Institut National de la Recherche Agronomique (INRA Nancy, France)
Treatment protocol
Seedlings of hazelnut tree inoculated with T. melanosporum were produced by ROBIN PEPINIERES SARL (France). For each biological replicate, 5 ECM root tips were collected 4 months after fungal inoculation. After harvesting, ECM root tips were immediately fixed on ice in Farmer’s solution containing 75% (v/v) ethanol and 25% (v/v) acetic acid. The fixative Farmer’s solution was 3 times 5 min vacuum-infiltrated (400 mm Hg) and then let in contact with the ECM root tips overnight at 4°C. After Farmer’s solution removal, samples were dehydrated in graded series of ethanol (45 min each on ice (v/v) 75%, 80%, 90% and 3 times in absolute ethanol), followed by ethanol:otix+ (Microm Microtech, Francheville, France) series (45 min each on ice (v/v) 75:25%, 50:50%, 25:75% and 3 times in 100% Otix+). Samples were then transferred in pre-warmed 37°C melting point wax (VWR, Fontenay-sous-Bois, France) 5 times and then embedded at 58°C in paraffin (Diapath, Martinengo, Italy). Fifteen μm-sections were cut on a rotary microtome (Microm Microtech, Francheville, France) and transferred onto a microscope membrane-slide (Zeiss, Bernried, Germany) without water bath impregnation of paraffin ribbons. In order to improve adhesion between microscope slide and ribbons, slides were kept at 45°C during 1 hour to spread out ribbons and stored at -20°C under dehydrating conditions as previously reported. Just before LCM, slides were deparaffinized once in Otix+ for 10 min and then in otix+:ethanol series (3 min each (v/v) 67:33%, 33:67% and 2 times in absolute ethanol).For each biological replicate, 80 transversal sections obtained from 5 ECM root tips were analysed. For each section, three areas corresponding to soil fungal interface (i.e. mantle, M), plant fungal interface (i.e. Hartig net, HN) and internal root cells (mainly containing vascular cells, VC) were isolated using LCM. A PALM® Robot-Microbeam system (P.A.L.M. Microlaser Technologies AG, Bernried, Germany) was used to microdissect cells from deparaffinized and dried tissue sections. For each of the three biological replicates, the three microdissected areas were collected in distinct adhesive caps of 500 μl tubes (Zeiss) for further nucleic acids isolation.
Extracted molecule
total RNA
Extraction protocol
Total RNA from microdissected samples were isolated using the PicoPure RNA isolation kit (Arcturus, Mountain View, CA, USA). A DNase treatment (Qiagen) was included during RNA isolation procedure to remove traces of genomic DNA. Elution was performed with 12 μl of pre-warmed DEPC Water (65°C). Quality and quantity of RNA samples were assessed using the Bio-Rad Experion analyzer and Experion RNA High-sens analysis kit (Bio-Rad). Total RNA from each sample was subjected to two rounds of amplification using the MessageAmp™ II aRNA amplification kit (Ambion, Austin, USA) following manufacturer’s instructions. For each sample, amplified RNA (aRNA) profiles were verified using the Experion analyzer and Experion RNA Standard-sens analysis kit (Bio-Rad). Double-stranded cDNA were synthesized from 3 μg of aRNA using the Superscript™ Double-Stranded cDNA Synthesis Kit (Life Technologies SAS, Saint Aubin, France).
Label
Cy3
Label protocol
Labelling by NimbleGen Company
Hybridization protocol
Hybridization by NimbleGen Company
Scan protocol
Scanning by NimbleGen Company
Data processing
Expression values reflect across-array quantile normalization and gene calls generated using the Robust Multichip Average (RMA). For information on RMA see Biostatistics 2003 Apr; 4(2): 249-264
Laser capture microdissection and microarray analysis of ectomycorrhizas formed by the ascomycete Tuber melanosporum reveal functional heterogeneity between mantle and Hartig net compartments
Data table header descriptions
ID_REF
VALUE
Gene expression value generated using the Robust multichip average (RMA) algorithm and quantile normalisation
SE_EXPRS
Coefficient of variation for the gene expression value
