The mycorrhizal fungus Rhizophagus irregularis (ex Glomus intraradices BEG141) was maintained on leek. Leek roots with a mycorhization rate of at least 85% (M value, Trouvelot et al., 1986) were thoroughly washed to remove any trace of soil, cut in 1 cm long pieces and used (50 mg FW/plant) to inoculate the mycorrhized (Myc) plants. Non colonized leek roots were used to inoculate non-mycorrhized (NM) plants
Growth protocol
Plants were grown in 16 h/8 h day/night cycle at of 23/21°C, respectively. Modified Long Ashton solution was used to fertilize the plants every second day.
Extracted molecule
total RNA
Extraction protocol
Colected leaf and root samples were immediately frozen in liquid nitrogen and stored at -80°C. Hundred mg of frozen samples were ground in liquid nitrogen using the Trizol method (Invitrogen, Carlsbad, CA). Total RNAs were purified using the RNeasy Plant kit (Qiagen) according to the manufacturer’s instructions. The genomic DNA was eliminated after treatment with Dnase I for 20 min at 37°C using the DNA-free kit (Ambion, Austin, TX, USA). RNA was checked for purity and degradation by capillary electrophoresis using the Bio-Analyzer Experion (Bio-Rad; RNA Standard Sens kit; RNA StdSens chips). RNA concentration was also determined by spectrometry and only RNAs with an OD260:OD280 ratio of ≥ 1.8 and no discernable degradation were used for microarray experiments.
Label
Cy3
Label protocol
Double-stranded cDNA (ds-cDNA) was synthesized from 10 μg of total RNA using an Invitrogen SuperScript ds-cDNA synthesis kit in the presence of 250 ng random hexamer primers. ds-cDNA was cleaned and labeled in accordance with the Nimblegen Gene Expression Analysis protocol (Nimblegen Systems, Inc., Madison, WI, USA). Briefly, ds-cDNA was incubated with 4 μg RNase A (Promega) at 37°C for 10 min and cleaned using 25:24:1 phenol:chloroform:isoamyl alcohol, followed by ice-cold absolute ethanol precipitation. For Cy3 labeling of cDNA, the Nimblegen One-Color DNA labeling kit was used according to the manufacturer’s guideline detailed in the Gene Expression Analysis protocol (Nimblegen Systems, Inc., Madison, WI, USA). One μg ds-cDNA was incubated for 10 min at 98°C with 2 OD of Cy3-9mer primer. Then, 100 pmol of deoxynucleoside triphosphates and 100U of the Klenow fragment (New England Biolabs, Ipswich, MA, USA) were added and the mix incubated at 37°C for 2h302.5 h. The reaction was stopped by adding 0.1 volume of 0.5 M EDTA, and the labeled ds-cDNA was purified by isopropanol/ethanol precipitation.
Hybridization protocol
Microarrays were hybridized at 38°C during 16 to 18 h with 6 μg of Cy3 labelled ds-cDNA in Nimblegen hybridization buffer/hybridization component A in a hybridization chamber (Hybridization System - Nimblegen Systems, Inc., Madison, WI, USA). Following hybridization, washing was performed using the Nimblegen Wash Buffer kit (Nimblegen Systems, Inc., Madison, WI, USA).
Scan protocol
Slides were scanned at 5 μm/pixel resolution using an Axon GenePix 4000 B scanner (Molecular Devices Corporation, Sunnyvale, CA, USA) piloted by GenePix Pro 6.0 software (Axon).
Description
Leaves NM +S
Data processing
Scanned images (TIFF format) were then imported into NimbleScan software (Nimblegen Systems, Inc., Madison, WI, USA) for grid alignment and expression data analyses. Expression data were normalized through quantile normalization and the Robust Multichip Average (RMA) algorithm included in the NimbleScan software.
