Unknown,Transcriptomics,Genomics,Proteomics

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Expression analysis of Aspergillus oryzae grown on different carbon sources

ABSTRACT: In this study, we focused on chemically defined inducers or substrates to drive expression of cellulases, hemicellulases and accessory enzymes in the model filamentous fungus Aspergillus oryzae. Cellohexaose (O-CHE), mannohexaose (O-MHE), xylopentaose (O-XPE), arabinoheptaose (O-AHP), 1,3:1,4-M-NM-2-glucohexaose (O-BGHEXA), 63-M-NM-1-D-glucosyl-maltotriosyl-maltotriose (O-GMH), 61-M-NM-1-D-galactosyl-mannotriose (O-GM3), xyloglucan (X3Glc4-borohydride reduced; O-X3G4R), turanose (TYR) and sophorose (SOP) were used to induce the plant polysaccharide degradation machinery of A. oryzae. The strain used in this study was the A. oryzae sequenced strain RIB40, obtained from IBT culture collection at Technical University of Denmark. To obtain a global view of the A. oryzae transcriptome activated for plant biomass conversion, mRNA from growth after 2 h on 10 different carbohydrate active enzyme inducers (di- and M-bM-^@M-^Soligo saccharides) was subjected to custom-designed Agilent microarray analysis.

ORGANISM(S): Aspergillus oryzae RIB40

SUBMITTER: D.B.R.K. Gupta Udatha

PROVIDER: E-GEOD-48864 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Rice blast is the most threatening disease to cultivated rice worldwide. Magnaporthe oryzae, its causal agent is likely to encounter environmental challenges during invasive growth in the host plants that require shifts in gene expression to establish a compatible interaction. Here, we tested the hypothesis that M. oryzae have similar gene expression patterns in planta, versus during in vitro stresses. Gene expression data was collected from in vitro experiments of heat shock, oxidative burst, and three different types of nutrient starvation. These data were compared to fungal gene expression during the invasive growth phase in compatible interactions on two grass hosts, rice and barley. We have identified 4,973 differentially expressed genes, from which 1,909 genes showed similar regulation patterns between at least one of the in vitro stresses and rice and/or barley. Hierarchical clustering of these 1,909 genes showed three major clusters in which growth in planta conditions closely grouped with the starving nutrient conditions. Out of these 1,909 genes, 55 and 129 genes were induced and repressed in all seven treatments, respectively. The functional categorization of those 55 induced genes revealed that most of them are either related to carbon metabolism, membrane proteins, or are involved in oxidoreduction reactions. The 129 repressed genes are putatively involved in vesicle trafficking, signal transduction, nitrogen metabolism, or molecular transport. These findings indicated that M. oryzae is likely coping with nutrient deprived environments in its hosts during the invasive growth stage about 72 hours post-inoculation, and not as much with host defense responses, or a temperature stress. Eight M. oryzae treatments: mycelia grown in liquid complete medium (set as the control); 72 hours post-incoulation (hpi) in rice leaves; 72 hours post-incoulation in barley leaves; mycelia under heat shock; mycelia under oxidative stress; mycelia under minimal medium; mycelia under nitrogen starvation; mycelia under carbon starvation. Three biological replicates had their total RNA pulled after RNA extraction. Dye-swaps used as technical replicates.

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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Expression analysis of Aspergillus oryzae grown on different carbon sources

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