Role of Epigenetic Modifications in a Plant Pathogen during Host Jump and Adaptation – EPI-PATH

Epigenetic modifications are heritable changes in gene expression that occur without changes in the DNA sequence. They are responsible of important phenotypic trait variations in all kingdoms of life. DNA methylation is the most common form of epigenetic modification in prokaryotes and eukaryotes and is catalyzed by DNA methyltransferases (MTases). The 5mC (5-methylcytosine) modification can be detected using treatment of DNA with bisulfite and various sequencing technologies. Recently, the development of the PacBio sequencing technology has enabled sequencing of single molecules in real time (SMRT) and detecting other methylated bases (e.g. 6mA, 4mC). In bacteria, the biological consequences of DNA methylation have not been extensively investigated yet. The impact of DNA methylation in virulence has been reported in some human and animal pathogens. However, the involvement of DNA methylation in host-pathogen interaction remains largely unexplored for most of the bacterial plant pathogens.
The main purpose of the EPI-PATH project is to investigate the role of DNA methylation in the plant pathogenic bacterium Ralstonia solanacearum during host jump and adaptation. This bacterium is a Species Complex (named RSSC) responsible of the bacterial wilt, one of the most destructive plant bacterial diseases affecting more than 250 plant species including many important crops (potato, tomato, banana, peanuts).
For that purpose, we will first study the MTases in the GMI1000 strain. In vitro and in planta expression of the MTases will be compared. The role of each GMI1000 MTase in virulence will be investigated by generating single-deleted mutants. The impact of MTase deletion on the GMI1000 methylome will be determined using both SMRT and bisulfite/Illumina sequencing. Here, protocols for library construction and DNA treatment will be optimized in order to detect the 6mA, 4mC and 5mC modifications. For data analysis, existing bioinformatics tools will be used and adapted to high GC content genomes, such as RSSC genomes.
The MTase repertoires and expression, and the methylomes will then be investigated in 20 wild-type strains representative of the RSSC genetic diversity. This should provide a global overview of the methylome diversity in RSSC and potential correlation with host specificity.
In a second time, we will address the methylome diversification in 30 experimentally evolved clones, derived from the GMI1000 strain after serial passages during 300 generations in a given host. These clones show a fitness gain in their experimental host (tomato, eggplant, bean, cabbage) but their genomes reveal only little even any mutation compared to the ancestral clone. The methylomes of the ancestral and the evolved clones will be compared in order to identify differential methylation marks. We will focus on differential methylation marks found in several independent evolved clones. The transcriptome profiles of the evolved clones will also be investigated in order to connect differential methylation marks in gene promoters with transcriptome variation. For the most interesting candidates, the effect of individual methylation marks on the in planta fitness will be characterized by conducting site-directed mutagenesis in the ancestral GMI1000 clone and then measure the fitness of the mutants in planta. This should validate the impact of DNA methylation in the expression of the adaptive phenotype of GMI1000 on different host plants.
The EPI-PATH project will provide a first step toward understanding the biological consequences of DNA methylation in RSSC, which will represent one of the first plant pathogen investigated for epigenetic modification impact. The knowledge on the MTases and the differential methylation marks within RSSC strains together with the tools optimized in this project will constitute solid roots for future studies aiming at identifying the role of epigenetic modifications in host adaptation in other plant pathogenic bacteria.