tissue: Root nodule culture condition: Cultivated without nitrogen, with 25 µM 1-NOA treatment: Inoculated with symbiotic Frankia bacteria in presence of 25µM 1-NOA developmental stage: Root nodule, 21 days regarding inoculation time
Treatment protocol
The starting point of the treatment was the day of inoculation of plant with the bacteria (0d). At 0d, a set of plants was inoculated with the bacteria in the BD medium without nitrogen (control condition) and one set of plants was inoculated with the bacteria in the BD medium without nitrogen containing 25 µM of 1-naphtoxy acetic acid (1-NOA), which is an auxin influx inhibitor (treated condition). Medium were replaced twice a week with freshly prepared medium (with or without 1-NOA according to treatment). 21 days after inoculation, nodules (specific organs that develop in symbiosis) were sampled of control and treated plants. All samples were frozen in liquid nitrogen and conserved at -80°C before RNA extraction
Growth protocol
Seeds were grown for one month in a soil/vermiculite substrate (1:1, vol/vol) in a greenhouse. Plantlets were transferred to a Broughton and Dilworth's modified liquid medium (BD medium) with nitrogen in plastic pots and grown for 3 weeks under these hydroponic conditions. Then they were grown for one week without nitrogen. After this starvation step which is necessary for optimal induction of symbiosis, plants were sub-divided according to the treatment.
Extracted molecule
total RNA
Extraction protocol
Total RNA was purified from sampled nodules by ultracentrifugation (Chirgwin et al., 1979). Residual DNA was removed from RNA samples using the Turbo DNA free kit (Ambion, USA), quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Courtaboeuf, France) and qualitatively assessed using a Bioanalyser 2100 (Agilent p/n G2938A) according to the manufacturer’s instructions (Agilent, Santa Clara, CA, USA).
Label
Cy3
Label protocol
Five hundred nanograms of RNA and Agilent One-Color RNA Spike-In RNA were labeled with the Agilent Low RNA Input Linear Amplification Kit PLUS, One-Color according to the manufacturer's instructions as follows: 1.2 µl of T7 Promoter Primer was added to 500 ng of RNA and 5 µl of spike-in control (in a 11.5-µl volume) and denatured at 65°C. First-Strand Buffer (to 1x), DTT (to 10 mM), dNTP (to 0.5 mM), Moloney murine leukemia virus (MMLV) reverse transcriptase (1 µl of stock provided in the kit in a 20-µl reaction), and RNaseOut (0.5 µl of stock provided in the kit in a 20-µl reaction) were added. The cDNA was synthesized during the following incubation step (2 h at 40°C). After 10-min denaturation at 65°C and the addition of Cy3-labeled CTP (to 0.3 mM), Transcription Buffer (to 1x), DTT (to 10 mM), NTP (8 µl of stock provided in the kit in a 80-µl reaction), RNaseOUT (0.5 µl of stock provided in the kit in a 80-µl reaction), inorganic phosphatase (0.6 µl of stock provided in the kit in a 80-µl reaction), and T7 RNA polymerase (0.8 µl of stock provided in the kit in a 80-µl reaction), the synthesis of the fluorescent labeled cRNA was performed during the second incubation step (2 h at 40°C). The labeled cRNA was purified with the RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol.
Hybridization protocol
The Agilent Hybridization Kit (catalog no. 5188-5242) was used . 600 ng of the labeled sample RNA was used for hybridization according to the Agilent One-Color Microarray-Based Gene Expression Analysis Protocol. The hybridization was performed for 17 h at 65°C. Slides were them washed for 1 min at room temperature in Wash Solution 1 (catalog no. 5188-5325) and 1 min at room temperature in Wash Solution 2, prewarmed to 37°C (catalog no. 5188-5326).
Scan protocol
Scanning was performed with the Agilent scanner (G2505B) using default parameters for 8 x 15 K formats and the one-color protocol. (scan area 61 X 21.6 mm; scan resolution 5µm; single pass; dye channel is set to green and green PMT is set to 100%).
Description
Gene expression in nodules sampled 21 days after inoculation with the bacteria Frankia on plants cultivated without nitrogen and with 25 µM 1-NOA
Data processing
Data were extracted with Feature Extraction 9.1 software (Agilent Technologies) according to the 1-color Agilent protocol (GE1-v5_95_Feb protocol). This software reads and processes microarray image files to prepare them for analysis. Feature Extraction automatically assigns a grid template and a protocol, based on the barcode of the slide. It determines feature intensities, rejects outliers and calculates statistical confidence intervals. Genespring sofware was then used for data processing. Normalization was done as follows: data transformation (Set measurements less than 5.0 to 5.0), per chip : normalized to 50th percentile (probes with presence flags only), per gene : normalized to median. Data were then filtered using flag parameter (Flags (Absent, Marginal, Present) : All control -> Absent, Saturated spot -> Marginal, Spot not uniform -> Marginal, Spot not positive and not significant -> Absent, Spot not above background -> Absent, Population outlier -> Marginal) and each probe must be present for 2/3 of the tested conditions.
Gene profiling during symbiosis between the actinorhizal tree Casuarina glauca and the actinobacteria Frankia CcI3 after treatment with 1-naphtoxy acetic acid, an auxin influx inhibitor.
