Project description:DDM1-mediated gene body DNA methylation is associated with inducible activation of defense-related genes in Arabidopsis [DNA methylation]
Project description:Background: Plants memorize previous pathogen attacks and are ‘primed’ to produce a faster and stronger defense response, which is critical for defense against pathogens. In plants, cytosines in transposons and gene bodies are reported to be frequently methylated. Demethylation of transposons can affect disease resistance by regulating the transcription of nearby genes during defense response, but the role of gene body methylation (GBM) in defense responses remains unclear. Results: Here, we find that loss of the chromatin remodeler decrease in DNA methylation 1 (ddm1) synergistically enhances resistance to a biotrophic pathogen under mild chemical priming. DDM1 mediates gene body methylation at a subset of stress-responsive genes with distinct chromatin properties from conventional gene body methylated genes. Decreased gene body methylation in loss of ddm1 mutant is associated with hyperactivation of these gene body methylated genes. Knockout of glyoxysomal protein kinase 1 (gpk1), a hypomethylated gene in ddm1 loss of function mutant, impairs priming of defense response to pathogen infection in Arabidopsis. We also find that DDM1-mediated gene body methylation is prone to epigenetic variation among natural Arabidopsis populations, and GPK1 expression is hyperactivated in natural variants with demethylated GPK1. Conclusions: Based on our collective results, we propose that DDM1-mediated GBM provides a possible regulatory axis for plants to modulate the inducibility of the immune response.
Project description:Background: Plants memorize previous pathogen attacks and are ‘primed’ to produce a faster and stronger defense response, which is critical for defense against pathogens. In plants, cytosines in transposons and gene bodies are reported to be frequently methylated. Demethylation of transposons can affect disease resistance by regulating the transcription of nearby genes during defense response, but the role of gene body methylation (GBM) in defense responses remains unclear. Results: Here, we find that loss of the chromatin remodeler decrease in DNA methylation 1 (ddm1) synergistically enhances resistance to a biotrophic pathogen under mild chemical priming. DDM1 mediates gene body methylation at a subset of stress-responsive genes with distinct chromatin properties from conventional gene body methylated genes. Decreased gene body methylation in loss of ddm1 mutant is associated with hyperactivation of these gene body methylated genes. Knockout of glyoxysomal protein kinase 1 (gpk1), a hypomethylated gene in ddm1 loss of function mutant, impairs priming of defense response to pathogen infection in Arabidopsis. We also find that DDM1-mediated gene body methylation is prone to epigenetic variation among natural Arabidopsis populations, and GPK1 expression is hyperactivated in natural variants with demethylated GPK1. Conclusions: Based on our collective results, we propose that DDM1-mediated GBM provides a possible regulatory axis for plants to modulate the inducibility of the immune response.
Project description:mRNA levels in Wild-type versus ddm1 Arabidopsis thaliana seedlings and bolting plants. Features found to be significantly enriched for DNA methylation were determined. This SuperSeries is composed of the following subset Series: GSE1324: EV23+24 mRNA levels in Wild-type versus ddm1/+ backcross bolting Arabidopsis thaliana plants GSE1325: EV33+34 mRNA levels in Wild-type versus ddm1 Arabidopsis thaliana seedlings GSE1326: VC109+111 mRNA levels in Wild-type versus ddm1 Arabidopsis thaliana seedlings GSE1327: EV39+40 mRNA levels in Wild-type versus ddm1 Arabidopsis thaliana seedlings GSE1328: VC110+112 mRNA levels in Wild-type versus ddm1 bolting Arabidopsis thaliana plants Refer to individual Series
Project description:Histone 3 lysine 4 and histone 3 lysine 9 methylation in wild type and ddm1 Arabidopsis thaliana seedlings. The purpose of the chromatin immunoprecipitation/microarray (ChIP/chip) experiment is to determine which regions of a genome are enriched for a particular histone modification in a single Arabidopsis thanliana genotype. Chromatin immunoprecipitation with antibodies raised against dimethyl histone-H3 lysine-9 (H3mK9) or dimethyl histone-H3 lysine-4 (H3mK4) is performed on a selected genotype. This purified DNA from each immunoprecipiation (mH3K9, mH3K4, no antibody control) is used for random amplification to increase the quantity of DNA for microarray hybridization. The amplified DNA from each experimental sample is then labeled with Cy5 and hybridized against total input DNA from the corresponding genotype, labeled in Cy3. In a single hybridization, the total input DNA serves as a baseline and is compared to the immunoprecipitated samples. Ratios of normalized signal intensities were calculated to identify enrichment of a particular sequence after immunoprecipitation, in comparison to the total input DNA. Dye swap analysis is carried out to take account of experimental variation by repeating the hybridization with identical samples labeled with Cy3 and Cy5, respectively. This SuperSeries is composed of the following subset Series: GSE1333: EV49+50, Histone 3 Lysine 4 methylation in wild-type Arabidopsis thaliana seedlings GSE1334: Histone 3 Lysine 4 methylation in ddm1 Arabidopsis thaliana seedlings GSE1335: EV104+105, Histone 3 Lysine 4 methylation in ddm1 Arabidopsis thaliana seedlings GSE1336: Ev106+107, Histone 3 Lysine 4 methylation in WT Arabidopsis thaliana seedlings GSE1337: EV51+52, Histone 3 Lysine 9 methylation in wild-type Arabidopsis thaliana seedlings GSE1338: EV59+60, Histone 3 Lysine 9 methylation in ddm1 Arabidopsis thaliana seedlings GSE1339: Histone 3 Lysine 9 methylation in wild-type Arabidopsis thaliana seedlings GSE1340: EV110+111, Histone 3 Lysine 9 methylation in ddm1 Arabidopsis thaliana seedlings Refer to individual Series
