Transcriptomic analysis of Medicago truncatula hairy
roots overexpressing 35S::MtHSFA9::GFP compared to 35S::GUS::GFP
For ectopic overexpression of MtHSFA9 ,35S::MtHSFA9::GFP/35::GUS and the control (plasmid containing
the GUS gene alone) plasmids were obtained by the gateway cloning
method according to Verdier et al (2013) and used forAgrobacterium rhizogenes transformation for subsequent hairy root
transformation in Medicago truncatula following the protocol of
(Boisson-Dernier et al. 2001). MtHSFA9 and GUSamplicons were obtained by PCR using primers listed in Table S1 and
cloned in pK7GW2D,1 (Karimi, Inzé & Depicker 2002) containing a 35S
cauliflower mosaic virus promoter and GFP reporter that was used as
transformation marker. Seedlings were grown in square Petri dishes with
Fahraeus medium two months at 20°C, 16h light, at an angle of 45°.
Transformed roots were selected by GFP fluorescence under UV microscope
(stereomicroscope, Olympus U-RFL-T) and harvested in liquid nitrogen.
RNA was extracted from three replicates of similar amounts of roots
using Nucleospin Extract II, Total RNA Purification Kit (Macherey
Nagel). RNA amplification, labeling, and hybridization of Nimblegen
Medtr_v1.0 12x135K arrays were performed according to Terrassonet al. (2013). Three biological replicates were analyzed per
comparison using the dye-switch method, and statistical analysis on the
gene expression data was performed according to Verdier et al. (2013).
Transcriptome data has been submitted to the Gene Expression Omnibus as
GSExxxxx.