Comment[ArrayExpressAccession]	E-GEOD-42833
MAGE-TAB Version	1.1							
Public Release Date	2013-03-23
Investigation Title	Analysis of  transcriptional response to mutated Potyvirus Potato virus A that induces necrotic symptoms.							
Comment[Submitted Name]	Analysis of  transcriptional response to mutated Potyvirus Potato virus A that induces necrotic symptoms.
Experiment Description	Mutations introduced to a highly variable region in HCpro reduced HCpro-HIP2 interactions and virulence of PVA, and induced novel types of necrotic symptoms in the systemically infected leaves of Nicotiana species. Microarray-based analysis of gene expression revealed induction of large numbers of defence-related genes including jasmonic acid and ethylene inducible pathways in the leaves systemically infected by the mutated PVA. Three treaments: infection with wild type PVA (WT), infection with mutated PVA (MUT), and mock-inoculation (Mock). Six plants were treated for each treatment. Experiment was repeated three times. WT and MUT samples from all three experiments were hybridised on microarray. Mock samples from experiments 1 and 2 were hybridised as healthy controls.							
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	Haikonen	Haikonen	Rajamaki	Tian	Valkonen			
Person First Name	Tuuli	Tuuli	Minna-Liisa	Yan-Ping	Jari			
Person Mid Initials					P			
Person Email	tuuli.haikonen@helsinki.fi							
Person Affiliation	University of Helsinki							
Person Address	Department of Agricultural Sciences, University of Helsinki, Latokartanonkaari 7, Helsinki, Finland							
Person Roles	submitter							
Protocol Name	P-GSE42833-1	P-GSE42833-6	P-GSE42833-3	P-GSE42833-8	P-GSE42833-7	P-GSE42833-2	P-GSE42833-4	P-GSE42833-5
Protocol Description	ID_REF =  VALUE = normalized log2 intensities	Labeled samples were combined pairwise, fragmented and hybridised to subarrays of tobacco 4x44K 60mer oligo array (Agilent, design ID 021113) according to instructions of Gene expression hybridisation kit (Agilent). During hybridisation, the array was rotated at 10rpm at +65C for 17h. Slide was washed with Gene expression wash buffers 1 and 2 (Agilent) with triton-X102 added.	Nicotiana tabacum cv. Samsun nn were grown from seeds and planted in soil prepared by mixing peat (KekkilM-CM-$ Horticulture Peat, KekkilM-CM-$ Oyj, Finland) and washed sand at 5:1 ratio (v/v) . Plants were grown in growth rooms (photoperiod 16 h, temperature 18/22M-0C, relative humidity 70%; light intensity 200 M-NM-<E mM-bM-^@M-^S2sM-bM-^@M-^S1; or temperature 20/24M-0C, relative humidity 40%, light intensity 250 M-NM-<E mM-bM-^@M-^S2sM-bM-^@M-^S1), with. fertilizer (N:P:K = 16:9:22, Yara, Espoo, Finland) mixed in water (0.3g/l) and given in every watering.	Fluorescence intensities were normalized using quantile normalization function. Differences in log-intensities were tested with moderated t-test and the resulting p-values were transferred to FDR-values using limma R/Bioconductor package (Smyth, 2004). To annotate the probe targets, probe sequences were blasted against tobacco unigene database (SOL genomics network, http://solgenomics.net/) using BLAST+ command-line applications (National Institutes of Health). The log2 mutant/wt data and additional annotations for all of the Samples combined are linked on the Series record.	The signals were scanned using GenePix 4200 AL microarray scanner and image analysis was done using GenePixPro 6.1 software (both Molecular devices (Axon), Sunnyvale, CA).	Tobacco plants were sap-inoculated with sap from healthy, mutPVA or wtPVA-infected N. benthamiana leaves. Samples were collected at 12 days post inoculation from the first systemically infected leaves and frozen in liquid nitrogen.	Leaf samples were ground with mortal and pestle in liquid nitrogen. RNA was extracted using home-made Trizol (Caldo et al., 2004, Plant Cell 16, 2514M-bM-^@M-^S2528) and purifed with LiCl precipitation. Equal amounts of RNA from 6 plants were pooled and further purified with Rneasy plant mini kit (Qiagen, Hilden, Germany) and quality of total RNA was assessed with Bioanalyzer (Agilent). mRNA was captured with oligo(dT)-primers, first and second strands of cDNA synthesized and resulting cDNA transcribed to antisense RNA to incorporate amino allyl-dUTP using Amino allyl MessageAmp kit (Life Technologies, Carlsbad, California). The quality of the resulting RNA was analysed with Bioanalyzer prior to labeling.	20M-NM-<g of the amino-allyl-UTP RNA were labelled with Cy3 or Cy5 (GE Healthcare), and purified (Amino allyl MessageAmp kit, Life Technologies). Incorporation of dyes to samples were assessed with Nanodrop. 825M-NM-<g of each labeled RNA was used in hybridisation.
Protocol Type	bioassay_data_transformation	hybridization	grow	feature_extraction	image_aquisition	specified_biomaterial_action	nucleic_acid_extraction	labeling
Experimental Factor Name	TREATMENT							
Experimental Factor Type	treatment							
Publication Title	Mutation of a Short Variable Region in HCpro Protein of Potato Virus A Affects Interactions with a Microtubule-Associated Protein and Induces Necrotic Responses in Tobacco.							
Publication Author List	Haikonen T, RajamM-oM-?M-=ki ML, Tian YP, Valkonen JP							
PubMed ID	23514111							
Publication DOI	10.1094/MPMI-01-13-0024-R							
Comment[SecondaryAccession]	GSE42833							
Comment[GEOReleaseDate]	2013-03-23							
Comment[ArrayExpressSubmissionDate]	2012-12-10							
Comment[GEOLastUpdateDate]	2013-03-24							
Comment[AEExperimentType]	transcription profiling by array							
Comment[AdditionalFile:Data1]	GSE42833_Haikonen_FC-values_annotations.txt							
SDRF File	E-GEOD-42833.sdrf.txt