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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.