Plantlets were carefully removed from slant agar plates and placed in 13 mL tubes wrapped with aluminium foil that were cut off at the 8 mL mark, using 20 plantlets per tube (Sarstedt, Nürnbrecht, Germany). Each tube contained 5 mL of the following solutions: sulphated Myc-LCO solution (half-strength Hoagland´s solution of pH 6.5 containing 10-8 M sulphated Myc-LCOs), non-sulphated Myc-LCO solution (half-strength Hoagland´s solution of pH 6.5 containing 10-7 M non-sulphated Myc-LCOs), mixed sulphated and non-sulphated Myc-LCO solution (half-strength Hoagland´s solution of pH 6.5 containing 10-8 M sulphated and 10-7 M non-sulphated Myc-LCOs), Myc control solution (half-strength Hoagland´s solution of pH 6.5), Nod-LCO solution (NH-mix of pH 7.5 containing 10-8 M Nod-LCOs), and Nod control solution (NH-mix of pH 7.5). After 6 h of incubation in the climate chamber, 10 plantlets per batch were removed from the treatment or control solutions and harvested, while the other 10 remained in the respective solutions for a total of 24 h.
Growth protocol
Medicago truncatula Gaertn cv Jemalong genotype A17 seeds were surface-sterilized and scarified. Plates with M. truncatula seeds were incubated for 24 hours in the climate chamber (humidity: 70 %; photosynthetic photon flux: 150 μmol m-2 s-1) at a 16 h light (23 °C) and 8 h dark (18 °C) regime. Subsequently, seedlings were moisturized with sterile water (pH 7.0) to allow careful removal of seed coats. In each case, eight seedlings were put on the upper quarter of a 2.5 % (w/v) phytoagar (Duchefa Biochemie, Haarlem, The Netherlands) slant plate (120 x 120 mm; DoctorLab, Jena, Germany). Seedlings used for treatment with Myc-LCOs and corresponding controls were put on plates with half-strength Hoagland´s solution (Arnon and Hoagland, 1940) set to pH 6.5 with KOH, whereas seedlings treated with Nod-LCOs and corresponding controls were grown on slant agar plates with an N-free nutrient solution of pH 7.5 as described in Baier et al. (2007), further on referred to as NH-mix. An approximately one cm strap of Whatman paper (Schleicher & Schuell, Dassel, Germany) soaked with appropriate nutrient solutions as described above was used to fix the seedlings on the plates beneath the cotyledons. Three plates at a time were sealed with transparent wrapping foil, which was cut at the upper side to allow gas exchange. The lower half of each plate stack was wrapped with aluminium foil for light protection to allow normal root development (Figure 1 E). Each triple pack of plates was placed in an approximately 70° angle in the climate chamber for five days.
Extracted molecule
total RNA
Extraction protocol
Ten root fragments per biological relicate were pooled and ground using lysing matrix D tubes (MP Biomedicals, Illkirch, France) in a FastPrep (MP Biomedicals, Illkirch, France) prior to RNA extraction. Total RNA isolation and DNaseI on-column digestion was performed via RNeasy Mini kits (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA preparations were quality-checked both via spectrophotometry (NanoDrop ND-1000, Peqlab, Erlangen, Germany) and capillary electrophoresis (RNA Nano chips, Agilent Bioanalyzer, Agilent, Böblingen, Germany), as recommended by the manufacturers.
Label
biotin
Label protocol
RNA was processed for use on Affymetrix (Santa Clara, CA, USA) GeneChip Medicago Genome Arrays, according to the manufacturer’s GeneChip 3’ IVT Express kit user manual. Briefly, 100 ng of total RNA with by a RIN number (Agilent 2100 Bioanalyzer, Agilent, Böblingen, Germany) of at least 9.0 containing spiked-in poly-A+ RNA controls was used in a reverse transcription reaction (GeneChip 3’ IVT Express Kit; Affymetrix) to generate first-strand cDNA. After secondstrand synthesis, double-stranded cDNA was used in a 16 h in vitro transcription (IVT) reaction to generate aRNA (GeneChip 3’ IVT Express Kit; Affymetrix). Size distribution of in vitro transcribed aRNA and fragmented aRNA, respectively, was assessed via an Agilent 2100 Bioanalyzer, using an RNA 6000 Nano Assay.
Hybridization protocol
12.5 ug of fragmented aRNA was added to a 250 ul cocktail also containing hybridization controls. 200 ul of the mixture was hybridized on GeneChips for 16 h at 45C. Standard post hybridization wash and double-stain protocols (FS450_0001; GeneChip HWS kit; Affymetrix, Santa Clara, CA, USA) were used on an Affymetrix GeneChip Fluidics Station 450.
Scan protocol
GeneChips were scanned on an Affymetrix GeneChip scanner 3000 7G.
Description
Gene expression data from Medicago truncatula plantlet roots treated with symbiotic LCOs
Data processing
Cel files obtained from Medicago GeneChip hybridizations were analysed using the Robin software (http://mapman.gabipd.org/web/guest/robin). Normalization was performed using the Robust Multichip Average (RMA) algorithm. Intensity values calculated for each probe set were log2-transformed and averaged across all three biological replicates. Log2 differences between the conditions studied were evaluated statistically via Students t-tests implemented in Robin.
