Analysis of the Interaction Transcriptome During Biotrophic Invasion of Rice by the Blast Fungus, Magnaporthe oryzae
Ontology highlight
ABSTRACT:
ORGANISM(S): Magnaporthe oryzae
SUBMITTER: Barbara Valent
PROVIDER: E-GEOD-8670 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Publications
Interaction transcriptome analysis identifies Magnaporthe oryzae BAS1-4 as Biotrophy-associated secreted proteins in rice blast disease.
Mosquera Gloria G Giraldo Martha C MC Khang Chang Hyun CH Coughlan Sean S Valent Barbara B
The Plant cell 20090407 4
Biotrophic invasive hyphae (IH) of the blast fungus Magnaporthe oryzae secrete effectors to alter host defenses and cellular processes as they successively invade living rice (Oryza sativa) cells. However, few blast effectors have been identified. Indeed, understanding fungal and rice genes contributing to biotrophic invasion has been difficult because so few plant cells have encountered IH at the earliest infection stages. We developed a robust procedure for isolating infected-rice sheath RNAs ...[more]
Similar Datasets
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.
2009-04-17 | E-GEOD-8518 | biostudies-arrayexpress
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. Keywords: Disease state analysis Our goal was to compare expression in biotrophic IH to expression in mycelium grown in nutrient medium, and to compare expression in infected rice sheath to expression in mock inoculated rice. Using version 2 of the fungal whole genome 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 rice microarray (see series GSE8518). 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.
2009-04-08 | E-GEOD-8517 | biostudies-arrayexpress
Project description:This series examines differential gene expression during germination and appressorium formation by the rice blast fungus Magnaporthe grisea. Conidia were germinated on either the hydrophobic (inductive) or hydrophilic (non-inductive) side of GelBond. RNA was extracted from conidia and following incubation for 7 and 12 hours. RNA from two biological replications of each treatment was pooled in equal amounts and labeled with both cy3 and cy5 dyes using the Agilent Technologies Low Input Linear Amplification Kit. Hybridizations were performed on Agilent Technologies whole genome oligo rice blast arrays (product G4137A) using manufacturer prescribed protocols and reagents in an interlaced loop design in which each treatment was paired with every other. This series contains a total of 10 hybridizations; each treatment was used in 4 hybridizations (2 with cy3 and 2 cy5). Spot fluorescence was normalized using Lowess within and between microarray slides in Bioconductor and gene expression profiles analyzed in GeneSpring. Keywords: other
2010-06-05 | E-GEOD-1945 | biostudies-arrayexpress
Project description:This experiment was designed to analyze transcriptional changes that occur when the fungal plant pathogen M. grisea is shifted from a complete media to a nitrogen-starved media. Comparisons were made in the following way between fungal mycelial grown in minimal media supplemented with a nitrogen source (MM+N) and minimal media without a nitrogen source (NS) Keywords: parallel sample
2010-07-01 | E-GEOD-2716 | biostudies-arrayexpress
Project description:3 genetic chicken lines (Leghorn G-B1, Fayoumi, broiler) were used. chicken were challenged with SE at day 1, spleen and cecum tissues were collected at 2 hours and 16 hours after post challenge
2010-06-11 | E-GEOD-2562 | biostudies-arrayexpress
Project description:LNCaP prostate cancer cells were treated with and without 100?g/ml Emetine Dihydrochloride Hydrate (Fluka, Buchs, Swizerland). Both Emetine-treated and untreated control cells were then incubated at 37°C for 10 hours. After the incubation, the first time point (0 min) was harvested for both treated and untreated cells. Simultaneously, Actinomycin D (Sigma-Aldrich, St Louis, MO) with the final concentration of 5ug/ml, was added to the remaining treated and untreated cells to stop new transcription. Time points of 10min, 30min, 1h, 2h, and 4h were harvested in both groups for most of the cell lines. Cell pellets were snap-frozen and mRNA extracted by using FastTrack kit (Invitrogen) according to the manufacturerâs instructions. For each time point, the untreated sample was hybridized against the Emetine-treated equivalent. Four ug of untreated mRNA was labeled with Cy5-dUTP and four ug of Emetine-treated mRNA with Cy3-dUTP (Amersham Biosciences, Piscataway, NJ) as previously described (Mousses et al, 2000). Image analysis was done by DeArray software. Average intensities of the tumor samples were divided by the average intensities of the reference sample at each microarray spot after background intensity subtraction. Within-slide normalization was performed with ratio statistics method using housekeeping genes as described previously (Chen et al, 1997). The data were quality filtered with ratio quality method (Chen et al, 2002), which computes a quality value for each ratio. The scale for the quality values is from zero (poor quality) to one (good quality). Here, all ratios having quality value below 0.5 were discarded from the subsequent analysis.
2010-06-09 | E-GEOD-1488 | biostudies-arrayexpress
Project description:GM00156 (Coriell) cells were treated with and without 100?g/ml Emetine Dihydrochloride Hydrate (Fluka, Buchs, Swizerland). Both Emetine-treated and untreated control cells were then incubated at 37°C for 10 hours. After the incubation, the first time point (0 min) was harvested for both treated and untreated cells. Simultaneously, Actinomycin D (Sigma-Aldrich, St Louis, MO) with the final concentration of 5ug/ml, was added to the remaining treated and untreated cells to stop new transcription. Time points of 10min, 30min, 1h, and 8h were harvested in both groups for most of the cell lines. Cell pellets were snap-frozen and mRNA extracted by using FastTrack kit (Invitrogen) according to the manufacturerâs instructions. For each time point, the untreated sample was hybridized against the Emetine-treated equivalent. Four ug of untreated mRNA was labeled with Cy5-dUTP and four ug of Emetine-treated mRNA with Cy3-dUTP (Amersham Biosciences, Piscataway, NJ) as previously described (Mousses et al, 2000). Image analysis was done by DeArray software. Average intensities of the tumor samples were divided by the average intensities of the reference sample at each microarray spot after background intensity subtraction. Within-slide normalization was performed with ratio statistics method using housekeeping genes as described previously (Chen et al, 1997). The data were quality filtered with ratio quality method (Chen et al, 2002), which computes a quality value for each ratio. The scale for the quality values is from zero (poor quality) to one (good quality). Here, all ratios having quality value below 0.5 were discarded from the subsequent analysis.
