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Transcript profiling of local and adjacent leaf responses in barley following inoculation with Pseudomonas syringae

ABSTRACT: Transcriptional changes were monitored in the barley cultivar Golden Promise 24 hours post inoculation (hpi) with the bacteria Pseudomonas syringae pv. tomato DC3000 avrRpm1 (PstavrRpm1) using the Affymetrix Barley genome array GeneChip®. Seedlings of Golden Promise were grown to growth stage 12-13 (Zadoks et al., 1974) before inoculating with either PstavrRpm1 or water (for the mock inoculation control) by infiltration. Plants were grown under a 18 °C / 16 h light period; 12 °C / 8 h dark period, with artificial lighting (100 µmol m-2 s-1) and a relative humidity of 75 – 85 %. Leaf samples from three seedlings were collected 24 hpi for RNA extraction and transcriptomics analysis from the area infiltrated (local) and from the area next to the infiltrated region (adjacent) from three biological replicates. Leaf tissue was ground under liquid nitrogen and total RNA extracted using the RNeasy miniprep kit (Qiagen), following the manufacturer’s instructions. RNA was DNase treated using Turbo DNase (Ambion) according to the manufacturer instructions. RNA integrity was confirmed using the Agilent 2100 Bioanalyzer (Agilent). The two cycle-target labeling method was used following the Affymetrix protocol. Affymetrix GeneChip processing, including RNA quality control, microarray hybridisation and data acquisition was performed through contract research services by Cogenics (North Carolina, U.S.A.). A total of twelve hybridisations were performed. ****[PLEXdb(http://www.plexdb.org) has submitted this series at GEO on behalf of the original contributor, Ellen Colebrook. The equivalent experiment is BB92 at PLEXdb.]

ORGANISM(S): Hordeum vulgare

PROVIDER: GSE43906 | GEO | 2013/01/31

SECONDARY ACCESSION(S): PRJNA188091

REPOSITORIES: GEO

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Project description:Transcriptional changes were monitored in roots of the barley cultivar Regina following inoculation with zoospores of the nonadapted plasmodiophorid virus vector Polymyxa betae using the Affymetrix Barley1 GeneChip®. Barley cv. Regina seeds were imbibed in distilled water over night and then transferred on to moist heat treated (90°C over night) sand in plastic trays that were sealed. Trays were incubated at room temperature for 4 days to allow the seeds to germinate to Zadocks stage 07 of the decimal code for the growth stages of cereals (Tottman et al., 1979 Annals of Applied Biology, 93: 221-234; Zadocks et al., 1974 Weed Research, 14: 415-421). 110 barley (cv. Regina) seedling (Zadocks stage 07) were placed into a 90 mm diameter plastic Petri dish (3 Petri dishes per treatment) and 60 mL of P. betae zoospore suspension added (1 x 10 6 spores mL-1). Control plants were treated exactly the same way except zoospore-free buffer (0.1 g L-1 Phostrogen, 0.5% Bovine serum albumen) was added. Zoospore challenge (and control) occurred at an ambient temperature of 22°C. Roots were excised from ten plants per treatment at ten time points (15’, 30’, 45’ 1h, 2h, 3h, 4h, 5h, 6h, 7h) and frozen in liquid nitrogen. Three independent biological replicate experiments were done. Total RNA was isolated from each sample using TRI-reagent following the manufacturer’s instructions (Invitrogen) and then treated with DNase I (Ambion, Texas, USA) according to the manufacturer’s protocol. RNA samples for microarray hybridisation were further purified using RNeasy Mini Spin column purification (Qiagen, Hilden, Germany). The integrity of the RNA samples was confirmed using the BioAnalyzer 2100 (Agilent Technologies). Affymetrix GeneChip processing, including RNA quality control, microarray hybridisation and data acquisition was performed through contract research services by Geneservice Ltd (www.geneservice.co.uk). A total of six hybridisations were performed. Publication: European Journal of Plant Pathology 123(1):5-15.<br> ****[PLEXdb(http://www.plexdb.org) has submitted this series at GEO on behalf of the original contributor, Graham McGrann. The equivalent experiment is BB75 at PLEXdb.]

Project description:Transcriptional changes were monitored in roots of the barley cultivar Regina following inoculation with zoospores of the adapted plasmodiophorid virus vector Polymyxa graminis using the Affymetrix Barley1 GeneChip®. Barley cv. Regina seeds were imbibed in distilled water over night and then transferred on to moist heat treated (90°C over night) sand in plastic trays that were sealed. Trays were incubated at room temperature for 4 days to allow the seeds to germinate to Zadocks stage 07 of the decimal code for the growth stages of cereals (Tottman et al., 1979 Annals of Applied Biology, 93: 221-234; Zadocks et al., 1974 Weed Research, 14: 415-421). 110 barley (cv. Regina) seedling (Zadocks stage 07) were placed into a 90 mm diameter plastic Petri dish (3 Petri dishes per treatment) and 60 mL of P. graminis zoospore suspension added (1 x 10 6 spores mL-1). Control plants were treated exactly the same way except zoospore-free buffer (0.1 g L-1 Phostrogen, 0.5% Bovine serum albumen) was added. Zoospore challenge (and control) occurred at an ambient temperature of 22°C. Roots were excised from ten plants per treatment at ten time points (15’, 30’, 45’ 1h, 2h, 3h, 4h, 5h, 6h, 7h) and frozen in liquid nitrogen. Three independent biological replicate experiments were done. Total RNA was isolated from each sample using TRI-reagent following the manufacturer’s instructions (Invitrogen) and then treated with DNase I (Ambion, Texas, USA) according to the manufacturer’s protocol. RNA samples for microarray hybridisation were further purified using RNeasy Mini Spin column purification (Qiagen, Hilden, Germany). The integrity of the RNA samples was confirmed using the BioAnalyzer 2100 (Agilent Technologies). Affymetrix GeneChip processing, including RNA quality control, microarray hybridisation and data acquisition was performed through contract research services by Geneservice Ltd (www.geneservice.co.uk). A total of six hybridisations were performed. Publication: European Journal of Plant Pathology 123(1):5-15 ****[PLEXdb(http://www.plexdb.org) has submitted this series at GEO on behalf of the original contributor, Graham RD McGrann. The equivalent experiment is BB74 at PLEXdb.]

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Transcript profiling of local and adjacent leaf responses in barley following inoculation with Pseudomonas syringae

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