Comment[ArrayExpressAccession] E-GEOD-51655 MAGE-TAB Version 1.1 Public Release Date 2013-10-25 Investigation Title Transcriptional profiling of the rust fungus Cronartium quercuum f. sp. fusiforme (Cqf) during vegetative growth on 2 different hosts. Comment[Submitted Name] Transcriptional profiling of the rust fungus Cronartium quercuum f. sp. fusiforme (Cqf) during vegetative growth on 2 different hosts. Experiment Description The purpose of this study was to make a single comparison between Cqf genes expressed during the vegetative stages of infection on the telial host (oak leaf) versus the aecial host (pine stem). A large proportion of genes were expressed in both hosts and significantly differentially expressed genes were enriched for candidate fungal effectors (small secreted proteins). These results suggest that the Cqf rust fungus uses a largely common set of genes to create two very different infection phenotypes. This study was based on hybridizations to custom microarrays containing features representing 8692 gene models from a Cqf genome sequencing project midpoint assembly. Two Agilent 4 X 44K microarray slides were populated with 60-mer probes (1 to 5 per transcript), designed using AgilentM-bM-^@M-^Ys web-based eArray software. Labeled target cRNA (complementary RNA) was generated using AgilentM-bM-^@M-^Ys Low Input Quick Amp Labeling Kit, such that oak and pine samples were labeled with either cy3 or cy5 an equal number of times across the experiment. Each microarray was hybridized with labeled cRNA target derived from a single oak sample and labeled cRNA target derived from a single pine sample. There were a total of eight oak sample replications and eight pine sample replications. Target hybridization and scanning were performed by the University of FloridaM-bM-^@M-^Ys Interdisciplinary Center for Biotechnology Research using standard procedures and an Agilent G250B Scanner. Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Davis Smith Davis Person First Name John Katherine John Person Mid Initials E M Person Email geo@ncbi.nlm.nih.gov Person Affiliation University of Florida Person Address School of Forest Resources and Conservation, University of Florida, 365 Newins-Ziegler Hall, Gainesville, FL, USA Person Roles submitter Protocol Name P-GSE51655-1 P-GSE51655-5 P-GSE51655-6 P-GSE51655-8 P-GSE51655-2 P-GSE51655-9 P-GSE51655-3 P-GSE51655-11 P-GSE51655-4 P-GSE51655-10 P-GSE51655-7 Protocol Description Agilent Feature Extraction Software (version 10.7) was used for local background subtraction and feature quality control. Background subtracted signal intensities were normalized by setting microarray means to zero with a standard deviation of 1. ID_REF = VALUE = Normalized signal intensity AgilentM-bM-^@M-^Ys Low Input Quick Amp Labeling Kit was used to label 200ng total RNA hybridized with rotation at 65M-0C for 17 hours.M- The arrays were washed according to the manufacturerM-bM-^@M-^Ys protocol Northern red oak seedlings were inoculated on the underside of the juvenile leaves using the single aeciospore isolate, SC20-21, at the USDA Forest Service Resistance Screening Center, Asheville, NC using standard operating procedures. 6 to 8 week old slash pine seedlings were spray inoculated (20,000 spores/ml) using basidiospores shed from the oak seedlings (inoculated with single aeciospore isolate SC20-21) in this experiment, in an inoculation chamber at the USDA Forest Service Resistance Screening Center, Asheville, NC using standard operating procedures. Northern red oak seedlings were grown in greenhouse conditions at the USDA Forest Service Resistance Screening Center, Asheville, NC using standard operating procedures. Open pollinated slash pine seedlings were grown in greenhouse conditions at the USDA Forest Service Resistance Screening Center, Asheville, NC using standard operating procedures. Pine stem gall samples were freeze dried for 4 days before extraction. They were broken into small pieces using a coffee grinder. The small pieces were further ground to a fine powder using three 5/32-inch stainless steel balls in microcentrifuge tubes processed in a Geno/Grinder 2000 homogenizer. Frozen harvested oak leaf samples (3 leaves pooled from an individual plant) were ground in liquid nitrogen to a fine powder. Total RNA was extracted from approximately 200mg of ground oak or pine sample using a previously described cetyltrimethylammonium bromide (CTAB) buffer method (Chang et al., 1993). Pine stem gall samples were freeze dried for 4 days before extraction. They were broken into small pieces using a coffee grinder. The small pieces were further ground to a fine powder using three 5/32-inch stainless steel balls in microcentrifuge tubes processed in a Geno/Grinder 2000 homogenizer. Total RNA was extracted from approximately 200mg of ground pine sample using a cetyltrimethylammonium bromide (CTAB) buffer method (Chang et al., 1993). Frozen harvested oak leaf samples (3 leaves pooled from an individual plant) were ground in liquid nitrogen to a fine powder. Total RNA was extracted from approximately 200mg of ground oak sample using a cetyltrimethylammonium bromide (CTAB) buffer method (Chang et al., 1993). Images were quantified using Agilent Feature Extraction Software (version 10.7) and an Agilent G250B scanner. Protocol Type normalization data transformation protocol labelling protocol hybridization protocol sample treatment protocol sample treatment protocol growth protocol growth protocol nucleic acid extraction protocol nucleic acid extraction protocol nucleic acid extraction protocol array scanning protocol Experimental Factor Name DAYS POST INOCULATION Experimental Factor Type days post inoculation Comment[SecondaryAccession] GSE51655 Comment[GEOReleaseDate] 2013-10-25 Comment[ArrayExpressSubmissionDate] 2013-10-24 Comment[GEOLastUpdateDate] 2013-10-30 Comment[AEExperimentType] transcription profiling by array Comment[AdditionalFile:Data1] GSE51655_README.txt SDRF File E-GEOD-51655.sdrf.txt