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| Status |
Public on Jun 22, 2022 |
| Title |
Plants at day 2 not treated and not inoculated _ repetition 3 |
| Sample type |
RNA |
| |
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| Source name |
Wheat plants at day 2 not treated and not inoculated (Ni) with Z. tritici
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| Organism |
Triticum aestivum |
| Characteristics |
cultivar: Alixan
tissue: leaves
age: three weeks after sowing at D0
treatment: at day 2 not treated
inoculation: not inoculated (Ni) with Z. tritici
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| Treatment protocol |
At day 0 (DO), corresponding to three weeks after sowing, plants were hand-sprayed with 30 mL of either a solution of 100 mg.L-1 rhamnolipid from Bacillus subtilis mock (control) treatment. At two days after treatment (D2), half of the plants treated with rhamnolipid, as well as control plants, were inoculated by hand-spraying 30 mL of Z. tritici spore suspension (106 spores.mL-1), mixed with 0.05 % of Tween 20, on the plants of each pot. Leaf samples were harvested at two (D2), five (D5) and fifteen (D15) days after treatment for transcriptomic analyses. For each condition, i.e. plants treated with rhamnolipid (M) or not (control, C), and inoculated with Z. tritici (i) or not (Ni), nine third leaves sampled from three different pots (three leaves per pot), were randomly harvested, bulked per pot, immediately frozen in liquid nitrogen and stored at -80°C for further analyses. Thereafter, 100 mg of frozen pooled leaves were ground in a mortar with liquid nitrogen for RNA extraction.
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| Growth protocol |
Seeds of the cultivar (cv.) Alixan (Limagrain, France), susceptible to STB, were pregerminated in square Petri dishes (12 × 12 cm) on moist filter paper as described by Siah et al. (2010), and germinated grains were delicately transferred into three-liter pots filled with universal loam (Gamm Vert, France). The prepared pots were then placed in the greenhouse at 21 ± 2°C with a 16/8 h day-night cycle. For each condition and each sampling modality, three pots harboring 12 wheat plants each (n=36), were used.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from wheat leaves using RNeasy Plant Mini Kit (Qiagen, Courtaboeuf, France). RNA quality was determined with Nanodrop by analyzing their absorbance ratios A260/280 and A260/230, which were found to range between 2.0 and 2.2. Moreover, RNA quality was also examined with Bioanalyzer 2100 (Agilent, France) and a minimal RNA integrity number (RIN) of 0.8 was required for all samples
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| Label |
Cy3
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| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.2 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
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| Hybridization protocol |
1.5 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Wheat Gene Expression Microarrays GE 4x44 (Agilent, Santa Clara, CA, USA) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
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| Scan protocol |
Slides were scanned immediately after washing on the Scanner GenePix 4200B (Molecular Device) using one color scan setting
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| Description |
Gene expression after 2 days of treatment
C3-D2-Ni
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| Data processing |
The scanned images were analyzed with GenePix Pro Software 6.0 (Molecular Device) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
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| Submission date |
Jun 22, 2021 |
| Last update date |
Jun 22, 2022 |
| Contact name |
Anca Lucau-Danila |
| Organization name |
University of Lille
|
| Department |
Biology
|
| Lab |
Charles Viollette Institute
|
| Street address |
Cité Scientifique SN2 303
|
| City |
Villeneuve d'Ascq |
| ZIP/Postal code |
59655 |
| Country |
France |
| |
|
| Platform ID |
GPL13627 |
| Series (1) |
| GSE178704 |
Deciphering defense mechanisms primed by a rhamnolipid in wheat towards Zymoseptoria tritici |
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