Hybridized slides were scanned on a GenePix 4100A scanner (Axon Instruments, Inc.). Images were acquired at 10-µm resolution, and the background-subtracted median spot intensities were determined using GenePix Pro 5.1 image analysis software (Axon Instruments, Inc.). Spots with anomalies were discarded and excluded from further analysis.
(Parameters: Scanning hardware = GenePix Personal 4100A [Axon Instruments], Scanning software = GenePix 4100A [Axon Instruments])
To identify reliable rhizosphere differentially expressed genes by Pseudomonas putida, populations of P. putida KT2440 previously exposed to a rhizospheric life style for seven days in the rhizosphere of corn (Zea mays var. Girona) were compared with populations previously exposed to a rhizospheric life style for a long period of 138 days (clone 6.3).
Corn seeds were surface-sterilized by rinsing with sterile deionized water, washing for 10 minutes with 70% (vol/vol) ethanol, then for 10 minutes with 20% (vol/vol) bleach two times, and followed by thorough rinsing with sterile deionized water. Surface sterilized seeds were pregerminated on MS medium (Murashige, T, & Skoog, F. 1962, Physiol Plant 15, 473) containing 0.2% (wt:vol) phytagel (Sigma P8169) and 0.5% (wt:vol) glucose at 30ºC in dark for 48 h.
Overnight bacterial cultures of Pseudomonas putida KT2440 were diluted in M9 salts (Sambrook et al., 1989) to an optical density at 600 nm of 1, and 10 ?l of the suspension (about 5,000,000 CFU/mL) were added to 10 ml of M9 and poured on tubes containing 40 g of sterile water-washed silica sand, where germinated seeds were then sown. Inoculated plants were maintained in a controlled chamber at 24°C and 55 to 65% humidity with a daily light period of 16 h.
(Parameters: start time = 7, stop time = 138, time unit = days, min temperature = 24, temperature unit = C)
Dried aminoallyl-labeled cDNA was resuspended in 9 µl of 0.1 M sodium carbonate buffer (pH 9.0), mixed with either Cy3 (KT2440-Smr) or Cy5 (clone 6.3) fluorescent dyes (mono-reactive NHS-esters; Amersham Biosciences, Cat No. PA23001 and PA25001, respectively), and allowed to couple for 2 h at room temperature in the dark. After coupling, the reaction was quenched with 4.5 µl of 4 M hydroxylamine for 15 min. Finally labeled cDNA probes were again purified with QIAquick PCR purification columns. Labeling efficiency was assessed using a NanoDrop ND1000 spectrophotometer (NanoDrop Technologies).
(Parameters: Mass unit = Micro gram, Amplification = none)
Total RNA was extracted by using TRI Reagent (Ambion, ref. 9738) as recommended by the manufacturer except that Tripure Isolation reagent was preheated at 70°C followed by treatment with RNase-free DNase I (10 U) (Roche) plus RNaseOUT (40 U) (Invitrogen) and purification with RNeasy columns (QIAGEN, cat no. 74104). RNA concentration was determined spectrophotometrically and its integrity was assessed by agarose gel eletrophoresis. Six extracts from the rhizosphere of six different corn plants were pooled and 25 µg of total RNA were primed with 7.5 µg of pd(N)6 random hexamers (Amersham, Cat. No. 27-2166-01).
cDNA synthesis was performed by overnight incubation at 42°C in a 30-µl reaction volume containing 0.5 mM (each) dATP, dCTP, and dGTP; 0.25 mM (each) dTTP and aminoallyl-dUTP (aa-dUTP; Sigma Cat. No. A0410); 10 mM dithiothreitol; and 400 U of SuperScript II reverse transcriptase (RT) (Invitrogen, ref. 18064-014) in RT reaction buffer. The reaction was stopped by adding 10 µl of 50 mM EDTA and the RNA template was hydrolyzed with the addition of 10 µl of 1 N NaOH followed by incubation at 65 ºC for 15 min. Samples were then neutralized by adding 25 µl of 1M HEPES (pH 7.5) and the hydrolyzed RNA and residual dNTPs were removed using QIAquick PCR purification columns (QIAGEN, ref. 28104) according to the manufacturer's recommendations, except that the Tris-based elution buffer (EB) supplied with the kit was replaced with phosphate elution buffer (4 mM KPO4 pH 8.5) to avoid interferences with the subsequent labeling. cDNA concentrations were measured by spectrophotometry (nanodrop). cDNA samples were dried in a Speed-Vac to completion.
(Parameters: Extracted product = total_RNA, Amplification = OTHER: Reverse transcriptase)
Plants were collected, shoots discarded and the roots placed in 50 ml Sterilin tubes containing 20 ml of M8 salts (Sambrook et al., 1989, Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) and 4 g of glass beads (diameter, 3 mm). Tubes were vortexed for 2 minutes, left standing for 15 seconds and cells from bacterial suspensions collected by centrifugation for 8 min at 6,700 x g (4ºC) in tubes precooled in liquid nitrogen. Pellets were immediately frozen in liquid nitrogen and conserved at -80 ºC.
Prior to the hybridization process, the microarray slides were blocked by immersion into 5 x SSC (1x SSC is 0.15 M NaCl; 15 mM sodium citrate, pH 7), 0.1% (wt/vol) SDS, 1% (wt/vol) bovine serum albumin for 1 h at 42 ºC. Then, the slides were washed by two successive immersions in MilliQ water at room temperature, followed by a final wash with isopropanol. The slides were spin-dried by centrifugation at 1,500 x g for 5 min, and used within the next hour. Equal amounts of Cy3- and Cy5-labeled cDNAs, one of them corresponding to the control and the other one to the problem to be analyzed, were mixed, dried in a Speed-Vac and reconstituted in 35 µl of hybridization buffer (5x SSC, 25% [vol/vol] formamide, 0.5% [wt/vol] SDS, 5x Denhardt’s solution, 5% [wt/vol] dextransulfate) preheated to 42ºC. The labeled probe was denatured at 98°C for 3 min, applied onto the microarray slide and covered with a glass coverslip. The slide was then introduced in a humidified hybridization chamber (AHC ArrayIt Hybridization Cassette; Telechem International, Inc.) and incubated for 18 to 20 h in a water bath at 42°C, preserved from light. Following hybridization, the microarrays were washed by gentle agitation in 2x SSC, 0.1% [wt/vol] SDS at 42 ºC for 5 min, followed by a 5-min wash at room temperature in 1x SSC, two 5-min washes in 0.2x SSC, and a final 5-min wash in 0.1x SSC. Finally, the slides were spin-dried in a centrifuge at 1,500 x g for 5 min, and scanned.
(Parameters: Chamber type = OTHER: AHC ArrayIt Hybridization Cassette, Quantity of label target used = 6, Mass unit = Micro gram, time = 18, Tiny time unit = hours, Volume = 35, Volume unit = Nano litre, temperature = 42)