Investigation Title	Transcription profiling of M.graminicola between growth in vitro and late-stage infection in planta	
Comment[Submitted Name]	Rothamsted-M.graminicola-Expt2-PDB v. 28d Infected Plant	
Experimental Design	cell_type_comparison_design	transcription profiling by array	
Experimental Design Term Source REF	mo	EFO	
Comment[ArrayExpressReleaseDate]	2006-02-28	
Comment[AEMIAMESCORE]	4	
Comment[ArrayExpressAccession]	E-MEXP-523	
Comment[MAGETAB TimeStamp_Version]	2010-08-11 15:12:37 Last Changed Rev: 13058	
Experimental Factor Name	cell type	
Experimental Factor Type	cell_type	
Experimental Factor Term Source REF	
Person Last Name	Hammond-Kosack	Antoniw	Keon	Rudd	Hargreaves	Skinner	
Person First Name	Kim	John	John	Jason	John	Wendy	
Person Mid Initials				J	
Person Email			john.keon@bbsrc.ac.uk	
Person Phone			01582 763133	
Person Fax	
Person Address			Harpenden, St Albans, Hertfordshire, AL5 2JQ, United Kingdom	
Person Affiliation	Rothamsted Research	Rothamsted Research	Plant Pathogen Interactions, Rothamsted Research	Rothamsted Research	Rothamsted Research	Rothamsted Research	
Person Roles			submitter	
Person Roles Term Source REF				
Quality Control Type	technical_replicate	
Quality Control Term Source REF	mo	
Replicate Type	
Replicate Term Source REF	
Normalization Type	
Normalization Term Source REF	
Date of Experiment	
Public Release Date	2006-02-28	
PubMed ID	
Publication DOI	
Publication Author List	John Keon; Jason Rudd; John Antoniw; Wendy Skinner; John Hargreaves; Kim Hammond-Kosack	
Publication Title	Metabolic and stress adaptation by Mycosphaerella graminicola during sporulation in its host revealed through microarray transcription profiling	
Publication Status	
Publication Status Term Source REF	
Experiment Description	Comparison of gene expression in M.graminicola between growth in vitro and late-stage infection in planta.	
Protocol Name	P-MEXP-7130	P-MEXP-7132	P-MEXP-7133	P-MEXP-7234	P-MEXP-8015	P-MEXP-15806	P-MEXP-15656	P-MEXP-15655	P-MEXP-15807	P-MEXP-15658	
Protocol Type	grow	grow	pool	pool	nucleic_acid_extraction	nucleic_acid_extraction	labeling	labeling	hybridization	feature_extraction	
Protocol Description	A virulent isolate of M. graminicola (LA951) recovered from the UK wheat cultivar Riband grown in experimental plots at IACR - Long Ashton Research Station was used. The isolate was stored at -80 C in 50% v/v glycerol. Fungal spores for plant and culture inoculation were harvested from 5 day old cultures growing in the budding form on potato dextrose agar plates (Oxoid Ltd., Hampshire, UK) at 17 C under near UV light (Sylvania, F8T5/BLB). <br> For fungal cultures grown in vitro, 100mls of growth medium, Potato Dextrose Broth [PDB](Sigma) was seeded with 4x10<sup>5</sup> cells/ml. Cultures were grown with shaking (220rpm) at 23 C. Tissue was harvested by filtration when the cell density reached c. 5x10<sup>6</sup> cells/ml. This density was achieved in c.72 hrs for cultures grown in PDB. Filtered tissue was stored at -80 C until required. Potato Dextrose Broth was used as a complex nutrient-rich condition typically containing 20g/L glucose and 4g/L potato extract. 	A virulent isolate of M. graminicola (LA951) recovered from the UK wheat cultivar Riband grown in experimental plots at IACR - Long Ashton Research Station was used. The isolate was stored at -80 C in 50% v/v glycerol. Fungal spores for plant and culture inoculation were harvested from 5 day old cultures growing in the budding form on potato dextrose agar plates (Oxoid Ltd., Hampshire, UK) at 17 C under near UV light (Sylvania, F8T5/BLB). To generate late stage plant infected material the second leaf of 17 day old wheat plants (cultivar Riband) was attached, adaxial side up to Perspex sheets, using double sided tape. The leaves were inoculated with 10 ul spore droplets containing 10<sup>5</sup> cells/ml, (5 drops per leaf). Following an initial 72 hour incubation at 100% relative humidity, inoculated plants were incubated at 16 C with a 16hr light period at 88% relative humidity for up to 28 days during symptom development. After 28 days many pycnidia were visible within diseased lesions, and the surrounding leaf tissue was senescent. Infected tissue was excised and stored at -80 C.	Each 100ml fungal culture yielded approximately 3-400mgs w/w after harvesting by filtration. Typically the fungus from 6 flasks was pooled to yield a single biological replicate of c. 1,800mgs which was used for RNA extraction.	