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Changes in DNA methylation contribute to rapid adaptation in bacterial plant pathogen evolution [1]

['Rekha Gopalan-Nair', 'Lipme', 'Université De Toulouse', 'Inrae', 'Cnrs', 'Castanet-Tolosan', 'Aurore Coissac', 'Ludovic Legrand', 'Céline Lopez-Roques', 'Get-Plage']

Date: 2024-09

Adaptation is usually explained by beneficial genetic mutations that are transmitted from parents to offspring and become fixed in the adapted population. However, genetic mutation analysis alone is not sufficient to fully explain the adaptive processes, and several studies report the existence of nongenetic (or epigenetic) inheritance that can enable adaptation to new environments. In the present work, we tested the hypothesis of the role of DNA methylation, a form of epigenetic modification, in adaptation of the plant pathogen Ralstonia pseudosolanacearum to the host during experimental evolution. Using SMRT-seq technology, we analyzed the methylomes of 31 experimentally evolved clones obtained after serial passages on 5 different plant species during 300 generations. Comparison with the methylome of the ancestral clone revealed a list of 50 differential methylated sites (DMSs) at the GTWWAC motif. Gene expression analysis of the 39 genes targeted by these DMSs revealed limited correlation between differential methylation and differential expression of the corresponding genes. Only 1 gene showed a correlation, the RSp0338 gene encoding the EpsR regulator protein. The MSRE-qPCR technology, used as an alternative approach for DNA methylation analysis, also found the 2 DMSs upstream RSp0338. Using site-directed mutagenesis, we demonstrated the contribution of these 2 DMSs in host adaptation. As these DMSs appeared very early in the experimental evolution, we hypothesize that such fast epigenetic changes can allow rapid adaptation to the plant stem environment. In addition, we found that the change in DNA methylation upstream RSp0338 remains stable at least for 100 generations outside the host and thus can contribute to long-term adaptation to the host plant. To our knowledge, this is the first study showing a direct link between bacterial epigenetic variation and adaptation to a new environment.

Funding: This work was supported by the French National Research Agency (ANR-17-CE20-0005-01 to RGN; AC; LL; XB; SG and AGu) and the "Laboratoires d’Excellence (LABEX)" TULIP (ANR-10-LABX-41 to RGN; AC; LL; XB; SG and AGu). RGN was funded by a PhD fellowship from the "Laboratoires d’Excellence (LABEX)" TULIP (ANR-10-LABX-41; ANR-11-IDEX-0002-02). AC was funded by the ‘Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement’ (INRAE), Plant Health and Environment Division (SPE) (INRAE-IB21-DOCT-SPE) and the LABEX TULIP (ANR-10-LABX-41; ANR-11-IDEX-0002-02). This work was performed in collaboration with the GeT core facility, Toulouse, France (DOI: 10.17180/nvxj-5333 ) ( http://get.genotoul.fr ) and was supported by France Génomique National infrastructure, funded as part of “Investissement d’avenir” program managed by the French National Research Agency (ANR-10-INBS-09 to CLR, CV and OB) and by the GET-PACBIO program (« Programme operationnel FEDER-FSE MIDI-PYRENEES ET GARONNE 2014-2020 » to CLR, CV and OB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2024 Gopalan-Nair et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

In this study, we analyzed the methylomes of 31 experimentally evolved clones using SMRT-seq. Comparison with the methylome of the ancestral GMI1000 clone revealed differential methylated sites (DMSs) at the GTWWAC motif in the evolved clones. Using site-directed mutagenesis, we demonstrated the contribution of 1 DMS in host adaptation, which, interestingly, turns out to be linked to a gene involved in the expression of a bacterial virulence determinant.

