The goal of this study is to compare the methylation state of galls formed in Arabidopsis roots infected by Meloidogyne javanica at 3 days post infection compared to uninfected root segments (RC).
Overall design: MethylC-seq samples were collected from galls induced by Meloidogyne javanica in Arabidopsis at 3 days post-infection (dpi) and from uninfected root segments (negative controls).
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 genomic DNA extraction. Genomic DNA 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).
The libraries were prepared using the NEXTflex Bisulfite-Seq Library Prep Kit (BiooScientific), 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 one lane of an Illumina HiSeq 4000 PE100 platform. Two of the libraries (RC2 and RC3) were sequenced in an Illumina® HiSeq X PE150 (San Diego, California, USA). Both steps were performed by the company AllGenetics & Biology SL. (Spain).
MethylC-seq data was processed using the pipeline available at https://github.com/seb-mueller/snakemake-bisulfite (commit f12bb6) in GitHub. The pipeline performed quality control using FastQC (version 0.11.8). Raw reads were trimmed for adapters, quality-trimmed and filtered using Trim Galore (version 0.5.0). A combined genome was constructed by concatenation the genomes (in fasta format) of A. thaliana (TAIR10, including the chloroplast and mitochondrial genome), M. javanica (Assembly GCA_900003945.1; Blanc-Mathieu et al., 2017) and Lambda phage into a single fasta format reference. Reads were then mapped to the constructed reference with Bismark (version 0.20.0; Krueger and Andrews, 2011), using the following parameters: L,0,-0.4. The SAM alignment generated by Bismark were further processed into BAM format and subsequently imported for further processing into the R package methylKit version 0.99.2 (Akalin et al., 2012). To identify differential methylated regions (DMRs), we defined various regions in form of adjacent non-overlapping window of 200 base pairs (referred to as bins), which were posteriorly checked for overlapping different genomic regions, including genes, promoters and transposable elements as specified in Araport11 (06/2016; Cheng et al., 2017). Promoters were considered as 1000 base pairs (bp) upstream until 200 bp downstream of the transcription start site. For each region, the containing methylated and unmethylated cytosines were counted (separately for each library, each region and each context [CHH, CHG, CG]) and tested for differential methylation using a logistic regression approach using the “MN” over-dispersion correction as implemented in MethylKit. DMRs are then defined if the difference of methylation proportion between the gall samples and the controls was higher than 15% and a FDR (q-value) of less than 0.05 and covered by at least two biological replicates of the treatment or control.
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