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Supplemental Figure S1: Schematic summary of the experimental design. Three pairs of host/nonhost pathogens of the genera Magnaporthe, Blumeria and Puccinia were inoculated on barley cultivar “Ingrid”. Mock-treated plants without inoculum served as controls. Plant material derived from peeled abaxial epidermis or whole leaves was harvested at the time-points indicated for mRNA extraction and 33P-labelled cDNA-probe preparation (see Materials and Methods for details). Macroarrays were hybridized with cDNA derived from 3-4 independent biological replicates as indicated. NH, nonhost; hai, hours after inoculation. For abbreviations of pathogens see Table I. Signal ratio E/L 100 Control 10 1 0,1 0,01 1 0 2001 2000 4001 4000 6001 6000 8001 8000 10001 10000 Signal ratio E/L Unigenes 100 Inoculated Bgh 10 1 0,1 0,01 01 2001 2000 4001 4000 6001 6000 8001 8000 10001 10000 Unigenes Supplemental Figure S2: Distribution of the ratio of signal intensities obtained from hybridization of epidermal versus whole leaf mRNA samples. RNA Was isolated from peeled epidermis and the remainder of leaves and used for hybridizaiton to the cDNA macroarray. Normalized mean signal intensities from all time points after inoculation with Bgh or from the corresponding non-inoculated controls in three biological replicates were calculated and compared to each other per spotted unigene. Supplemental Figure S3: Reproducibility of macroarray experiments. (A) Two aliquots of one RNA sample from control plants were reverse-transcribed, 33P-labelled and hybridized to different macroarray membranes in two independent experiments. The Pearson correlation of signal intensities obtained from both membranes is shown. (B) Gene regulation induced by Bgh attack was compared in two independent inoculation experiments by using the barleyPGRC1 macroarray. First leaves of Ingrid BC mlo5 were inoculated or left un-treated, followed by peeling of abaxial epidermis and RNA extraction 12 h after inoculation. 33P-Labelled cDNA samples were hybridized to different sets of macroarray membranes. The Pearson correlation of gene regulation factors obtained from both membranes is shown. Similar results were obtained by comparing log(2)-transformed regulation factors (data not shown). (C) Gene regulation induced by Bgh attack was compared in two independent inoculation experiments by using different array technology. First leaves of Ingrid BC mlo5 were inoculated or left un-treated, followed by peeling of abaxial epidermis and RNA extraction 12 h after inoculation. 33P-labelled cDNA and fluorescently labeled RNA was hybridized to the barleyPGRC1 macroarray and the Affymetrix Barley1 array, respectively (Close et al., 2004). The Pearson correlation of regulation factors of unigenes present on both array types is shown. (B and C) Only those unigenes on the barleyPGRC1 array were analyzed that could be linked by clustering (Stackpack) two corresponding Affymetrix exemplar sequences. Unigenes not producing signals >2-fold above background in at least two hybridizations were eliminated from the analysis. This resulted in a total number of 7004 unigenes that could be compared. PM Epidermis Mag Epidermis PM Leaf Rust Leaf 12h 24h 6h 12h 24h 24h 6h PC1 = 56% 12h 24h 48h 12h 6h PC2 = 13% Supplemental Figure S4: Principal component analysis (PCA) of all transcript-derived, log(2)-transformed signals on the barleyPGRC1 array hybridized with non- or mock-treated control samples from inoculation series of PM, Mag or Rust pathogens. Mean values of 3-4 biological replicates were used for PCA calculation. PM Rust Bgt 6h Phor 24h Bgh 6h Ptrit 24h Bgh 12h Bgt 12h Ptrit 12h PC1 = 30% Phor 12h Phor 48h Bgh 24h Bgt 24h Ptrit 48h PC2 = 18% Supplemental Figure S5: Principle component analysis of log(2)-transformed regulation factors (Inocated/Control) of all spotted unigenes in leaf samples of barley inoculated with PM or rust. Mean values from 3 biological replicates were used for PCA analysis. Supplemental Figure S6: Break-down of the functional transcript category “miscellaneous” from the MapMan binning file. A total of 55 pathogen UP-regulated genes were assigned to this superbin.