Project description:To systematically investigate viral sRNA production and sRNA-target interaction, we sequenced sRNAs from Tobacco Rattle Virus (TRV)-infected Nicotiana benthamiana at an early (1 week post infection) and late time point (3 weeks post infection). The N. benthamiana 16c plants were infected with TGS-inducing viruses (TRV-35S and TRV-35-2M) and PTGS-inducing viruses (TRV-GFP and TRV-GFP-2M), respectively. TRV-35S is a recombinant TRV containing a 120 nt segment of the 35S promoter. Its derivative, TRV-35S-2M, carrying single nucleotide substitutions (SNS) at every 10 nt within the 120 nt 35S target segment. Same strategy was used to create recombinant TRV-GFP and TRV-GFP-2M targeting GFP coding sequence. According to SNS content, sRNAs from TRV-35S-2M/TRV-GFP-2M infected plants can be separated to yield primary (containing SNSs) and secondary sRNAs (lacking SNSs). Wild Type TRV was used along as viral infection control. Libraries were indexed during PCR amplification (16 cycles) according to the Illumina protocol. See individual sample information for specific index primers used.
Project description:sRNAs were cloned and sequenced from the wild type Arabidopsis fwa epimutant, and the Arabidopsis dcl2/4 mutant harbouring the fwa epimutant. Both wild type and dcl2/4 were infected with mock conditions or modified Tobacco Rattle Virus (TRV) containing a portion of the FWA promoter sequence (TRV:FWAtr). Libraries were indexed during PCR amplification (12 cycles) according to the Illumina protocol. See individual sample information for specific index primers used.
Project description: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.
Project description:To investigate how high temperature affects sRNA production during virus induced gene silencing (VIGS), we sequenced sRNAs from Tobacco Rattle Virus (TRV)-infected Nicotiana benthamiana kept at 29°C at an early (1 week post infection) and a late time point (3 weeks post infection). To compare sRNA production between virus induced transcriptional gene silencing (ViTGS) and virus induced post-translational gene silencing (ViPTGS) at 29°C, the N. benthamiana 16c plants were infected with TGS-inducing viruses (TRV-35S and TRV-35-2M) and PTGS-inducing viruses (TRV-GFP and TRV-GFP-2M). TRV-35S is a recombinant TRV containing a 120 nt segment of the 35S promoter. Its derivative, TRV-35S-2M, carrying single nucleotide substitutions (SNS) at every 10 nt within the 120 nt 35S target segment. Same strategy was used to create recombinant TRV-GFP and TRV-GFP-2M targeting GFP coding sequence. According to SNS content, sRNAs from TRV-35S-2M/TRV-GFP-2M infected plants can be separated to yield primary (containing SNSs) and secondary sRNAs (lacking SNSs). Wild Type TRV was used along as viral infection control. Libraries were indexed during PCR amplification (16 cycles) according to the Illumina protocol. See individual sample information for specific index primers used.
Project description:Purpose: The goal of this study is to compare the transcriptome profilling (RNA-seq) 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: Here we report a significant repression of DNA methylation genes in inflorescences of Arabidopsis plants infected with Tobacco rattle virus (TRV) that coincides with dynamic changes in methylation at the whole genome level. Arabidopsis mutants deficient in DNA methylation were more resistant to this virus in early colonized tissues but more susceptible at later time points of infection, indicating that DNA methylation was critical to control both proliferation and antiviral defense. We found that TRV interference with DNA methylation leads to changes in the methylation and trancriptional status of transposable elements (TEs), including TEs located in the promoter of disease resistance genes that were significantly repressed in plants exposed to TRV. Activation of both TEs and their nearby disease resistance genes was altered in a range of hypo- and hyper-methylated Arabidopsis mutants, indicating that perturbations in DNA methylation contributes to modulate their expression in infected plants. 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.
Project description:Field-grown tubers of potato were examined for infection by Tobacco rattle virus and consequent production of corky ringspot or spraing symptoms. A microarray study identified tuber genes that are differentially expressed in response to TRV infection and to spraing production, showing that hypersensitive response (HR) pathways are activated in spraing-symptomatic tubers. This was confirmed by quantitative RT-PCR (Q-RT-PCR) of a selected group of HR-related genes and by histochemical staining of excised tuber tissue with spraing symptoms. Q-RT-PCR of TRV in different areas of the same tuber slice showed that non-symptomatic areas contained higher levels of virus than did spraing-symptomatic areas. This suggests that spraing formation is associated with an active plant defence that reduces the level of virus in the infected tuber. Expression of two plant defence genes was similarly upregulated in spraing-symptomatic tubers that were infected with another virus, Potato mop-top-virus, suggesting that spraing is a generalised response to virus infection of tubers.
Project description:Construction of Parallel analysis of RNA ends (PARE) libraries was done as described by German et al., 2009. Raw reads consisting of short sequences of 16 to 21 nts after MmeI digestion.