Convergent Epigenetic Mechanisms Avoid Constitutive Expression of Immune Receptor Gene Subsets

Abstract

The gene pool encoding PRR and NLR immune receptors determines the ability of a plant to resist microbial infections. Basal expression of these genes is prevented by diverse mechanisms since their hyperactivity can be harmful. To approach the study of epigenetic control of PRR/NLR genes we here analyzed their expression in mutants carrying abnormal repressive 5-methyl cytosine (5-mC) and histone 3 lysine 9 dimethylation (H3K9me2) marks, due to lack of MET1, CMT3, MOM1, SUVH4/5/6, or DDM1. At optimal growth conditions, none of the mutants showed basal expression of the defense gene marker PR1, but all of them had greater resistance to Pseudomonas syringae pv. tomato than wild type plants, suggesting they are primed to stimulate immune cascades. Consistently, analysis of available transcriptomes indicated that all mutants showed activation of particular PRR/NLR genes under some growth conditions. Under low defense activation, 37 PRR/NLR genes were expressed in these plants, but 29 of them were exclusively activated in specific mutants, indicating that MET1, CMT3, MOM1, SUVH4/5/6, and DDM1 mediate basal repression of different subsets of genes. Some epigenetic marks present at promoters, but not gene bodies, could explain the activation of these genes in the mutants. As expected, suvh4/5/6 and ddm1 activated genes carrying 5-mC and H3K9me2 marks in wild type plants. Surprisingly, all mutants expressed genes harboring promoter H2A.Z/H3K27me3 marks likely affected by the chromatin remodeler PIE1 and the histone demethylase REF6, respectively. Therefore, MET1, CMT3, MOM1, SUVH4/5/6, and DDM1, together with REF6, seemingly contribute to the establishment of chromatin states that prevent constitutive PRR/NLR gene activation, but facilitate their priming by modulating epigenetic marks at their promoters.

Keywords: 5-mC/H3K9me2 and H2A.Z/H3K27me3 marks; PRR/NLR immune receptor genes; defense cascades; epigenetics; priming.

FIGURE 1

Expression of defense marker gene and resistance to Pst infection in chromatin mutant plants. (A) PR1 transcript levels in non-infected or Pst-infected plants of the indicated genotypes analyzed at 8 and 24 h post inoculation (hpi). (B) Pst content in Col-0 wild type and mutant plants at 3 days after infection showing no basal PR1 expression (bottom). Values represent mean ± standard deviation (SD) of two technical replicates. Similar results were obtained in two independent infection experiments. *p < 0.05 from two-tailed unpaired t-test.

FIGURE 2

Activation of defense and PRR/NLR genes in chromatin mutants. (A) Intersection of all defense genes (top) or all PRR/NLR genes (bottom) activated (FDR < 0.05, FC > 1) in mutant plants. Transcriptome data were analyzed in three groups, corresponding to low, moderate, or high defense activation conditions. The number of activated genes in each sample is described at the left and represented with blue bars. Intersections between mutants are indicated by black lines linking black dots, and the number of genes in the intersection is described above vertical bars. The number of genes shared (Sh. by) or not shared (Not Sh.) among mutants are indicated at the top. (B) Clustering of PRR/NLR genes (base mean > 10, n = 326) in mutant plants under low defense activation conditions. (C) Pearson correlation between pair of mutants (top right panels), distribution of FC values in each sample (diagonal), and dot plot and linear regression of pairs of mutants (bottom left) for data shown in (B). (D) Clustering of the PRR/NLR genes (base mean > 10, n = 359) in all the datasets of the different mutants. In (B,D), a Z-score was use to scale the data by mutants (columns). Transcriptomes data analyzed (see Supplementary Table 3): *1: (Stroud et al., 2012), *2:(Stroud et al., 2014), *3:(Bourguet et al., 2018), *4:(Choi et al., 2020), *5:(Zhang C. et al., 2018), *6:(Ning et al., 2020), *7:(Shook and Richards, 2014), *8: (Le et al., 2020), *9:(Moissiard et al., 2014), *10:(Han et al., 2016). Transcriptomes with “low,” “moderate,” or “high” defense activation are indicated with different colors as indicated in Supplementary Table 5.

FIGURE 3

Mutant-specific PRR/NLR genes spontaneously activated in each mutant. (A) Intersection of PRR/NLR genes activated (FDR < 0.05, FC > 1) in each mutant plotted as in Figure 2A. Red intersections and numbers indicate genes that are consistently activated in different transcriptomes (upregulated at “low”/“moderate” -or “low” condition for met1- and also at a subsequent condition), and green intersections genes also included in (B). All transcriptome datasets were used to reach at least four mRNA-seq data for each mutant. Transcriptomes with “low,” “moderate,” or “high” defense activation are indicated with different colors. (B) Intersection of mutant-specific PRR/NLR genes among the different mutants (see section “Materials and Methods”). The number of genes shared (Sh. by) or not shared (Not Sh.) among mutants are indicated at the top. (C) Heatmap of the chromatin state of promoters of the 37 PRR/NLR genes activated in the mutants (B), described for wild type plants. Clustering is shown in the left. (D,E) Heatmap of the chromatin states of promoters (D) or coding sequences (E) of induced genes shown in Figure 2B (base mean > 10, FC > 0.2, n = 193) described for wild type plants. A Z-score was used in all heatmaps to scale the data by mutants (row). (F) Venn diagram showing the intersection of 37 mutant-specific PRR/NLR from (B) (“M.S.” PRR/NLR), and the PRR/NLR hypermethylated (H3K27 hyp.) in ref6, elf6, and ref6/elf6. Red numbers indicate the 18 mutant-specific PRR/NLR genes targeted by REF6 and/or ELF6. Fisher’s exact test was applied between mutant-specific PRR/NLR genes (this study) and PRR/NLR genes hypermethylated in the mutants (Antunez-Sanchez et al., 2020): mutant-specific PRR/NLR and H3K27 hyp ref6: p = 0.011; mutant-specific PRR/NLR and H3K27 hyp erf6/elf6: p = 0.033; mutant-specific PRR/NLR and H3K27 hyp elf6: p = 1. (G) Venn diagram showing the intersection of all PRR/NLR genes, PRR/NLR genes with decreased amount of H2A.Z in pie1 and 37 PRR/NLR genes from (B).