2010-06-09 | E-GEOD-1490 | biostudies-arrayexpress
Project description:PC3 prostate cancer cells were treated with and without 100?g/ml Emetine Dihydrochloride Hydrate (Fluka, Buchs, Swizerland). Both Emetine-treated and untreated control cells were then incubated at 37°C for 10 hours. After the incubation, the first time point (0 min) was harvested for both treated and untreated cells. Simultaneously, Actinomycin D (Sigma-Aldrich, St Louis, MO) with the final concentration of 5ug/ml, was added to the remaining treated and untreated cells to stop new transcription. Time points of 10min, 30min, 1h, 2h, and 4h were harvested in both groups for most of the cell lines. Cell pellets were snap-frozen and mRNA extracted by using FastTrack kit (Invitrogen) according to the manufacturerâs instructions. For each time point, the untreated sample was hybridized against the Emetine-treated equivalent. Four ug of untreated mRNA was labeled with Cy5-dUTP and four ug of Emetine-treated mRNA with Cy3-dUTP (Amersham Biosciences, Piscataway, NJ) as previously described (Mousses et al, 2000). Image analysis was done by DeArray software. Average intensities of the tumor samples were divided by the average intensities of the reference sample at each microarray spot after background intensity subtraction. Within-slide normalization was performed with ratio statistics method using housekeeping genes as described previously (Chen et al, 1997). The data were quality filtered with ratio quality method (Chen et al, 2002), which computes a quality value for each ratio. The scale for the quality values is from zero (poor quality) to one (good quality). Here, all ratios having quality value below 0.5 were discarded from the subsequent analysis.
2010-06-09 | E-GEOD-1489 | biostudies-arrayexpress
Project description:High-density phage epitope microarray from 31 samples were used for unsupervised analysis (GSM36153...GSM36183). 129 samples from prostate cancer patients and controls were screened on small focused epitope chips, which contained 180 phage elements. These data were used to train GA/KNN program (GSM36184...GSM36312). 128 samples from localized prostate cancer patients and controls were screened on small focused epitope chips. These independent data were used to validate the epitomic profile (GSM36313...GSM36375, GSM40203...GSM40213, GSM40216, GSM40218, GSM40219, GSM40222, GSM40225, GSM40227, GSM40229, GSM40233, GSM40237, GSM40246...GSM40294). Three subgroups of samples were used as test sets to validate the specificity of epitomic profile (GSM36376...GSM36410, GSM40214, GSM40215, GSM40217, GSM40220, GSM40221, GSM40224, GSM40226, GSM40228, GSM40231, GSM40234...GSM40236, GSM40238...GSM40244). Project----Identification of humoral signature for prostate cancer diagnosis We constructed a prostate cancer cDNA phage display library. cDNAs were reverse-synthesized from mDNA pool isolated from prostate cancer tissues. Enzyme-digested cDNA fragments were then inserted into phage vector to make a whole prostate cancer phage expressed cDNA library. In order to select cancer specific phage epitope from this library, we performed several cycles of affinity enrichment. We used the bounded IgG pool isolated from prostate cancer patient sera to select the tumor specific phage epitope clones. Once we had the enriched phage epitope library, we cultured the phage library on LB-agar dish for individual phage colonies. About 2300 phage colonies from agar dish were picked up using toothstick and cultured in 96-well microtiter plates. Each clone was labeled as microtiter plate #, column #, row#, i.e. clone ID. These 2300 clones were then spotted on slides in single spot (no any duplicate), i.e. each spot (labeled by clone ID) represents a single phage clone. The phage epitope microarrays were then screened using cancer or control sera. We employed two color system. Cy5-anti human IgG was to detect human IgG. For green color, we used Cy3-labeled anti-phage capsid protein as internal reference to normalize the ammount difference of phage particles spotted on each spot. Thus the ratio of Cy5/Cy3 would count for the immune response in cancer or control sera. Once we identified humoral signature in prostate cancer patients, we could sequence the phage clone to characterize the nature of the genes or proteins.
2005-10-04 | E-GEOD-2010 | biostudies-arrayexpress
Project description:We examined the major patterns of changes in gene expression in mouse splenic B cells in response to stimulation with 33 single ligands for 0.5, 1, 2, and 4 h. We found that ligands known to directly induce or costimulate proliferation, namely, anti-IgM (anti-Ig), anti-CD40 (CD40L), LPS, and, to a lesser extent, IL-4 and CpG-oligodeoxynucleotide (CpG), induced significant expression changes in a large number of genes. The remaining 28 single ligands produced changes in relatively few genes, even though they elicited measurable elevations in intracellular Ca2+ and cAMP concentration and/or protein phosphorylation, including cytokines, chemokines, and other ligands that interact with G protein-coupled receptors. A detailed comparison of gene expression responses to anti-Ig, CD40L, LPS, IL-4, and CpG indicates that while many genes had similar temporal patterns of change in expression in response to these ligands, subsets of genes showed unique expression patterns in response to IL-4, anti-Ig, and CD40L.
2004-12-08 | E-GEOD-2050 | biostudies-arrayexpress