In our previous works, we conducted an experimental evolution of the R. pseudosolanacearum GMI1000 strain in order to better understand the molecular bases of adaptation. In this experiment, strain GMI1000 was maintained in a fixed plant line during 300 generations by serial passages from stem to stem. This experiment was conducted on 6 different plant species including susceptible hosts (tomato var. Marmande, eggplant var. Zebrina, pelargonium var. Maverick Ecarlate) and tolerant hosts (bean var. Blanc Précoce, cabbage var. Bartolo, tomato var. Hawaii 7996) [ 37 , 39 ]. Most of the evolved clones showed a better fitness (better growth rate) in their experimental host than the ancestral clone. Whole genome sequence analysis revealed between 0 and 3 mutations in the adapted clones, and the role of some mutations in host adaptation was demonstrated [ 37 , 39 – 41 ]. However, in several adapted clones, no mutation could be detected, suggesting that epigenetic modifications may play a role in host adaptation. In addition, transcriptomic analysis of these clones revealed important differential gene expression compared to the ancestral clone, thus reinforcing the hypothesis of a role of epigenetic modification in gene expression change [ 39 , 42 ].

A recent study compared the methylomes using SMRT-seq of 2 RSSC strains belonging to distant phylogenetic groups, the GMI1000 strain from phylotype I (R. pseudosolanacearum) and the UY031 strain from phylotype II (R. solanacearum) [ 38 ]. This work identified a commonly methylated motif in the 2 strains, the GTWWAC motif, 6mA methylated, associated with an MTase, M.RsoORF1982P, that is conserved in all RSSC genomes and across the Burkholderiaceae [ 38 ]. Analysis of the methylated regions in RSSC genomes identified genes involved in global and virulence regulatory functions, thus suggesting a role for DNA methylation in regulation of their expression.

R. pseudosolanacearum is part of the Ralstonia solanacearum species complex (RSSC), a soil-born plant pathogen responsible of the lethal bacterial wilt disease on more than 250 plant species including economically important crops such as tomato, potato, or banana [ 28 ]. This bacterium is worldwide distributed and represents a major threat in agriculture. It is characterized by a strong adaptive capacity, with no effective control method available today, and new strains capable of colonizing new hosts are continuously emerging [ 29 – 33 ]. Numerous works have been conducted with the aim of better understanding adaptive processes in RSSC. The role of genetic modifications of the bacterial genome such as mutation, transposable elements (TEs) movement, recombination, or horizontal gene transfer were reported [ 34 – 37 ]. However, the contribution of epigenetic modifications in RSSC adaptation has not yet been addressed.

A well-documented epigenetic mechanism known to be involved in the modification of the phenotype is DNA methylation. DNA methylation consists in the addition of a methyl group (CH 3 ) on the adenine or cytosine base of DNA catalyzed by DNA methyltransferases (MTases) that recognize specific DNA motifs. In bacterial genomes, methylated DNA is found in the forms of 6mA (6-methyladenine), which is the most prevalent form, 4mC (4-methylcytosine), and 5mC (5-methylcytosine) [ 14 , 15 ]. Many works demonstrated the role of DNA methylation in the regulation of important cellular functions in bacteria, including DNA replication, DNA repair, chromosome segregation, transcriptional regulation, phenotypic heterogeneity, and virulence [ 16 – 22 ]. Nowadays, thanks to the Pacbio sequencing technology, which enables the sequencing of single molecules in real time (SMRT-seq) without amplification, it is possible to analyze the 6mA and 4mC methylation profile of bacteria [ 14 , 15 , 23 – 27 ]. Here, we used SMRT-seq technology to explore the DNA methylation profile (methylome) of the model bacterial plant pathogen Ralstonia pseudosolanacearum. The purpose of this study was to test the hypothesis of methylome variation during the experimental adaptation of the bacteria to various host plants and investigate the potential role of methylome changes in the generation of adapted phenotypes.

