5 protocols
nucleic acid sequencing protocol
Sequenced in next generation sequencing platform SOLiD 4 (Life Technologies) using the ToP Sequencing Kit (1 x50bp).
nucleic acid library construction protocol
The rRNA was depleted using the Microbe Express kit (Ambion®), the libraries for sequencing were prepared using the Whole Transcriptome Analysis kit (Life Technologies).
nucleic acid extraction protocol
Attached bacteria were recovered by vigorous vortexing of roots from 80 plantlets, 3 days after inoculation in Hoagland’s medium followed by centrifugation. Planktonic cells were collected by centrifugation from the hydroponic medium at the same time. The pellet was resuspended in RNA latter (Ambion®) and stored at - 20oC. The total RNA of H. seropedicae cells (~ equivalent to 106 CFU/g of fresh wheat root) was extracted using Trizol (Invitrogen®) and treated with DNAseI (Ambion®) following the manufacturer instructions. The
growth protocol
H. seropedicae growth conditions The H. seropedicae strain SmR1 was grown routinely at 30° C with shaking at 120 rotations per minute (rpm) in NFbHP-malate medium (Klassen et al. 1997) with 20 mM of NH4Cl added (NFbHPN-Malate). Streptomycin was added as needed at the concentrations of 80 μg/mL. The bacterial strains for plant inoculation were pre- cultured overnight in 5 mL of NFbHPN-malate medium containing NH4Cl and antibiotics as needed. The overnight culture was used to inoculate 10mL NFbHPN-malate medium, which was grown as previously described to an OD600 = 1.0. The bacterial cells were collected by quick centrifugation (14.000 rpm for 20 seconds at room temperature) to ensure that the bacteria remained viable. Cell pellets were re-suspended in the same volume of Hoagland’s medium (Hoagland 1950) to a cell density of 107 cells/mL. A volume of 250 μL of this bacterial suspension was added to glass tube containing 25 mL of Hoagland’s medium and the wheat plantlets to give 105 bacteria per mL. Germination, inoculation and growth of plantlets Seeds of Triticum aestivum (cv. CD104) were disinfected as described previously (Dobereiner, Baldani e Baldani, 1995; (Camilios-Neto et al. 2014). In vitro plant cultivation was done under hydroponic and axenic conditions. Surface sterilized seeds were pre-germinated in agar/water petri dishes in the dark for 24 h at 30°C. The plantlets were then transferred to glass test tubes (two plantlets in each tube) containing 25 mL of Hoagland’s medium and 10 cm3 of polypropylene spheres were added to each tube to serve as support for the plantlets (Fig. S1). On the second day after germination, plantlets were inoculated with H. seropedicae to a final density of 105 H. seropedicae cells per mL of Hoagland’s medium.
high throughput sequence alignment protocol
'The sequences obtained were mapped on the H. seropedicae genome using the software CLC Genomics Workbench (v. 6.5.1). The following parameters were used: reads were trimmed to minimum of 40 pb, 90% alignment to the reference sequence and 80% similarity required for inclusion as a mapped read, number of hits equal to 1, number of additional bases down and upstream of the CDS equal to 50 pb. Expressed genes were those that had more than 3 times of coverage. The differencial gene expression analyses was performed using DESeq (DESeq using R-package from Bioconductir’s project) and were considered regulated those with a fold- change greater than or equal to two and significance level in the Baggerley''s test higher than 95%, FDR ≤ 0.05. RPKM values (Mortazavi et al. 2008) were calculated using CLC Genomics Workbench (v. 6.5.1).'