Project description:Mitogen-activated protein kinases (MAPKs) target a variety of protein substrates to regulate cellular signaling processes in eukaryotes. In plants, the number of identified MAPK substrates that control plant defense responses is still limited. Here, we generated transgenic Arabidopsis thaliana plants with an inducible system to simulate in vivo activation of two stress-activated MAPKs, MPK3 and MPK6. Metabolome analysis revealed that this artificial MPK3/6 activation (without any exposure to pathogens or other stresses) is sufficient to drive the production of major defense-related metabolites, including various camalexin, indole glucosinolate and agmatine derivatives. An accompanying (phospho)proteome analysis led to detection of hundreds of potential phosphoproteins downstream of MPK3/6 activation. Besides known MAPK substrates, many candidates on this list possess typical MAPK-targeted phosphosites and in many cases, the corresponding phosphopeptides were detected by mass spectrometry. Notably, several of these putative phosphoproteins have been reported to be associated with the biosynthesis of antimicrobial defense substances (e.g. WRKY transcription factors and proteins encoded by the genes from the “PEN” pathway required for penetration resistance to filamentous pathogens). Thus, this work provides an inventory of candidate phosphoproteins, including putative direct MAPK substrates, for future analysis of MAPK-mediated defense control.
Project description:DNA methylation in wild type bolting plants, wild type seedlings, and ddm1 seedlings. The purpose of the McrBC methylation microarray assay is to determine which regions of a genome are methylated versus those that are unmethylated in a single Arabidopsis thanliana genotype. McrBC is a methylation-sensitive enzyme that restricts DNA only at purine-Cmethyl half sites when separated between 50bp and 3kb. A designated amount of DNA from a particular genotype is sheared to a size range of 1kb-10kb using nebulization. We restrict half of the nebulized DNA with McrBC, and the methylated fraction is then removed from the unmethylated fraction through gel purification of DNA fragments greater than 1kb.* The remaining nebulized DNA is subjected to the same gel purification scheme, but with no McrBC treatment. In a single hybridization, the untreated sample is labeled with Cy5 and the McrBC-treated sample with Cy3. Thus, after labeling and microarray hybridization, the ratio of normalized Cy5 to normalized Cy3 represents the relative methylation at the sequence represented by the spot on the microarray. Dye swap analysis is carried out to take account of experimental variation by repeating the hybridization with identical samples labeled with Cy3 and Cy5, respectively. This SuperSeries is composed of the following subset Series: GSE1329: DNA methylation in wild-type bolting Arabidopsis thaliana plants GSE1330: DNA methylation in ddm1 seedling Arabidopsis thaliana plants GSE1331: VC133+137, DNA methylation in ddm1 seedling Arabidopsis thaliana plants GSE1332: VC134+136, DNA methylation in wild-type seedling Arabidopsis thaliana plants Refer to individual Series
Project description:Despite of the paramount importance of heterosis in agriculture, the molecular mechanisms underlying heterosis remain elusive. Recent studies in Arabidopsis suggested possible involvement of DNA methylation in heterosis. We tested this hypothesis genetically by crossing homozygous mutants in DNA methylation-related genes in the Columbia (Col) ecotype with homozygous mutants in the same DNA methylation-related genes in the C24 ecotype. When genes in the RNA-directed DNA methylation (RdDM) pathway were mutated, the resultant F1 hybrids did not show appreciable loss of best parent heterosis (BPH) performances in early seedling growth. In contrast, mutations in the putative chromatin remodeling protein DECREASE OF DNA METHYLATION 1 (DDM1) caused ddm1-Col like growth pattern of ddm1-F1 hybrids, a mid-parent heterosis (MPH) performance. To understand the underlying molecular mechanisms, we compared the transcriptomes of the parental plants and reciprocal F1 hybrids in the wild type and ddm1 mutant backgrounds, and identified 183 non-additively expressed (NAE) genes, which were functionally enriched in defense response, a group of genes negatively associated with plant size. Interestingly, for the expression levels of the NAE genes, WT-F1 hybrids were enriched in the Golden Ration between mid-parent values (MPVs) and Col parents in WT, but ddm1-F1 hybrids were comparable to ddm1-Col parent in ddm1 mutant, which explained the MPH performance of ddm1-F1 hybrids. DNA methylation analyses revealed only one third of the NAE genes with highly methylated promoters whose expression is negatively associated with DNA methylation of promoters, strongly suggesting that DDM1 regulates heterosis by multiple epigenetic modifications.
Project description:mRNA levels in Wild-type versus ddm1 Arabidopsis thaliana seedlings and bolting plants. Features found to be significantly enriched for DNA methylation were determined. This SuperSeries is composed of the SubSeries listed below.