Faced with the selection pressure imposed by their environment, pathogens must continuously adapt to survive and multiply. Many works aim to better understand the adaptive processes of pathogens in order to better apprehend the sustainability of the control strategies. Adaptation, the modification of the phenotype as a result of natural selection, is usually explained by beneficial genetic mutations that are transmitted from parents to offspring and become fixed in the adapted population [ 1 – 3 ]. However, more and more studies show that genetic mutation analysis alone is not sufficient to fully explain the processes of adaptive evolution and report the role of nongenetic (or epigenetic) inheritance in the generation of adapted phenotypes [ 4 , 5 ]. Models suggest that epigenetic inheritance of the parental phenotype can be adaptive in slowly fluctuating and correlated environments, since the parent and offspring will most often share the same environmental conditions [ 6 , 7 ]. However, direct tests of this prediction are, so far, lacking. Epigenetic changes were described to be more involved in short-term adaptation, or acclimation, by inducing phenotypic plasticity [ 8 ]. This was supported by the observation that epigenetic changes occur at a faster rate than genetic mutations but may be less stable [ 9 , 10 ]. However, recent works also support the hypothesis that epigenetic modifications could impact long-term adaptive responses to changing environments through the transgenerational inheritance of epigenetic signatures [ 5 , 8 , 10 – 13 ].

Results

Defining the methylation profile of strain GMI1000 In order to detect potential changes in the methylation profile of evolved clones, we first established the methylated motifs in the wild-type ancestor GMI1000 using SMRT-seq technology. In order to limit the number of cells in division and avoid a bias towards hemimethylated marks, genomic DNA was prepared from bacterial cells collected at the beginning of stationary phase. Growth was performed in synthetic medium with glutamine to mimic xylem environment of the plant, glutamine being the main compound of xylem sap in most plant species [43]. The global analysis of all modification marks on the GMI1000 DNA identified a total number of 45,831 modification marks above default thresholds. This number was much lower than that found in our previous work that reported 229,207 modification marks [38]. This difference probably results from several factors such as changing SMRT-seq technology and analysis pipelines. In the present study, 2 methylated motifs were detected in the GMI1000 genome, GTWWAC and YGCCGGCR. As the YGCCGGCR motif was detected with a very low percentage of methylation, although the sequencing depth was very high (160×), it suggested that this motif is associated to 5mC modification, which is difficult to detect by SMRT-seq. The third motif reported previously, CCCAKNAVCR [38], was not detected in the present work. As this motif was very degenerate and detected with a very weak signal, it was probably a false positive detection. For the comparative methylation analysis using SMRT-seq technology, we thus investigated the methylation profile of the GTWWAC motif in the ancestral and evolved clones. A total of 392 GTWWAC motifs are present in the GMI1000 genome and affect 366 genes either in the promoter region (i.e., <300 bp upstream from a start codon) or in the gene ORF, thus affecting 7% of all GMI1000 genes, whose number has been estimated at 5,129 [44]. In our culture and growth phase conditions and according to SMRT-seq data, 10 GTWWAC motifs were detected unmethylated and 9 motifs were hemimethylated (DNA methylation of either strand–or strand +) in the GMI1000 genome (Tables 1 and S1). The analysis of the distribution of methylated and unmethylated GTWWAC motifs with respect to genes or putative promoter regions showed that most (82%) of the unmethylated GTWWAC motifs were located in putative promoter regions, while only 42% of the methylated motifs were located in these regions (Fig 1). These unmethylated sites could be associated to potential regulatory regions where a competition between the MTase and a DNA binding protein could occur [45]. These sites specifically concerned the RSc0958 gene encoding a type VI secretion system tip VgrG family protein [46], the epsR gene (2 motifs) encoding the negative regulator of exopolysaccharide (EPS) production [47] and the efe gene encoding the ethylene-forming enzyme [48] (Table 1). PPT PowerPoint slide

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TIFF original image Download: Fig 1. Distribution of the methylated and unmethylated GTWWAC motifs in the GMI1000 genome. Numbers and proportions of GTWWAC motifs located in gene body (blue), in putative promoter region (i.e., <300 bp upstream from a start codon) (orange) or in intergenic region (grey). When the same GTWWAC motif affects 2 genes, it was counted twice. The data underlying this figure can be found in S1 Data. https://doi.org/10.1371/journal.pbio.3002792.g001 PPT PowerPoint slide

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TIFF original image Download: Table 1. Genomic regions of the GMI1000 strain of R. pseudosolanacearum with a GTWWAC motif detected unmethylated or hemimethylated at the beginning of the stationary phase during growth in synthetic medium with glutamine, according to SMRT-seq data. https://doi.org/10.1371/journal.pbio.3002792.t001

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002792

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