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DNA demethylases are required for myo-inositol-mediated mutualism between plants and beneficial rhizobacteria

Abstract

Root-associated soil bacteria can strongly influence plant fitness. DNA methylation is an epigenetic mark important to many fundamental biological processes; however, its roles in plant interactions with beneficial microbes remain elusive. Here, we report that active DNA demethylation in Arabidopsis controls root secretion of myo-inositol and consequently plant growth promotion triggered by Bacillus megaterium strain YC4. Root-secreted myo-inositol is critical for YC4 colonization and preferentially attracts B. megaterium among the examined bacteria species. Active DNA demethylation antagonizes RNA-directed DNA methylation in controlling myo-inositol homeostasis. Importantly, we demonstrate that active DNA demethylation controls myo-inositol-mediated mutualism between YC4 and Solanum lycopersicum, thus suggesting a conserved nature of this epigenetic regulatory mechanism.

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Fig. 1: Active DNA demethylation is required for the mutualism between Arabidopsis and B. megaterium YC4-R4.
Fig. 2: Myo-inositol supplementation to the Arabidopsis DNA demethylation mutants restored plant mutualism with B. megaterium YC4-R4.
Fig. 3: Plant biosynthesis of myo-inositol is required for the mutualism between Arabidopsis and B. megaterium YC4-R4.
Fig. 4: Myo-inositol preferentially stimulates B. megaterium colonizing activities through transcriptional regulation.
Fig. 5: Plant transcriptomic responses to YC4-R4 and myo-inositol homeostasis are disrupted in rdd compared with Col-0.
Fig. 6: ROS1-mediated DNA demethylation counteracts RdDM in controlling myo-inositol homeostasis genes.
Fig. 7: Myo-inositol mediates DNA demethylation-dependent mutualism between B. megaterium YC4-R4 and plants, including the tomato.

Data availability

The mRNA-Seq and WGBS data from this publication have been deposited in the NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) and are accessible through Gene Expression Omnibus Series accession number GSE83802 (ref. 23). Source data are provided with this paper.

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Acknowledgements

We thank the core facilities of Genomics, Plant Proteomics and Metabolomics, and Plant Cell Biology at the Shanghai Center for Plant Stress Biology for sequencing, GC–MS and microscopic analyses, respectively. We also express special thanks to A. Álvarez for artistic collaboration in the design of the model presented in this manuscript. Research in the laboratory of H.Z. has been supported by the Chinese Academy of Sciences and Thousand Talents Program for Young Scientists, China. J.I.V. was supported by the Chinese Academy of Sciences President’s International Fellowship Initiative fellowship.

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Contributions

H. Zhang designed the project. J.I.V. performed or led all of the experiments and coordinated with Y.Y. for the RNA-Seq and WGBS data analyses. H.Zi, L.P., R.L. and K.T. analysed RNA-Seq and/or WGBS raw data for the DEG and DMR lists. D.H., S.L., R.K., W.W., W.H., Z.L. and D.M. participated in the experiments and/or data analyses. H. Zhang and J.I.V. wrote the manuscript with input from P.W.P., C.-P.S. and J.-K.Z.

Corresponding author

Correspondence to Huiming Zhang.

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The authors declare no competing interests.

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Peer review information Nature Plants thanks Glenda Gillaspy, Mingbo Wang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Bacillus megaterium YC4-R4 induces growth-promotion in various plant species (Related to Fig. 1).

a, Arabidopsis thaliana and Brachypodium distachyon showed YC4-R4-induced growth-promotion. Plant images are of the same scale, as indicated by the vertical rulers. These experiments were repeated three times with similar results (n = 6 biological independent samples). b–d, YC4-R4 induced plant growth-promotion in Solanum lycopersicum, Brassica napus and Capsicum annuum, respectively. The lower panels show quantification of whole plant or shoots dry weight. The boxplots and the pictures show representative data from three independent experiments (Solanum lycopersicum and Capsicum annuum, n = 18; Brassica napus, n = 16 biological independent samples, respectively). Whiskers represent the min to max data range; the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. All sets of data were compared by t-student test by two-tailed way and 95% confident intervals (p < 0.05), in which * means statistical differences. e, YC4-R4 induced plant growth-promotion in Arabidopsis mutants (nrpd1-3 and nrpe1-11) with defective RNA-directed DNA methylation, but not in the rdd mutant that is defective in DNA demethylation. Plants were pictured at 14 DAT (left panel) and their dry weights were quantified (right panel). The boxplots show representative data from three independent experiments (n = 30). Whiskers represent the min to max data range; the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. All sets of data were compared by t-student test by two-tailed way and 95% confident intervals (p < 0.05), in which * means statistical differences. In all data sets described above, error bar means SD.

Source data

Extended Data Fig. 2 myo-Inositol supplementation to the Arabidopsis DNA demethylation mutants restored root association with B. megaterium YC4-R4 (Related to Fig. 2).

a, Quantification of root-associated bacteria from plants that were grown in sterile growth medium, treated with YC4-R4 and colonization rate was recorded at 14 DAT. The boxplots show representative data from three independent experiments (n = 30 biological independent samples). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Sets of data were compared by t-student test by two-tailed way and 95% confident intervals (p < 0.05), in which * means statistical differences. In all data sets described above, error bar means SD. b, Visualization of root-associated bacteria from plants that were grown in sterile growth medium and were treated with YC4-R4 at 5 DAT. The green arrows point to example spots where bacteria are present. This experiment was carried out three times with similar results (n = 5 biological independent samples).

