Cells were grown to mid-exponential phase (OD600 of 0.4-0.5) and stationary phase (OD600 of 2) in VMM minimal medium. For harvesting, cultures were rapidly transferred to cold tubes containing one tenth of the culture volume of “stop solution” (10% Tris-HCl-buffered phenol [pH 8] in ethanol), centrifuged for 5 min (10,800 × g; 4°C), the supernatant was decanted, and cells were frozen in liquid nitrogen and stored at −80°C. Nodules from 5 soybean plants were collected for each GeneChip hybridization experiment. Nodules were harvested 21 post inoculation (dpi) and immediately frozen in liquid nitrogen.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated using the hot phenol extraction procedure described previously (Babst et al. 1996). For RNA isolation from symbiotically grown bacteria, approximately 10 frozen nodules were placed in a 2-ml Eppendorf tube, disrupted and homogenized using a TissueLyzer (Qiagen, Valencia, CA) (3 times for 50 sec; 30 Hertz) with a tungsten carbide bead (3 mm; Qiagen) (Pessi et al. MPMI, 2007). RNA integrity was checked by agarose gel electrophoresis. Precipitated RNA (100 µg) was treated with 20 units of RQ1 DNase I (Promega, Madison, USA) for 30 min at 37°C in a reaction volume of 200 µl. SUPERase•InTM (100 units; Ambion,Huntingdon, UK) was included in the reaction to inhibit potential RNase activity. RNA samples were cleaned up with RNeasy spin columns (Qiagen). cDNA was synthesized. according to the Affymetrix antisense genome array protocol for E. coli (http://www.affymetrix.com). For reverse transcription, MMLV reverse transcriptase RNase H minus (Promega, Madison, USA) was used in the supplied reaction buffer. The resulting cDNA was spectrophotometrically quantified and fragmented according to the Affymetrix manual except that the time for fragmentation by DNase I was shortened to 3 min. For control, 200 ng of DNase I-treated cDNA were separated on a 4-to–20% acrylamide gradient gel and stained with SYBR green II (Molecular Probes, Inc., Eugene, OR, USA). Ideally, the fragmented cDNA migrated in a range that corresponded to 50–200 bp of the 50-bp ladder (Fermentas International Inc., Burlington, Canada). RNA was isolated from the roots of soybean plants as described above, and cDNA was fragmented and labeled according to the protocol for free-living bacteria.
Label
biotin
Label protocol
The fragmented cDNA was end-labeled with biotin-ddUTP in a final volume of 50 µl using terminal dexoynucleotidyl transferase (Promega) in combination with the gene chip labeling reagent (Affymetrix). The double amounts of labeling reagent and terminal deoxynucleotide transferase was used for fragmented cDNA from bacteroids. The reaction was incubated for 75 min at 37°C and stopped by the addition of 2 µl of 0.5 M EDTA.
Hybridization protocol
Hybridisation, washing, staining, and scanning were done according to the Affymetrix manual using a gene chip fluidics station 450 (Affymetrix) and a gene chip scanner 3000 (Affymetrix). For hybridization of individual gene chips, 2–2.5 µg of labeled cDNA from culture-grown bacteria, and 6 -7.5 µg of total bacteroid cDNA were used in a total volume of 150 µl hybridization solution, which contained 7% DMSO (Sigma-Aldrich, St. Louis, USA) in addition to the described composition. Hybridization was done overnight at 48°C. The amount of bacteroid cDNA was estimated from the proportion of bacterial:plant RNA in nodules (Bioanalyzer, Agilent technologies, Palo Alto CA).
Scan protocol
Standard Affymetrix procedures
Description
B. japonicum blr1880 mutant grown aerobically in VMM to stationary phase
Data processing
Signal intensities were detected and analyzed using the mas5 algorithm described in the Affymetrix statistical algorithms description document (http://www.affymetrix.com). Normalized signal values from the arrays were then analyzed using Genespring GX 7.3 (Agilent Technologies,CA, USA). For culture-grown bacteria or bacteroids, only those genes which are “present” (i.e., expressed, based on the detection P-value ≤ 0.06) in 80% of all replicates were further considered. For experiments comparing different conditions amongst culture-grown bacteria or bacteroids, each chip was normalized to the median. Values below 0.01 were set to 0.01. For statistical comparisons, the student t-test with a P-value threshold of 0.01 was applied. Genes were considered as differentially expressed if the fold change (FC) was higher than 2, when comparing different conditions amongst culture-grown bacteria or symbiotic bacteria.
