Computational protocol: A Naturally Associated Rhizobacterium of Arabidopsis thaliana Induces a Starvation-Like Transcriptional Response while Promoting Growth

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Protocol publication

[…] One or two Col-0 seedlings were grown in 1.5 ml Eppendorf tubes, from which the bottom was cut off. The tubes contained sandy soil and were placed on peat substrate. Six hours before harvesting 18-day old seedlings, 500 µl of P. sp. G62 (OD 0.5 in 10 mM MgSO4), or MgSO4 without bacteria (mock) for controls were applied to the medium. Twenty-five seedlings were pooled for each of three replicates per treatment. RNA was extracted from whole seedlings with RNeasy Mini Kit from Quiagen according to the manufacturer's protocol. RNA quality check and microarray hybridization was carried out by Atlas Biolabs (Berlin, Germany). Chip quality was analysed and raw data (.cel files) were evaluated and normalised with the ROBIN software (criteria: robust multi array averaging, nested F-test, Benjamini-Hochberg correction) . The orignal data can be downloaded from the GEO database (http://www.ncbi.nlm.nih.gov/geo/) with the GEO accession GSE24552. Microarrays were evaluated with ROBIN, the Genevestigator online-tool and visualized with the TM4 software tool . Microarray comparison was further carried out with the MapMan tool . [...] Total RNA was extracted from 3 and 4 week old plants that were continuously colonized by bacteria and grew in vertical agar plates (control plants were not inoculated). Five plants were pooled for each of the 5 replicates per treatment. RNA was extracted using the RNeasy kit (Qiagen, http://www.qiagen.com/) for shoots and using the InviTrap Spin Plant RNA Mini Kit (Invitek, http://www.stratec-biomedical.de/) for roots. RNA was subjected to DNase treatment using the TURBO DNA-free kit (Ambion, http://www.ambion.com/). Five micrograms of RNA were reverse transcribed into cDNA using the Superscript III reverse transcriptase kit (Invitrogen). RT-qPCR amplification was carried out with the ABI Prism 7900 sequence detection system (Applied Biosystems, http://www.appliedbiosystems.com/), using a power SYBR green master mix (Applied Biosystems) and the primers as described in . UBQ10 (At4g05320), Pex 4 (At5g25760), bHLH (At4g38070), SAND (At2g28390) and PP2A (At1g13320) were used as reference genes (). Relative quantification of the expression of each individual gene was performed using the comparative threshold cycle method, as described in the ABI PRISM 7900 sequence detection system user bulletin no. 2 (Applied Biosystems). Primer efficiencies were calculated with the LinReg software (http://LinRegPCR.HFRC.nl). [...] Statistical analysis was carried out with SigmaPlot software version 11.0 from Systat Software, San Jose, USA. […]

Pipeline specifications

Software tools Genevestigator, TM4, MapMan, LinRegPCR, SigmaPlot
Applications Miscellaneous, qPCR, Transcription analysis
Organisms Arabidopsis thaliana, Phytoplasma sp.
Chemicals Carbohydrates, Sucrose