Source data

Extended Data Fig. 3 myo-Inositol restored B. megaterium YC4-R4 biofilm formation on the roots of rdd and ros1 (Related to Fig. 4).

Roots of Col-0, rdd and ros1 were shown in bright field images (a) and Scanning Electronic Microscopy (SEM) images (b). For samples with no visualized biofilm, less amplified images were used to show no visual findings. Red area or arrows indicate bacteria in the bright field images. Blue area indicates biofilm in the SEM images. In both cases, images show representative pictures from three independent experiments with similar results (n = 5 biological independent samples).

Extended Data Fig. 4 myo-Inositol preferentially influences Bacillus megaterium (Related to Fig. 4).

a, Root colonization of the bacteria including B. megaterium YC4-R4, B. megaterium TG1-E1, B. amyloliquefaciens GB03 and P. syringe DC3000. The effects of myo-inositol on bacteria colonization to rdd roots were statistically analyzed based on each bacteria strain. The bar graph shows representative data from three independent experiments (n = 3 biological independent samples). Sets of data were analyzed by one-way ANOVA including a Tukey’s test, where * means statistical difference by p < 0.05. b, Gene Ontology (GO) categorization of DEGs that were up-regulated in myo-inositol-treated YC4-R4. c, GO categorization of DEGs that were down-regulated in myo-inositol-treated YC4-R4. d, Relative expression levels of a group of key genes involved in bacteria colonization-related activities. The bar graphs show representative quantitative real-time PCR results from three independent experiments (n = 3 biological independent samples). Sets of data were compared by t-student test by two-tailed way and 95% confident intervals (p < 0.05), in which * means statistical differences with mock set. In all data sets described above, error bar means SD.

Source data

Extended Data Fig. 5 Transcriptional regulation of plant responses to YC4-R4 and myo-inositol homeostasis are altered by defective DNA demethylation (Related to Fig. 5).

a, Gene Ontology (GO) categorization of YC4-R4-induced DEGs in rdd. Diagrams are designed based on Cytoscape Software. The size of solid-lined circles represents the number of genes in each GO category. Scale color bar indicates the significance of gene expression in each GO category. Clustered biological processes were indicated manually by dash-lined gray circles, and the GO term is underlined if the cluster is present only in either the YC4-R4-induced or YC-repressed DEGs. b, GO categorization of YC4-R4-repressed DEGs in rdd. c, Relative expression levels of myo-inositol homeostasis genes in Col-0, rdd and ros1. The bar graph shows representative quantitative real-time PCR results from three independent experiments (n = 3 biological independent samples). Sets of data were compared by t-student test by two-tailed way and 95% confident intervals (p < 0.05), in which * means statistical differences with Col-0 set. In all data sets described above, error bar means SD.

Source data

Extended Data Fig. 6 ROS1 counteracts RdDM in controlling DNA methylation at gene-associated loci (Related to Fig. 6).

a, Snapshots of WGBS results that show DNA hyper methylation at the vicinity regions of myo-inositol homeostasis genes in rdd with (T) and without (M) YC4-R4 treatment. b, Snapshots of WGBS results showing that ROS1-mediated DNA demethylation counteracts RdDM at the vicinity regions of myo-inositol homeostasis genes. c, Categorization of Arabidopsis genomic loci where DNA methylation is co-regulated by ROS1 and RdDM. Gene-associated regions were further subject to GO analysis, revealing an enrichment of genes related to cell wall organization.

Extended Data Fig. 7 Tomato myo-inositol homeostasis genes are subject to the regulation by SlDML2-dependent DNA demethylation (Related to Fig. 7).

a, Quantification of plant dry weights at 14 DAT. The boxplots show representative data from three independent experiments (n = 18 biological independent samples). Whiskers represent the min to max data range; the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. In both cases (C and D), sets of data were compared by t-student test by two-tailed way and 95% confident intervals (p < 0.05), in which * means statistical differences. b, Relative expression levels of three tomato homologs of Arabidopsis myo-inositol homeostasis genes. The bar graphs show representative quantitative real-time PCR results from three independent experiments (n = 3 biological independent samples). Sets of data were compared by t-student test by two-tailed way and 95% confident intervals (p < 0.05), in which * means statistical differences with WT set. c, Snapshots of WGBS results that show DNA hyper methylation associated with myo-ionsitol homeostasis genes in tomato. d, A heat map of myo-inositol catabolism DEGs identified by RNAseq in myo-inositol-treated YC4-R4 compared to YC4-R4 under the mock condition. Values are representatives from three independent experiments (n = 4 biological independent samples). e, Relative expression levels of myo-inositol catabolism genes in YC4. The bar graph shows representative quantitative real-time PCR results from three independent experiments (n = 3 biological independent samples). Sets of data were compared by t-student test by two-tailed way and 95% confident intervals (p < 0.05), in which * means statistical differences with mock set. f, The effects of L-threonin on YC4-R4 chemotaxis responses. The bar graph shows representative RFU (relative fluorescent unit) values from three independent experiments (n = 9 biological independent samples). Sets of data were compared by t-student test by two-tailed way and 95% confident intervals (p < 0.05), in which * means statistical differences with mock set. In all data sets described above, error bar means SD.

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Vílchez, J.I., Yang, Y., He, D. et al. DNA demethylases are required for myo-inositol-mediated mutualism between plants and beneficial rhizobacteria. Nat. Plants 6, 983–995 (2020). https://doi.org/10.1038/s41477-020-0707-2

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