transcriptome of beet cyst nematode induced syncytial cells in wild-type and clv1-101 clv2-101 rpk2-5 mutant in Arabidopsis

The goal of this project is to identify Arabidopsis CLE signaling downstream genes involved in syncytium formation during beet cyst nematode (Heterodera schachtii) infection.Wild-type (Col-0) and clv1-101 clv2-101 rpk2-5 mutant seedlings were grown on modified Knop's media in square plates (100 x 100 x 15 mm) vertically for 7 days. The growth chamber was set at 24 Degree Celsius with 12 h light/12 h dark light cycle, with light intensity of about 100 umol/m2/s. About 10-15 sterilized BCN (Heterodera schachtii) pre-parasitic 2nd stage juveniles were inoculated to each root. Developing syncytium (about 8 mm root segments) and root segments from uninfected roots were collected from primary roots at 4 dpi (days-post-inoculation) under stereoscope, fixed in ice-cold EAA (ethanol: acetic acid = 3:1) overnight, and embedded in paraffin wax. Samples were sectioned on Leica HM 325 rotary microtome, and syncytial cell content or control tissue (central vasculature of uninfected root segments) were collected on a ArcturusXT Laser Capture Microdissection System (ABI). RNA was isolated with a picoPure RNA Isolation Kit (ThermoFisher Scientific, cat# KIT0204). RNA concentration was determined by Qubit flourometer (Invitrogen) using the Qubit HS RNA assay kit (Invitrogen, cat# Q32852), and the RNA integrity was checked using the Fragment Analyzer automated electrophoresis system. rRNA was then depleted by Ribominus plant kit (ThermoFisher, cat# A1083808), and ethanol precipitated with glycogen. Libraries were constructed with Illumina's TruSeq stranded Total RNA Library Prep kit and sequenced on Illumina NextSeq 500.