Transcriptome of galls formed by Meloidogyne javanica in Arabidopsis thaliana at 3 days post infection

The goal of this study is to compare the transcriptome profilling (RNA-seq) of galls (G) formed in Arabidopsis roots infected by Meloidogyne javanica at 3 days post infection compared to uninfected root segments (RC). Meloidogyne javanica-induced galls and control roots mRNA profiles were generated by deep sequencing with Illumina HiSeq X PE150. Arabidopsis thaliana plants were grown in controlled environment chambers under 16 h/8 h of light/dark at 23ºC. Arabidopsis plants were inoculated at 5 days post germination at the root tip with approximately 20 freshly-hatched Meloidogyne J2 each plant. The inoculated and non-inoculated plants were then kept in the dark for 2 days, in plates horizontally orientated. During the first 48 hours, the infections and control roots growth were assessed. After 2 days, the plates containing both inoculated and non-inoculated plants were maintained in the light, vertically orientated and covered by a gauze. For more details concerning this protocol please see Olmo et al. (2017; doi: 10.1007/978-1-4939-6831-2_5). Galls and control root segments were collected at 3 days post infection and frozen on liquid nitrogen. Galls and control root segments (RC) from at least three independent experiments were pooled together per sample. For each treatment, three independent biological samples (G1, G2 and G3 for galls; RC1, RC2 and RC3 for RC) were used for total RNA extraction. Total RNA was extracted using the AllPrep DNA/RNA/miRNA Universal Kit from Qiagen with several adaptations (Silva et al., 2019; doi: 10.3389/fpls.2019.00657). Illumina's TruSeq Stranded mRNA Library Prep Kit was used to prepare the libraries, strictly following the manufacturer's instructions. The fragment size distribution of the libraries were checked in the Agilent 2100 Bioanalyzer using the Agilent High Sensitivity DNA Kit. The libraries were then sequenced in a fraction of an Illumina HiSeq X PE150. Both steps were performed by the company AllGenetics & Biology SL.(Spain). For the bioinformatic analysis, the reads were firstly checked for quality using FastQC (version 0.11.8). Trimming was then performed using BBDuk (version 38.39). Trimmed reads were mapped to the combined A. thaliana (TAIR10) and M. javanica (Assembly GCA_900003945.1; Blanc-Mathieu et al., 2017) genomes using STAR (version 2.7.0d; Dobin et al., 2013) and the following parameters: --outFilterMismatchNmax 7, --outFilterMultimapNmax 5. The BAM files produced with STAR where then used by HTSeq (version 0.11.2), in order to produce the count tables used for the differentially expressed analysis. The normalized count tables and the posterior differentially expression analysis were performed with DESeq2 (version 1.20.0; Love et al., 2014) using R (version 3.5.2) and RStudio (version 1.1.463). Genes with a |log2-fold change|>0.5 and an adjusted p value <0.01 were considered as differentially expressed.