Illumina HiSeq 2500 sequencing; Transcriptional response of Arabidopsis thaliana in systemic acquired resistance: critical roles for pipecolic acid and salicylic acid
Transcriptional response of Arabidopsis thaliana in systemic acquired resistance: critical roles for pipecolic acid and salicylic acid
In each SAR experiment, at least 6 upper (2) leaves from 6 different plants pre-treated in 1 leaves with Psm (MgCl2) were pooled for one biological Psm- (mock-control) replicate. In this way, 3 biologically independent, replicate samples per treatment and plant genotype were obtained within each SAR set. Arabidopsis thaliana plants were grown individually in pots containing a mixture of soil, vermiculite and sand (8:1:1) in a controlled cultivation chamber with a 10-h day (9 AM to 7 PM; photon flux density 100 mol m-2 s-1) / 14-h night cycle and a relative humidity of 70 %. Day and night temperatures were set to 21C and 18C, respectively. Experiments were performed with 5- to 6-week-old, naive plants exhibiting a uniform appearance. To activate SAR, plants were infiltrated between 10 AM and 12 AM into three lower (1) leaves with suspensions of the bacterial pathogen Pseudomonas syringae pv. maculicola (OD600 = 0.005). Infiltration with 10 mM MgCl2 served as the mock-control treatment. Upper (2) leaves were harvested 48 h after the primary treatment for the determination of systemic gene expression by RNA-seq analyses. Three biologically independent SAR induction experiments were performed with Col-0 and ald1 plants. RNA was extracted from leaf sample replicates with the Plant RNeasy extraction kit (Qiagen, Germany), and treated on-column (Qiagen, Germany) and in solution with RNA-free DNase (New England Biolabs, MA, USA). RNA integrity, sequencing library quality and fragment size were checked on a 2100 Bioanalyzer (Agilent, CA, USA). Libraries were prepared using the TruSeq RNA Sample Prep Kit v2 (Illumina, San Diego, CA) and library quantification was performed with a Qubit 2.0 (Invitrogen, Germany). Leaf samples were multiplexed 12 libraries per lane yielding approximately 150 million reads per lane.
Experimental Factor: Pseudomonas syringae pv. maculicola
stimulus
Experimental Factor: ald1
genotype