Nicotiana_pauciflora MSOS 3
Total RNA was extracted from ~100 mg aliquots of homogenized leaves that were used for phy-tohormone analysis using Trizol (Life Technologies) according to the manufacturer's protocol.   All RNA samples were subsequently treated with RNase-free DNase-I (Fermentas) to remove all genomic DNA contamination. The mRNA was enriched using mRNA-seq sample preparation kit (Illumina),   and ~ 200bp insertion size libraries were constructed using Illumina whole transcriptome analysis Kit following the manufacturer’s standard protocol (Illumina, HiSeq system).   All libraries were then sequenced on the Illumina HiSeq 2000 at the sequencing core facility at Max Planck Institute for Molecular Genetics, Berlin.   Manduca sexta and Spodoptera littoralis oral secretions (OS) were collected on ice from larvae reared on N. attenuata plants until the 3rd-5th instar as previously described.   To simulate herbivore attack, one leaf of each plant was wounded with a pattern wheel and 20 μL of 1:5 diluted OSMs, OSSl or FAC (138 ng μL-1 C18:3-Glu) or water was added to the puncture wounds.   All leaves were collected 30 minutes after elicitation, their middle veins rapidly excised, flash frozen in liquid nitrogen and stored at -80°C until analysed.
Max Planck Institute for Chemical Ecology
Npau_WMS03