Genome-wide DNA hypomethylation shapes nematode pattern-triggered immunity in plants

Abstract

A role for DNA hypomethylation has recently been suggested in the interaction between bacteria and plants; it is unclear whether this phenomenon reflects a conserved response. Treatment of plants of monocot rice and dicot tomato with nematode-associated molecular patterns from different nematode species or bacterial pathogen-associated molecular pattern flg22 revealed global DNA hypomethylation. A similar hypomethylation response was observed during early gall induction by Meloidogyne graminicola in rice. Evidence for the causal impact of hypomethylation on immunity was revealed by a significantly reduced plant susceptibility upon treatment with DNA methylation inhibitor 5-azacytidine. Whole-genome bisulphite sequencing of young galls revealed massive hypomethylation in the CHH context, while not for CG or CHG nucleotide contexts. Further, CHH hypomethylated regions were predominantly associated with gene promoter regions, which was not correlated with activated gene expression at the same time point but, rather, was correlated with a delayed transcriptional gene activation. Finally, the relevance of CHH hypomethylation in plant defence was confirmed in rice mutants of the RNA-directed DNA methylation pathway and DECREASED DNA METHYLATION 1. We demonstrated that DNA hypomethylation is associated with reduced susceptibility in rice towards root-parasitic nematodes and is likely to be part of the basal pattern-triggered immunity response in plants.

Keywords: Meloidogyne graminicola; Oryza sativa; DNA hypomethylation; RdDM; basal defence; nematodes; pattern-triggered immunity; rice.

Fig. 1

Nematode (Meloidogyne graminicola) infection causes strong hypomethylation in rice plants, which is associated with plant defence. (a) Enzyme‐linked immunosorbent assay reveals global DNA hypomethylation upon nematode infection in young (3 d post‐inoculation) galls induced in rice roots (n = 12). (b) Foliar application of 5‐azacytidine (50 µM) 24 h before nematode inoculation makes plants less susceptible to nematode infection, while root and shoot length were unaffected (n = 20); galls and nematodes were counted 2 wk post‐inoculation. *, P < 0.1; **, P < 0.05. Error bars indicate SEM.

Fig. 2

Nematode‐associated molecular pattern (NAMP) and bacterial pathogen‐associated molecular pattern (PAMP) treatments cause strong hypomethylation in plants 3 d post‐treatment. (a) Treatment of rice plants with NAMP obtained from Meloidogyne graminicola compared with untreated plants (n = 9). (b) Treatment of tomato plants with NAMP obtained from Meloidogyne incognita compared with untreated plants (n = 10). (c) Treatment of rice plants with NAMP obtained from Pratylenchus zeae compared with untreated plants (n = 10). (d) Treatment of rice plants with bacterial PAMP (flg22) compared with untreated plants. (e) Treatment of tomato plants with bacterial PAMP (flg22) compared with untreated plants. *, P < 0.1; **, P < 0.05. Error bars indicate SEM. flg22, flagellin 22.

Fig. 3

Overview of promoter/terminator methylation and genome‐wide distribution of genomic regions in 3 d post‐inoculation (dpi) galls induced by Meloidogyne graminicola in rice. (a) DNA methylation of promoter and terminator regions in 3 dpi galls and control root tips, 2 kb upstream and 2 kb downstream from start (left) or end (right), of genes and transposable element (TE) classes RLC, RLG, RLX, RSU, and DTM (bin size of 100 bases). (b) Genome‐wide overview. Outer circle represents the 12 rice chromosomes. Black bands represent the centromere regions. Inner circles represent the distribution of differentially methylated regions across the (A) chromosomes, (B) genes, (C) TE class DTM, (D) TE class RLG, (E) TE class RLX, (F) TE class RLC, (G) TE class RSU. DTM, DNA transposon mutator; RLG, Retroelement long tandem repeat Gypsi; RLC, Retroelement long tandem repeat Copia; RLX, Retroelement long tandem repeat ‘Unknown’; RSU, Retroelement short interspersed nuclear element.

Fig. 4

Differentially methylated regions (DMRs) in 3 d post‐inoculation (dpi) galls induced by Meloidogyne graminicola in rice. (a) Methylation pattern of DMRs. (b) Significance of associations between DMRs and genomic elements. The violin plots show the distribution of the number of the overlaps between DMRs and randomly scattered transposable element (TE) or gene regions (1000 simulations). The dots show the observed number of overlaps between DMRs and TE gene regions in the whole‐genome bisulphite sequencing data set of nematode‐induced galls. Asterisks indicate significant over or underrepresentation (P < 0.05). DTM, DNA transposon mutator; RLG, Retroelement long tandem repeat Gypsi; RLC, Retroelement long tandem repeat Copia; RLX, Retroelement long tandem repeat ‘Unknown’; RSU, Retroelement short interspersed nuclear element.

Fig. 5

DNA methylation association with gene expression in galls induced by Meloidogyne graminicola in rice. (a) Significance of associations between differentially methylated (DM) regions (DMRs) and differentially expressed (DE) genomic elements. The violin plots show the distribution of overlaps between DMRs and a randomly sampled group of genes/promoters. The dots show the observed number of overlaps between DMRs and DE genes, or promoters of DE genes, in the whole‐genome bisulphite sequencing data set of nematode‐induced galls. *, P < 0.05. (b) Venn diagrams showing the number of genes that overlap with either their gene body or their promoter with 3 d post‐inoculation (dpi) CHH DMRs (DM genes) and the number of DE genes at 3 or 7 dpi. (c) DNA methylation at 3 dpi (percentage) in galls and roots for interval 24 655 000–24 662 000 on chromosome 4 (bin size of 100 bases). Note the similar methylation levels between galls and root for CG and CHG methylation, the CHH hypomethylation in the promoter region of gene OsbHLH65 at 7 dpi. The DMR is indicated with an orange box. (d) Quantitative reverse transcription PCR‐based expression profile of five genes that contain a DMR in their promoter in 3 dpi and 7 dpi gall tissue; OsbHLH65 is indicated with a red dashed box. Error bars indicate SEM. *, P < 0.05. bHLH, basic helix–loop–helix.

Fig. 6

DNA hypomethylation confers reduced susceptibility to Meloidogyne graminicola infection in rice plants. Mutants of (a) DICER‐LIKE 3b (dcl3b, n = 20), (b) ARGONAUTE 4a/b (ago4a/b, n = 19), (c) WAVY LEAF 1 (waf1, n = 20). (d) DOMAINS REARRANGED METHYLTRANSFERASE (drm2, n = 23) are less susceptible to nematode infection. Galls and nematodes were counted 2 wk post‐inoculation. *, P < 0.1; **, P < 0.05. Error bars indicate SEM.