title: WRA1
organism: Herbaspirillum seropedicae SmR1
description: Protocols: 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. 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 The rRNA was depleted using the Microbe Express kit (Ambion®), the libraries for sequencing were prepared using the Whole Transcriptome Analysis kit (Life Technologies).
attributes.tag: ENA first public
attributes.value: 2016-01-01
attributes.tag: ENA last update
attributes.value: 2015-10-06
attributes.tag: External Id
attributes.value: SAMEA3593401
attributes.tag: INSDC center alias
attributes.value: UFPR
attributes.tag: INSDC center name
attributes.value: UFPR
attributes.tag: INSDC first public
attributes.value: 2016-01-01T17:01:23Z
attributes.tag: INSDC last update
attributes.value: 2015-10-06T10:43:05Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-3646_1:WRA1
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: growth condition
attributes.value: attached to wheat root
attributes.tag: sample name
attributes.value: E-MTAB-3646_1:WRA1