Plants were grown in half seed trays with 20 leaves inoculated in one experimental treatment. Typically 18 leaf segments c. 7cm long were harvested and pooled for extraction. 7cm uninfected leaf segments weighed approximately 100mg each, although the weight was lower following infection.	Total RNA was isolated from freeze-dried M. graminicola cells or senescent wheat leaves containing M. graminicola using the TRIZOL procedure (Invitrogen), following the suppliers protocol and incorporating the suggested additional procedures for polysaccharide containing tissues. TRIZOL was used at the ratio of 1ml. per 100mg fresh/weight of tissue. Tissue was disrupted by 4x 1minute bursts using a Polytron over a 20 minute incubation. Further purification of the total RNA was achieved by precipitation from a solution of 4M Lithium Chloride. Total RNA was used for all microarray analyses.	Total RNA was isolated from freeze-dried M. graminicola cells or senescent wheat leaves containing M. graminicola using the TRIZOL procedure (Invitrogen), following the suppliers protocol and incorporating the suggested additional procedures for polysaccharide containing tissues. TRIZOL was used at the ratio of 1ml. per 100mg fresh/weight of tissue. Tissue was disrupted by 4x 1minute bursts using a Polytron over a 20 minute incubation. Further purification of the total RNA was achieved by precipitation from a solution of 4M Lithium Chloride. Total RNA was used for all microarray analyses.	Labelling of total RNA isolated from fungal cultures or from 28 day infected plant material was by indirect incorporation of reactive AlexaFluor dyes into amine-modified cDNA synthesised using Superscript III reverse transcriptase (SuperScript Indirect cDNA Labelling System, Invitrogen), following the suppliers protocols. 20 ug of total RNA was used in each labelling reaction. AlexaFluor 555 (equivalent to Cy3 dye) was used in the green channel and AlexaFluor 647 (equivalent to Cy5 dye) was used in the red channel. Following purification, the labelled probe was reduced to dryness in a centrifugal evaporator.	Labelling of total RNA isolated from fungal cultures or from 28 day infected plant material was by indirect incorporation of reactive AlexaFluor dyes into amine-modified cDNA synthesised using Superscript III reverse transcriptase (SuperScript Indirect cDNA Labelling System, Invitrogen), following the suppliers protocols. 20 ug of total RNA was used in each labelling reaction. AlexaFluor 555 (equivalent to Cy3 dye) was used in the green channel and AlexaFluor 647 (equivalent to Cy5 dye) was used in the red channel. Following purification, the labelled probe was reduced to dryness in a centrifugal evaporator.	Detailed hybridisation and washing conditions were as described at the COGEME website (http://www.cogeme.man.ac.uk). Specifically; Slides were pre-hybridised for 45min. at 42C (5xSSC, 0.1%w/v SDS, 1%w/v BSA) followed by washing in de-ionised water (5x) and briefly in isopropanol before drying in a centrifuge (2,000g for 1min.). Each dried purified probe was dissolved in 20ul of hybridisation buffer (25% formamide, 5xSSC, 0.1%w/v SDS).These were mixed, denatured for 3minutes at 90C and cooled on ice. The 40ul of mixed probe was applied to the slide and covered with a Hybri-slip (SIGMA 22mmx60mm). Slides were incubated overnight at 42C before washing with (1.) 2xSSC, 1%SDS, for 15min. (2.) 1xSSC, 0.2%SDS, for 8min. &amp; (3.) 0.1%SSC, 0.2%SDS, for 5min. (all washes were at room temperature).	The fluorescently labelled slides were scanned using an Axon 4000B scanner with a spot size of 5um. Laser power (PMT) values for each block were initially set by eye and refined using the analysis supplied with the scanner. Maximum PMT values in each channel were set to produce fewer than 5 saturating spots	
Protocol Parameters	media;	media;			Amplification;Extracted product;	Extracted product;Amplification;	Amount of nucleic acid labeled;Label used;Amplification;	Amplification;Amount of nucleic acid labeled;Label used;	Quantity of label target used;Chamber type;temperature;	
Protocol Hardware										Axon- GenePix4000B	
Protocol Software										GenePix [Axon Instruments]	
Protocol Contact	
Protocol Term Source REF	
SDRF File	E-MEXP-523.sdrf.txt	
Term Source Name	ncbitax		ArrayExpress	mo	EFO	
Term Source File	http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html		http://www.ebi.ac.uk/arrayexpress	http://mged.sourceforge.net/ontologies/MGEDontology.php	http://www.ebi.ac.uk/efo/	
Term Source Version