Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Expression Analysis of Carotenoid Biosynthesis Pathway Genes in Tomato

ABSTRACT: Tomato pericarp tissues at three different stages (green, breaker and ripening) of fruit development from both the above genotypes were collected using a sterile surgical blade and flash frozen in liquid nitrogen. High quality RNA was extracted from the frozen pericarp tissue using TRI Reagent (Ambion, INC. USA) and pooled at least from three independent samples and treated with DNase-I (QIAGEN GmbH, Germany). RNA cleanup was carried out using RNeasy Plant Mini Kit (QIAGEN GmbH, Germany) and 5ug of total RNA from each sample with three biological replications were reverse-transcribed to double stranded cDNA using the GeneChipR One-Cycle cDNA Synthesis Kit. The biotin-labelled cRNA was made using the GeneChipR IVT Labelling Kit (Affymetrix, CA, USA). 15 ug of cRNA samples was fragmented and was hybridized for 16 hours at 45C to the Affymetrix tomato GeneChip Array (Santa Clara, CA, USA). After washing and staining with R-phycoerythrin streptavidin in a Fluidics Station, using the GenechipR Fluidics Station 450, the arrays were scanned by the GenechipR 3000 Scanner. The chip images were scanned and extracted using default settings and the .CEL files were produced by using the Affymetrix GeneChip Operating Software (GCOS 1.2). Samples that passed through various quality checks (data not shown) were normalized with RMA algorithm using GeneSpring GX 10 (Agilent Technologies Inc., Santa Clara, CA, USA). Probes showing twofold up- or down-regulation (in Log2 transformed values) compared to corresponding control condition were taken only into consideration for further analysis

ORGANISM(S): Lycopersicon esculentum

SUBMITTER: SHUCHI SMITA

PROVIDER: E-MEXP-2934 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Colletotrichum coccodes secretes ammonium during germination and colonization of host tissue as a mechanism for dual activation of fungal pathogenicity factors and plant cell death via activation of host NADPH oxidase. Ammonium accumulation during colonization of tomato fruits by C. coccodes induced host nucleus integrity changes and program cell death (PCD). Transcriptome analysis at the leading edge of colonized tissue and ammonium treated tomato fruits revealed 82 and 237 common up-regulated and common down-regulated genes, respectively. The common-up-regulated genes showed over representation of pathogen related (PR) proteins, salicylic acid (SA) dependent and systemic acquired resistance (SAR) related genes and genes related to biotic stress. The down regulated genes showed over representation of Jasmonic acid (JA) dependent genes. Consistent with this observation the direct application of SA enhanced C. coccodes colonization, whereas the application of JA delayed colonization. Candidate genes were selected for QRT-PCR analysis of transcripts in normal and transgenic RBOH antisense plants and showed dependence on RBOH regulation. In all, the results suggest that ammonia accumulation during C. coccodes colonization activates SA and suppress JA pathways through activation of RBOH. The effects of ammonium on the expression of the selected up-and down regulated genes were examined in fruit of different maturation stages. Red, susceptible and green resistant, tomato fruit displayed similar behavior, suggesting that resistance of green fruits to C. coccodes colonization is not modulated by the JA and SA pathways but depends on other factors Ten micrograms of total RNA was processed for the microarray hybridizations using the Affymetrix GeneChip one-cycle target-labeling kit (Affymetrix). The four treatments were: C. coccodes "mat inoculation" compared to a wound control (24 hours after the tomato fruit peel was removed). 5 mM Ammonium treatment in PBS compared to PBS treatments as a control for ammonium treatment, the treatments applied every four hours. Each treatment had a duplicate. The biotinylated complementary RNA was fragmented and hybridized to the GeneChip Tomato Genome Array (10,038 tomato probe sets for more than 9,200 tomato genes). The arrays were washed, stained, and scanned at the biological services (Weizmann institute of science, Rehovot, Israel). CEL files for the Affymetrix microarray data arising from the microarray experiments were analyzed utilizing the Partek genomics software (Downey, 2006).

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Expression Analysis of Carotenoid Biosynthesis Pathway Genes in Tomato

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