Investigation Title	Transcription profiling of ectomycorrhizal fungus Paxillus involutus grown with different sources of nitrogen representing various degrees of complexity
Comment[Submitted Name]	FHIG DW2 PaxBet Mcr
Experimental Design	loop_design	dye_swap_design	co-expression_design	growth_condition_design	transcription profiling by array
Experimental Design Term Source REF		mo	mo	mo	EFO
Comment[ArrayExpressReleaseDate]	2008-06-01
Comment[AEMIAMESCORE]	3
Comment[ArrayExpressAccession]	E-MEXP-1257
Comment[MAGETAB TimeStamp_Version]	2011-06-28 19:00:16 Last Changed Rev: 14857
Experimental Factor Name	Nitrogen source
Experimental Factor Type	nutrients
Experimental Factor Term Source REF
Person Last Name	Johansson
Person First Name	Tomas
Person Mid Initials	-
Person Email	tomas.johansson@mbioekol.lu.se
Person Phone	+46 46 2224549
Person Fax
Person Address	Ecology Building
Person Affiliation	Microbial Ecology
Person Roles	submitter
Person Roles Term Source REF
Quality Control Type
Quality Control Term Source REF
Replicate Type
Replicate Term Source REF
Normalization Type
Normalization Term Source REF
Date of Experiment
Public Release Date	2008-06-01
PubMed ID
Publication DOI
Publication Author List	Tomas Johansson, Derek P. Wright, Antoine Le Quéré, Carl Troein, Carsten Pettersson, and Anders Tunlid
Publication Title	Nutrient assimilation by the ectomycorrhizal fungus Paxillus involutus – Transcriptional responses in the extrametrical mycelium due to complexity of substrate
Publication Status
Publication Status Term Source REF
Experiment Description	A microarray analysis to investigate transcriptional responses in the ectomycorrhizal fungus Paxillus involutus, after providing various sources of nitrogen. Five different sources of nitrogen representing various degrees of complexity were provided as patches in peat microcosms for fungal ingrowth: ammonium phosphate (APO), ammonium sulphate (ASU), glutamine (GLN), bovine serum albumin (BSA), and chitin (CHI). After fungal establishment of patches total RNA was isolated and used for global transcriptional analyses using cDNA microarrays. The entire design involved 9 slides (in the range DW2_01--DW2_13), 14 labelled extracts, and 3 biological replicates (R1-R3). The experiment was designed as a loop combining the 5 diffrent samples and including dye-swaps and biological replication.  COMMENT: In our analysis of the raw dataset, 3 channels were removed due to poor signal quality: array 12707648b, Cy5 (635nm) channel (GLN R1 sample); array 12707650c, Cy5 (635nm) channel (APO R1 sample); array 12707651b, Cy5 (635nm) channel (APO R1 sample).
Protocol Name	P-MEXP-89960	P-MEXP-5516	P-MEXP-89961	P-MEXP-5508	P-MEXP-5509	P-MEXP-5510	P-MEXP-5511
Protocol Type	grow	specified_biomaterial_action	nucleic_acid_extraction	labeling	labeling	hybridization	image_acquisition
Protocol Description	Stocks of Paxillus involutus (Batsch) Fr. (ATCC 200175) were maintained aseptically on agar containing modified Melin-Norkrans (MMN) medium. The complete MMN medium contained: (all as mM) glucose (13.9), KH2PO4 (3.67), (NH4)2HPO4 (1.89), MgSO4 7H2O (0.61), NaCl (0.43), CaCl2 (0.34), FeCl3 6H2O (0.044) and 3 µM thiamine hydrochloride. Birch (Betula pendula Roth.) seeds (Domsjöänget, Sweden) were stored at -20°C until use. An ECM association between birch and P. involutus was synthesized using the cellophane-agar Petri-dish system (Brun et al., 1995). Seeds of birch were surface-sterilized in 245 mM calcium hypochlorite for 60 min, washed with four changes of sterile distilled water (250 ml), each of 15 min, then transferred aseptically to water agar plates (7 g L-1) until germination occurred. Nine day old seedlings were aseptically transferred to the edge of 9 day old colonies of P. involutus growing on sheets of autoclaved cellophane (Erik S Ekman AB, Stockholm, Sweden) placed over MMN agar containing 5.55 mM glucose. The plates were maintained in a growth chamber (S10H, Conviron, Winnipeg, Canada) at 22/15C day/night temperature with a 16 h photoperiod (70 µmol m-2 s-1 photon flux density). After four weeks, by which time inoculated plates supported heavily mycorrhizal seedlings, mycorrhizal seedlings were transferred to pots (392 cm3, 4 seedlings per pot) containing unamended sphagnum peat (pH 3.5 – 4.5, Hasselfors Naturtorv Original Solmull, Hasselfors Garden AB, Hasselfors, Sweden) to which four plugs of P. involutus mycelium were added. These pots were enclosed within a propagator, to maintain high humidity, and placed in a growth chamber at 18/15C day/night temperature with a 16 h photoperiod (130 µmol m-2 s-1 photon flux density) and 80% relative humidity for four weeks. During this period three additions of an Ingestad nutrient solution were supplied to each pot. Each nutrient addition consisted of 4 ml of a solution comprising 0.1 ml L-1 Ingestad stock A and 0.1 ml L-1 Ingestad stock B (Nylund & Wallander, 1989). Plants were otherwise irrigated with distilled water. Individual mycorrhizal plants were transferred to Perspex microcosms (20 x 20 x 1 cm) containing a 5 mm layer of sieved (2 mm mesh) unamended sphagnum peat. Perspex blocks (1 x 1 x 1 cm), placed at the edges of the microcosm, supported the upper Perspex sheet of the microcosm, maintaining an air-space of 5 mm above the surface of the peat. Each microcosm was wrapped in aluminium foil to obscure light from the root system