Jumonji C domain protein JMJ705-mediated removal of histone H3 lysine 27 trimethylation is involved in defense-related gene activation in rice

Abstract

Histone methylation is an important epigenetic modification in chromatin function, genome activity, and gene regulation. Dimethylated or trimethylated histone H3 lysine 27 (H3K27me2/3) marks silent or repressed genes involved in developmental processes and stress responses in plants. However, the role and the mechanism of the dynamic removal of H3K27me2/3 during gene activation remain unclear. Here, we show that the rice (Oryza sativa) Jumonji C (jmjC) protein gene JMJ705 encodes a histone lysine demethylase that specifically reverses H3K27me2/3. The expression of JMJ705 is induced by stress signals and during pathogen infection. Overexpression of the gene reduces the resting level of H3K27me2/3 resulting in preferential activation of H3K27me3-marked biotic stress-responsive genes and enhances rice resistance to the bacterial blight disease pathogen Xanthomonas oryzae pathovar oryzae. Mutation of the gene reduces plant resistance to the pathogen. Further analysis revealed that JMJ705 is involved in methyl jasmonate-induced dynamic removal of H3K27me3 and gene activation. The results suggest that JMJ705 is a biotic stress-responsive H3K27me2/3 demethylase that may remove H3K27me3 from marked defense-related genes and increase their basal and induced expression during pathogen infection.

Figure 1.

JMJ705 Is a Histone H3K27me2/3 Demethylase. (A) Schematic presentation of the vector 35S-JMJ705-FLAG-HA. The relative positions of JmjN, JmjC, and zinc-finger (ZnF) domains are indicated. Arrow indicates the position of the substitution mutation H244A. (B) In vitro demethylase activity of JMJ705. Bulk histone was incubated with (+) or without (−) tobacco cell-expressed JMJ705-FLAG-HA fusion protein and analyzed by protein gel blots using antibodies against specific histone modification modules indicated on the left. The same blots were analyzed by anti-H3. JMJ705-FLAG-HA was revealed by anti-HA. (C) In vivo histone demethylase activity of JMJ705. The 35S-JMJ705-FLAG-HA construct was transfected into tobacco leaf cells. Nuclei isolated from leaves were inspected for expression of the fusion protein (stained with anti-HA and indicated by arrows) and then examined for histone methylation levels (stained by DAPI) by using antibodies against specific histone H3 methylation modules indicated on the left. The H244A substitution mutation was tested similarly with anti-H3K27me2. At least 30 nuclei expressing JMJ705 fusion per transfection were observed and imaged. Bar = 25 μm. (D) Histone H3K27me2/3, H3K4me3 and H3K9me3 methylation levels in wild type (ZH11, HY), JMJ705 overexpression (OX-5) and T-DNA mutant (jmj705) plants revealed by protein gel blots. Only one set of several repeated data is shown. H3 was detected as loading control. Mean signals ± sd (from three replicates) relative to the wild type (set at 1) are indicated below the bands. Significance of differences was determined using t tests. *P < 0.05; **P < 0.01.

Figure 2.

JMJ705 Overexpression Produces a Leaf Lesion–Mimic Phenotype at the Mature Stage. (A) Leaf phenotype and JMJ705 transcript levels revealed by RNA gel blot in the wild type and five transgenic lines. rRNAs are shown as loading controls. (B) Comparison of wild-type (left) and overexpression (right) plants at the mature stage. (C) mRNA levels of defense-related genes detected by real-time RT-PCR in wild-type (ZH11) and overexpression (OXJMJ705-5 and OXJMJ705-14) flag leaves. Bar indicates mean ± sd from three biological repeats. [See online article for color version of this figure.]

Figure 3.

