GSE194090
RNA-seq time course in response to lipo-chitooligosaccharides in hybrid poplar roots
We treated Populus tremula x alba roots with rhizobial LCOs. We analyzed gene expression by RNA sequencing at seven time-points: 0 hr (control treatment), 15, 30 min, 1, 2, 4, 8, 24 hours over the following 24 hours. Overall design: Shoot tips (about 10-15 mm long) cut from in vitro grown Populus tremula × alba INRA clone 717-1B4 were placed aseptically on Murashige and Skoog media with vitamins (PhytoTech Labs-M519) supplemented with 0.1 g/L myo-inositol (PhytoTech Labs-I703), 0.25 g/L MES (PhytoTech Labs-M825), 2% (w/v) sucrose (PhytoTech Labs-S391) and solidified with 0.8% agar (PhytoTech Labs-A296). The pH of the medium was adjusted to 5.8 with KOH before autoclaving. Explants were placed in a growth chamber under long-day conditions (16 hours light/8 hours darkness, 24 °C, 65% relative humidity, and 100 µmol m-2 s-1 photosynthetic photon flux). Once the shoot tips were rooted, and the root was at least 25 mm long, plantlets were transferred to square plates (22.5 x 22.5 cm) containing Woody Plant Medium with reduced nitrogen (0.5 mM) and phosphate (7 µM), and 1.3 g/l calcium gluconate (Lloyd and McCown 1980). Plantlets were grown vertically for ten days in the growth chamber. Next, 3 hours after growth chamber dawn, plates were flooded to immerse roots in a 10-8 M solution of purified Rhizobium sp. IRBG74 LCOs or a control solution (0.005% ethanol) for 1 hour. After 1 hour, the solution was poured off, and plants returned to the growth chamber. Roots were collected immediately (control), 15 minutes, 30 minutes, 1, 2, 4, 8, and 24 hours after the LCO treatment. Roots were snap-frozen in liquid nitrogen, and roots from four plants were pooled for each of three biological replicates used in RNA sequencing. For each RNA extraction, roots from four plants were pooled and ground while keeping the sample frozen. RNA extraction was performed as described previously (Chang et al. 1993). Libraries were prepared using 0.5 µg of RNA in the NEBNext® UltraTM Directional RNA Library Prep Kit following the supplier's instructions (New England Biolabs, Ipswich, MA, USA). Sequencing was conducted with an Illumina HiSeq3000 (2×100 cycles) at the Interdisciplinary Center for Biotechnology Research at the University of Florida (Gainesville, FL, USA).