and a polythene bag placed loosely over the microcosm to maintain humidity and returned to the growth chamber. Each microcosm received a weekly addition of 3 ml Ingestad nutrient solution (0.1 ml L-1 stock A and 0.1 ml L-1 stock B). After approximately three weeks, once the extramatrical mycelium had colonized approximately two-thirds of the microcosm, two nutrient patches were placed into each chamber in advance (1 cm) of the mycelial front. Each nutrient patch consisted of a shallow plastic dish (3 x 3 x 0.5 cm) filled with autoclaved fine quartz sand (Sigma-Aldrich Sweden AB, Stockholm, Sweden) to which 1.5 ml 1mM (NH4)2SO4 was applied initially. Subsequently, either 1mM (NH4)2SO4, or 1mM (NH4)2PO4, or 1mM glutamine, or 0.1g BSA or 0.1g chitin was applied weekly for four weeks to allow for extensive hyphal development within the patch. Harvesting occurred within a 3 h period, approximately 5 h into the photoperiod and within 24 h of an nutrient application to each nutrient patch. Mycelium was harvested from within the nutrient patch using chilled forceps. In order to obtain sufficient sample for total RNA extraction, material of each tissue type was pooled from three microcosms to produce one biological replicate. All material were immediately frozen in liquid N2 and stored at -80C until use. 	See Growth condition protocol description	Approximately 50 mg frozen tissue sample mycelium from each nutrient patch was transferred to a microfuge tube containing a 3 mm-tungsten carbide bead (Qiagen GmbH, Hilden, Germany), which was pre-chilled in dry ice in a -80C freezer. The tubes were inserted into a polypropylene tube holder, pre-chilled at -80C, for a bead mill (Type MM2, Retsch GmbH, Haan, Germany) to grind the samples for 2 x 90 s. The tube holder and samples were returned to the -80C freezer for 5 min between each grinding run. Total RNA was immediately isolated from each sample using the RNeasy Plant Mini Kit (Qiagen) according to the manufacturer’s instructions except that PEG6000 (20 mg mL-1) and â-mercaptoethanol (10 µl L-1) were added to the RLC buffer. Total RNA was eluted in 60 ul H2O and stored at -80C until use. Antisense RNA (aRNA) was synthesized by in vitro T7 polymerase transcription using two rounds of amplification with the MessageAmp aRNA kit (Ambion (Europe) Ltd, Huntingdon, Cambridgeshire, UK) according to the manufacturer’s instructions. The aRNA was quantified spectrophotometrically.	For dual color labeling (Cy3 or Cy5), a total of 1 µg aRNA for each target was used, including exogenous ArrayControl spikes (Ambion), using the CyScribe post-labeling kit (Amersham Biosciences, Uppsala, Sweden). After cDNA synthesis and labeling, targets were finally purified using the QIAquick PCR Purification kit (Qiagen). After purification, the Cy3- and Cy5-labeled targets were combined in an amber microfuge tube (Eppendorf AG, Hamburg, Germany) and after evaporation labeled cDNA was dissolved in 6 µL H2O.	For dual color labeling (Cy3 or Cy5), a total of 1 µg aRNA for each target was used, including exogenous ArrayControl spikes (Ambion), using the CyScribe post-labeling kit (Amersham Biosciences, Uppsala, Sweden). After cDNA synthesis and labeling, targets were finally purified using the QIAquick PCR Purification kit (Qiagen). After purification, the Cy3- and Cy5-labeled targets were combined in an amber microfuge tube (Eppendorf AG, Hamburg, Germany) and after evaporation labeled cDNA was dissolved in 6 µL H2O.	After denaturation of the labelled target (6 µL) at 95°C for 2 min and a 30-s incubation on ice, 1.5 µL of dT80 (1 mg/ml) was added followed by incubation at 75°C for 45 min. cDNA was mixed with an equal volume of hybridization buffer (CyScribe post-labeling kit, Amersham Biosciences) and then an equal volume (15 µL) of formamide was added. The hybridization mixture was pipetted onto the slide and sealed under a LifterSlip coverslip (Eire Scientific Company, Portsmouth, NH, U.S.A.). Slides were placed in humidified CMT hybridization chambers (Corning) and incubated in a hybridization oven at 42°C for 18 h. After hybridization the slides were washed twice (1 min, 5 min) with 2 x SSC and 0.1% SDS (42°C), once (10 min) with 0.1 x SSC and 0.1% SDS (20°C), three times (15 s, 2 min, 1min) with 0.1 x SSC (20°C), and once (10 s) with 0.01 x SSC (20°C). The slides were dried by a short centrifugation and stored in a dry, dark chamber prior to scanning.	Fluorescence intensities were measured using an Axon 4000A laser scanner followed by conversion of images into digital values using GenePix Pro software (3.0.6.89) (Axon Laboratories, Union City, CA, U.S.A.). Data images were inspected manually and low-quality spots were flagged.
Protocol Parameters			Amplification;Extracted product;	Label used;Amplification;Amount of nucleic acid labeled;	Amplification;Label used;Amount of nucleic acid labeled;	Volume;time;Quantity of label target used;Chamber type;temperature;
Protocol Hardware							Axon GenePix 4000A scanning hardware
Protocol Software							Default scanner software
Protocol Contact
Protocol Term Source REF							mo
SDRF File	E-MEXP-1257.sdrf.txt
Term Source Name	mo		ArrayExpress	mo	EFO
Term Source File	http://mged.sourceforge.net/ontologies/MGEDontology.php		http://www.ebi.ac.uk/arrayexpress	http://mged.sourceforge.net/ontologies/MGEDontology.php	http://www.ebi.ac.uk/efo/
Term Source Version