The Arabidopsis elongator complex subunit2 epigenetically regulates plant immune responses

Abstract

The Arabidopsis thaliana Elongator complex subunit2 (ELP2) genetically interacts with NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1), a key transcription coactivator of plant immunity, and regulates the induction kinetics of defense genes. However, the mechanistic relationship between ELP2 and NPR1 and how ELP2 regulates the kinetics of defense gene induction are unclear. Here, we demonstrate that ELP2 is an epigenetic regulator required for pathogen-induced rapid transcriptome reprogramming. We show that ELP2 functions in a transcriptional feed-forward loop regulating both NPR1 and its target genes. An elp2 mutation increases the total methylcytosine number, reduces the average methylation levels of methylcytosines, and alters (increases or decreases) methylation levels of specific methylcytosines. Interestingly, infection of plants with the avirulent bacterial pathogen Pseudomonas syringae pv tomato DC3000/avrRpt2 induces biphasic changes in DNA methylation levels of NPR1 and PHYTOALEXIN DEFICIENT4 (PAD4), which encodes another key regulator of plant immunity. These dynamic changes are blocked by the elp2 mutation, which is correlated with delayed induction of NPR1 and PAD4. The elp2 mutation also reduces basal histone acetylation levels in the coding regions of several defense genes. Together, our data demonstrate a new role for Elongator in somatic DNA demethylation/methylation and suggest a function for Elongator-mediated chromatin regulation in pathogen-induced transcriptome reprogramming.

Figure 1.

Pathogen-Induced Transcriptome Changes in elp2. (A) Dynamic changes in the numbers of genes that are differentially expressed between elp2 and the wild type (WT) and between npr1 and the wild type after Pst DC3000/avrRpt2 infection. (B) Overlaps between the genes that are differentially expressed at 4 hpi between elp2 and the wild type and those between npr1 and the wild type. (C) Dynamic changes in the numbers of genes that are up- or downregulated in the wild type, npr1, and elp2 after Pst DC3000/avrRpt2 infection. (D) Overlaps among the genes that are up- or downregulated at 4 hpi in the wild type, npr1, and elp2. (E) Expression of eight major defense genes in Pst DC3000/avrRpt2-infected wild-type and elp2 plants. The y axes indicate relative expression levels monitored by qPCR (results in [A] to [D] were from microarray analysis). Expression levels were normalized against UBQ5. The x axes indicate hours after Pst DC3000/avrRpt2 infection. Data represent the mean of three independent samples with sd. An asterisk indicates a significant difference between the wild type and elp2 (P < 0.05, t test).

Figure 2.

Epistasis between elp2 and the 35:NPR1-GFP Transgene (A) Expression of NPR1 and nine NPR1 target genes in Pst DC3000/avrRpt2-infected wild-type (WT), elp2, and 35S:NPR1-GFP elp2 plants. The y axes indicate relative expression levels. Expression levels were monitored using qPCR and normalized against UBQ5. The x axes indicate hours after Pst DC3000/avrRpt2 infection. Data represent the mean of three independent samples with sd. (B) Growth of Psm ES4326 in wild-type, elp2, and 35S:NPR1-GFP elp2 plants. Data represent the mean of eight independent samples with sd. Different letters on the right of the bars indicate significant differences (P < 0.05, t test). Experiments were repeated three times with similar results.

Figure 3.

Histone H3 Acetylation Levels in Several Defense Genes. The position of the primers is relative to the initiation ATG codon. The relative amount of immunoprecipitated chromatin fragments (as determined by real-time qPCR) from elp2 was compared with that from the wild type (WT; arbitrarily set to 1). Data represent the mean of three independent samples with sd. An asterisk indicates a significant difference between elp2 and the wild type (P < 0.05, t test). The experiment was repeated four times with similar results.

Figure 4.

DNA Methylation Levels in Several Defense Genes. (A) and (B) DNA methylation levels at CG sites (A) and CHG sites (B) of the NPR1 promoter region in elp2 and the wild type (WT). (C) and (D) DNA methylation levels at CG sites in the coding region of NPR1 (C) and PAD4 (D) in elp2 and the wild type. DNA samples were extracted from three biological replicates of each genotype. After bisulfite conversion and PCR amplification, the PCR products were cloned into pGEM-T easy vector. A total of 45 independent clones were sequenced for each genotype (15 for each DNA sample). The 15 clones from the same DNA sample were used to calculate methylation levels, which were then used for statistical analysis. Data represent the mean of three independent samples with sd. An asterisk indicates a significant difference between elp2 and the wild type (P < 0.05, t test).

Figure 5.

Genomic DNA Methylation Profiles of elp2. (A) Average genome-wide DNA methylation levels of elp2 and the wild type (WT). (B) to (D) Distribution of methylation percentage in the sequence context of CG (B), CHG (C), and CHH (D) in elp2 and the wild type The x axes are divided into 10 individual bins that correspond to methylation levels. The y axes are the percentage of total counts for each respective bin. (E) and (F) Regions on chromosome 1 where more methylcytosines are hypermethylated in either elp2 (E) or the wild type (F). Regions 1 to 4 correspond to nucleotides 8,390,001 to 8,392,000, 568,001 to 570,000, 9,756,001 to 9,758,000, and 3,876,001 to 3,878,000, respectively (E), or nucleotides 19,962,001 to 1,996,400, 11,318,001 to 11,320,000, 16,434,001 to 16,436,000, and 27,932,001 to 27,934,000, respectively (F). (G) to (L) Methylation levels of the methylcytosines in all sequence contexts in two regions on chromosome 1.

Figure 6.

Pathogen-Induced Dynamic DNA Methylation Changes in NPR1 and PAD4. (A) to (C) Average methylation levels of methylcytosines at CG sites (A), CHG sites (B), and CHH sites (C) of the NPR1 promoter region in elp2 and the wild type (WT). (D) Average methylation levels of methylcytosines at CG sites of the PAD4 coding region in elp2 and the wild type. DNA samples were extracted from three biological replicates of each genotype/time point. After bisulfite conversion and PCR amplification, the PCR products were cloned into pGEM-T easy vector. A total of 45 independent clones were sequenced for each genotype/time point (15 for each DNA sample). The 15 clones from the same DNA sample were used to calculate methylation levels, which were then used for statistical analysis. Data represent the mean of three independent samples with sd. Different letters above the bars indicate significant differences (P < 0.05, t test). Note that the comparison was made separately among time points for each genotype.