JMJ705 Overexpression Enhances Rice Resistance to the Bacterial Pathogen Xoo. (A) and (B) Three wild-type (ZH11) plants and the T2 segregates of line 5 (OXJMJ705-5) and line 14 (OXJMJ705-14) were inoculated with the Xoo strain PXO99 that causes rice blight disease. Genotyping of the T2 plants for the presence of the transgene is shown for each line. The percentages of the leaf lesion areas were measured 14 d after inoculation. Bar indicates mean ± sd from four to five replicates for the lesion area. (C) Leaf phenotype after PXO99 inoculation. (D) Bacterial growth rate (log [COLONY-FORMING UNITS/leaf]) measured at 0 (2 h post inoculation) and 3 to 12 d postinoculation (dpi). Significance of bacterial growth differences between wild-type and overexpression plants was determined by Student’s t tests. Bar indicates mean ± sd from five inoculated plants. *P < 0.05; **P < 0.01. [See online article for color version of this figure.]

Figure 4.

Characterization of a T-DNA Insertion Mutation of JMJ705. (A) Schematic representation of the gene structure and position of the T-DNA insertion (open triangle). The positions of the primers used for genotyping and RT-PCR are indicated. (B) Genotyping of nine segregates and the wild type (HY) using the two primer sets as indicated. (C) DNA gel blot analysis of copy number of the T-DNA insertion. (D) RT-PCR detection of JMJ705 transcripts. (E) Mature stage and panicle phenotype comparison between the wild type (left) and mutant plant (right). (F) Leaf lesion area (%) in three wild-type (HY) and nine T-DNA (jmj705) plants 14 d after inoculation with the Xoo strain PXO99. Bar indicates mean ± sd from four to five replicates. (G) Leaf phenotype. (H) Bacterial growth rate on jmj705 mutant leaves compared with HY as described in Figure 3. [See online article for color version of this figure.]

Figure 5.

H3K27me3 and H3K4me3 ChIP-seq Intensities of Genes that Are Upregulated or Downregulated in JMJ705 Overexpression Plants Compared with the Genome-Wide Levels. Numbers of sequenced tags (y-axis) per each 5% of the genic region (black box) or per 100-bp intervals in the 2 kb upstream and 2 kb downstream regions (line, x-axis) are shown. Arrow indicates the direction of transcription.

Figure 6.

H3K27me2/3 Removal Is Related to JMJ705-Mediated Gene Upregulation. Chromatin fragments isolated from wild-type (ZH11) and overexpression plants (OXJMJ705) were immunoprecipitated with antibodies against H3K4me3, H3K9me3, H3K36me3, H3K27me2, and H3K27me3 as indicated, and analyzed by real-time PCR using primer sets corresponding to transcriptional start site regions of 12 upregulated genes (numbered as in Supplemental Table 4 online). Chromatin fragments of JMJ705-FLAG overexpression plants were immunoprecipitated by anti-FLAG and analyzed by PCR using the same primer sets (right bottom).

Figure 7.

JMJ705 Enhances JA Induction of Gene Expression. mRNA levels of JA-responsive genes JAMYB, PR10, TPS3, and Os07g11739 (genes 5 and 7 in Supplemental Table 4 online) in wild type, two lines of overexpression and one line of RNAi plants (all in ZH11 background) treated by MeJA (0.2 mM) for 0 to 24 h were analyzed by quantitative RT-PCR. Relative mRNA levels are presented with wild type at 0 h set as 1. Because of great variation between induction experimental repeats, data from three biological repeats are presented individually. Insets show relative mRNA levels during the first hours of induction for TPS3 and 07g11739. Bar indicates mean ± sd from three technical repeats.

Figure 8.

JA-Induced H3K27me3 Removal from Responsive Genes Is Dependent on JMJ705. Anti-H3K27me3 ChIP assays were performed to analyze transcriptional start site of JA-responsive genes PR10, JAMYB, TPS3, and Os07g11739 in wild-type (ZH11) plants, overexpression (OXJMJ705), and JA-treated RNAi plants during 0, 8, and 12 h. Bar indicates mean ± sd from three biological repeats.