Study : Next Generation Sequencing Facilitates Quantitative Analysis of mock and tobacco ratle virus (TRV) Arabidopsis inflorescences Methylome. [MethylC-seq]
Next Generation Sequencing Facilitates Quantitative Analysis of mock and tobacco ratle virus (TRV) Arabidopsis inflorescences Methylome. [MethylC-seq]
Purpose: The goal of this study is to compare the whole genome bisulfite sequencing of inflorescences infected with tobacco ratle virus (TRV) to mock inoculated inflorescences (negative controls), in Arabidopsis plants Methods: Inflorescences of systemically TRV infected or mock-inoculated plants were collected from more than 40 independent Arabidopsis plants, at 14 days post-inoculation (dpi). TRV and mock mRNA profiles were generated by deep sequencing by Illumina HiSeq 2000. The sequence reads that passed quality filters (SOAPnuke) were analysed by Burrows-Wheeler (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. Genes and isoforms were quantified by RSEM sofware package. qRT-PCR validation was performed using TaqMan and SYBR Green assays. Results: We show a significant repression of DNA methylation genes in Arabidopsis infected with Tobacco rattle virus (TRV) that coincides with changes in methylation at the whole genome level. Loss of de novo methylation and/or maintenance of CHH methylation caused a phenotype of high resistance in early colonyzed tissues, whereas defects in CHG methylation correlated with hypersusceptibility to TRV as the infection progresses. Reactivation of several transposable elements (TEs) during TRV infection inversely correlated with the expression of nearby disease resistance genes. Transcript accumulation of both TEs and TRV-responsive disease resistance genes was altered in hypo- and hyper-methylated mutants. Conclussion: Our study showed that TRV interferes with DNA methylation to alter the transcriptional silencing of TEs, which in turn compromises the expression of neighboring disease resistance genes. Overall design: TRV and mock mRNA profiles were generated from Arabidopsis inflorescences by deep sequencing with Illumina HiSeq 2000. MethylC-seq samples were collected from inflorescences of systemically tobacco ratle virus (TRV) infected plants or mock-inoculated plants (negative controls) at 14 days post-inoculation (dpi). Inflorescences from more than 40 independent Arabidopsis plants were pooled per sample.