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Sample GSM5336222 Query DataSets for GSM5336222
Status Public on Sep 12, 2023
Title Leaf-inoculated-ozone-1 [L-I-O-1]
Sample type SRA
 
Source name Leaf-inoculated-ozone
Organisms Populus trichocarpa; Laccaria bicolor
Characteristics clone: Populus trichocarpa (Washington clone 101-74)-Laccaria bicolor S238N
inoculation: Roots inoculated with Laccaria bicolor
treatment: Ozone treatment
tissue: Leaves
Treatment protocol Three treatments were applied on randomly assigned batch of inoculated and non-inoculated poplars. One third of trees were submitted to ozone exposure (“O” modality), or to stop watering (“SW” modality) while the remaining trees were kept in the initial growth conditions (“C” modality). To ensure that trees were submitted to only one treatment, O and C plants were watered to field capacity twice a day and SW and C plants were cultivated under coal-filtered air. In details, O plants were exposed to 200 nmol mol-1 O3 using the control device previously published (Dusart et al., 2019) during the 8 hours preceding the harvest (from 5h to 13h in the light time). For SW plants, the watering was withheld 43h before harvest. At the end of treatments, trees were harvested following a random sampling to avoid batch effects between inoculated or not (I or NI) control and treated poplars. For the molecular analysis, two mature leaves per plant (9 and 10th from the apex) were sampled and immediately flash frozen. Then, root system was rapidly washed; samples of bare fine roots and mycorrhizae-enriched fine roots (denoted “myc”) were rapidly (in less than 30 min) sampled under binocular and flash-frozen in liquid nitrogen. We pooled three or four plants per biological replicate and we made four replicates per modality. The biological replicates were paired-distributed among organs (i.e. leaf, root or myc libraries with the same index were constructed from the same pool of plants)."
Growth protocol Cuttings of the western poplar Populus trichocarpa (Washington clone 101-74) were rooted for two weeks in distillated water under climate-controlled greenhouse conditions, under a 16-h photoperiod and a temperature of 22°C (day). A random batch of young rooted poplars were then transplanted on well-dampened calcined attapulgite clay (Turf-Pro, Toul, France) and correspond to non-inoculated poplar roots (“NI” modality). Another random batch of poplar was transplanted in a mix of the ectomycorrhizal (ECM) fungal inoculum Laccaria bicolor into well dampened calcinated attapulgite clay (inoculated poplar roots “I” modality). The fungal inoculum was prepared as described in Plett et al., 2011. Regardless to the colonization modality, each pot was given 20 ml of a diluted nutrient solution (0.8 mM KNO3, 0.8 mM Ca (NO3)2, 4 H2O, 0.3 mM NaH2PO4, 0.3 mM MgSO4,7 H2O, and the commercial trace-element solution Kanieltra (CO- FAZ, BP 198-08, Paris, France). To allow plant rooting and symbiosis development, poplars were kept three months under climate-controlled greenhouse conditions, maintaining a 16-h photoperiod, with a photosynthetic photon flux density (PPFD) of 450–500 μmol m–2s-1 and a day temperature of 22°C. In June 2019, I and NI were transferred into phytotron chambers with a PPFD of 470–500 μmol m–2s-1 and a day temperature of 22°C for acclimation two weeks before the application of the short-term environmental challenges.
Extracted molecule total RNA
Extraction protocol Total RNA of each sample was extracted separately. The RNA extraction was performed using the RNeasy Plant mini kit (Qiagen, Courtaboeuf, France) per the manufacturer’s instructions, with the addition of 25 mg of polyethylene glycol 8000 per milliliter of RLC buffer (RNeasy, Qiagen). A DNA digestion step with DNAse I (Ambion, Invitrogen) was also included to remove DNA contamination. RNA quality was verified by Nanodrop (Thermo Fisher Scientific) and Fragment Analyzer (Agilent, Santa Clara, USA).
Stranded cDNA sequencing libraries were generated from 1-3 µg of total pooled RNA following the manufacturer’s instructions (Illumina Inc., USA) followed by Illumina HiSeq 3000 mRNA sequencing (RNA-Seq) at the Genotoul sequencing facilities (Toulouse, France).
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 3000
 
Description OZMYC5
This sample is from leaves of plants with roots inoculated with Laccaria bicolor and treated with ozone . It is the first of four biological replicates used in this experiment.
Data processing Illumina software was used by the Genotoul sequencing facilities (Toulouse, France) to generate fastq raw data files
Raw reads were filtered and trimmed using erne-filter command (Erne v2.1.1, default parameters except --min-size=70; Del Fabbro et al., 2013). Filtered reads from each library were aligned to P. trichocarpa (https://phytozome-next.jgi.doe.gov/info/Ptrichocarpa_v4_1) using TopHat2 v2.12 (Trapnell et al., 2009; D. Kim et al., 2013). The genome was indexed with the ‘bowtie-build’ command from the genome sequence fasta file. TopHat2 default parameters were used except: -r 60 --mate-std-dev 25 --read-mismatche 15 --read-gap-length 15 --read-edit-dist 30 -I 20000 -i 20 --read-realign-edit-dist 10 --max-insertion-length 19 --max-deletion-length 19 -g 10 --segment-mismatches 3 --segment-length 25 --min-segment-intron 20 --max-segment-intron 20000 --b2-very-sensitive. Reads that mapped once were extracted from BAM files (accepted_hits.bam, TopHat2 output) using the NH:i:1 tag as described by Loraine et al. (2013). We considered only genes that contained a minimum of 10 reads in the four replicates of a treatment as expressed genes.
DESEQ2 was used for normalisation of data and co-expression network analysis (WGCNA) to identify differentially expressed genes
Genome_build: reference genome sequence of Populus tricocarpa (https://phytozome-next.jgi.doe.gov/info/Ptrichocarpa_v4_1)
Supplementary_files_format_and_content: tab-delimited text file including aligned reads (raw counts) and normalised expression values (DESEQ2).nd; not used in DESEQ2 due to no or background expression.
 
Submission date May 25, 2021
Last update date Sep 12, 2023
Contact name Annegret Kohler
E-mail(s) annegret.kohler@inrae.fr
Phone +33 (0)383 394072
Organization name INRAE
Department UMR 1136
Lab Interactions Arbres/Micro-organismes
Street address Centre INRAE Grand Est Nancy
City Champenoux
ZIP/Postal code 54280
Country France
 
Platform ID GPL30192
Series (1)
GSE175506 Gene expression changes in leaves, roots and Laccaria bicolor mycorrhiza of Poplar treated with ozone or stopped watering.
Relations
BioSample SAMN19330498

Supplementary file Size Download File type/resource
GSM5336222_L-I-O-1-OZMYC5.txt.gz 272.4 Kb (ftp)(http) TXT
Raw data are available in SRA
Processed data provided as supplementary file

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