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Open Access Research article

Co-expression analysis identifies putative targets for CBP60g and SARD1 regulation

William Truman* and Jane Glazebrook

Author Affiliations

Department of Plant Biology, Microbial and Plant Genomics Institute, University of Minnesota, 1500 Gortner Avenue, Saint Paul, MN, 55108, USA

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BMC Plant Biology 2012, 12:216  doi:10.1186/1471-2229-12-216

Published: 16 November 2012

Additional files

Additional file 1:

Table S1. Significance of various motifs within promoters of genes clustered with SID2 Motifs are derived from the 10mer oligo found to bind CBP60g and SARD1 in vitro [19]. POBO analyses of a cluster of 11 genes including SID2 identified during pilot co-expression analysis are reported as t-values from two-tailed t-tests. Positive values indicate an enrichment of motifs compared to the genome background and negative values an under-representation with increasing magnitude indicating greater significance. Promoter regions were defined as starting at the transcription start site.

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Additional file 2:

Table S2. Overview and cluster contents of co-expression experiment #1.

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Additional file 3:

Table S3. Overview and cluster contents of co-expression experiment #2.

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Additional file 4:

Figure S1. CBP60g and SARD1 exert antagonist effects on the expression of phylogenetically related calmodulin-like genes. qRT-PCR measurement of gene expression 24 hpi Pma ES4326 (OD600 =0.01). Data from five biological replicates were merged using a mixed linear model and the mean log2 ratio to Actin2 expression plotted along with the standard error. Asterisks denote a significant differential expression between wildtype and the cbp60g sard1 mutant with p-value ≤ 0.05 from a two-tailed t-test.

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Additional file 5:

Figure S2. Distribution of selected motifs in the SID2 regulon. Visualisation of the distribution of GAAATT and CCTN7TCC motifs throughout cluster 2 of experiment #2. Plot created using the feature map function of the RSAT suite of tools (http://rsat.ulb.ac.be/ webcite). Red ticks denote CCTN7TCC and blue ticks represent GAAATT motifs, ticks above the promoter line are in the sense orientation and ticks below the line antisense. There is a significant bias of the CCTN7TCC motif towards the 750 bp proximal to the transcription start site (p-value=0.005).

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Additional file 6:

Table S4. POBO analysis of motif enrichment in the promoters of clusters from experiment #1 and experiment #2. POBO analysis of the abundance of assorted motifs in the 1500 bp promoter region of clustered genes. 1000 pseudoclusters of size matched to the given cluster were generated both from within the cluster and the genome background. Significance is recorded as t-values from two-tailed t-tests. Positive values indicate an enrichment of motifs compared to the genome background and negative values an under-representation with increasing magnitude indicating greater significance. Motifs are derived from various sources. (1) The 10mer oligo found to bind CBP60g and SARD1. (2) Known W-box consensus sequences involved in binding WRKY transcription factors. (3) A novel dyad motif identified in a cluster containing SARD1 and SID2. (4) An uncharacterised motif enriched in several clusters containing CBP60g. (5) The binding site for the SID2 repressor EIN3. (6) The core binding site for NAC transcription factors including the negative regulator of SID2, ANA019. (7) The binding site for SR1 (CAMTA3), the negative regulator of EDS1 and other defence signalling components.

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Additional file 7:

Figure S3.CBP60g forms a SID2 independent co-expression network in response to abiotic stress. A network was created from the genes co-expressed with CBP60g and SARD1 across 27 selected abiotic stress microarray datasets where CBP60g was induced by stress but not strongly correlated with SID2 expression. Edges represent a Spearman rank correlation coefficient of at least 0.7; the width is proportional to the correlation between two genes.

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Additional file 8:

Table S5. Primer sequences used for qRT-PCR.

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Additional file 9:

Table S6. Overview of microarray data used for co-expression analysis. For each experiment the Spearman rank correlation coefficient between SID2 and WRKY28, CBP60g and the two SARD1 probesets is followed by the p-values associated with these correlations. Experiments used for the abiotic stress co-expression network are marked.

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