXPRSS: an Illumina based high-throughput expression-profiling method to reveal transcriptional dynamics.

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