Unknown,Transcriptomics,Genomics,Proteomics

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Characterization of Gene Expression in Rice Shoot and Root

ABSTRACT: The rice OneArray® 60-mer, oligonucleotide microarray consists of a total of 21,179 probes covering 20,806 genes of japonica and 13,683 genes of indica. Through a validation study, total RNA isolated from rice shoots and roots were used for comparison of gene expression profiles via microarray examination. A list of significantly differentially expressed genes was generated; 438 shoot-specific genes were identified among 3,138 up-regulated genes, and 463 root-specific genes were found among 3,845 down-regulated genes. The Phalanx microarray platform is based upon the hybridization of a single labeled sample (derived from RNA), followed by one-channel detection. The intensity of the hybridization signal is used to determine target concentration. In order to validate the technical quality of each probe in our arrays, we carried out 10 independent hybridizations on samples representing two different rice tissues – root and shoot. To examine the gene expression profiles between rice root and shoot development, total RNA extracted from rice root and shoot were processed on the rice OneArray® microarray following the standard protocol using five arrays for each tissue type. Raw expression data from 10 microarrays (e.g. 5 arrays × 2 tissues) were normalized and Pearson’s correlation coefficients were calculated for the data sets of hybridization signal intensities.

ORGANISM(S): Oryza sativa Japonica Group

SUBMITTER: Jia-Shing Chen

PROVIDER: E-GEOD-50844 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The hemibiotrophic fungus Magnaporthe oryzae produces specialized biotrophic invasive hyphae (IH) that alter membrane structure and defense responses in invaded rice cells. IH successively invade live neighbor cells, apparently through plasmodesmata. Understanding fungal and rice genes that contribute to biotrophic invasion has been a challenge because so few plant cells have encountered IH at the earliest infection stages. Using a rice sheath inoculation method, we successfully enriched for infected tissue RNA that contained ~20% fungal RNA at a point when most IH were still growing in first-invaded rice cells. The RNAs were analyzed using the whole-genome M. oryzae oligoarray and a rice oligoarray. Rice genes that were induced >50-fold during infection were enriched for genes involved in transferring information from sensors to cellular responses. Fungal genes that were induced >50-fold in IH included the PWL2 avirulence gene and genes encoding hypothetical secreted proteins. The IH-specific secreted proteins are candidate effectors, proteins that the fungus secretes into live host cells to control cellular processes. Gene knock-out analyses of three putative effector genes failed to show major effects on pathogenicity. Details of the blast interaction transcriptome will provide insights on the mechanisms of biotrophic plant disease. Experiment Overall Design: Our goal was to compare expression in infected rice sheath to expression in mock inoculated rice, and to compare expression in biotrophic IH to expression in mycelium grown in nutrient medium. Using the rice microarray (Agilent Technologies), we analyzed samples from three biological replicates of rice leaf sheath at 36 hours after inoculation with the rice blast fungus M. oryzae. The same samples were used with the Agilent M. oryzae microarray (see series GSE8517). To estimate the ratio of fungal to rice RNAs in the infected sheaths, we compared RT-PCR amplification of fungal genes in infected tissue to amplification in standards produced by mixing known ratios of pure mycelial RNA with mock-inoculated rice RNA. Using this assay, fungal RNA content in infected tissues were approximately 20% of the total RNAs. We prepared control mixtures by pooling 20% mycelial RNA and 80% RNA from mock-inoculated rice sheath. Complementary RNAs from infected tissues were labeled with Cy3 or Cy5 and hybridized together with the control mixture RNA labeled with the other dye. Three independent biological replications were performed, with separate hybridizations for 2 technical replications and corresponding dye swap experiments. Data were analyzed by Rosetta Resolver® and showed a correlation of over 80% between biological replications.

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Characterization of Gene Expression in Rice Shoot and Root

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