Comparative Analysis of Ralstonia solanacearum Methylomes

Ralstonia solanacearum is an important soil-borne plant pathogen with broad geographical distribution and the ability to cause wilt disease in many agriculturally important crops. Genome sequencing of multiple R. solanacearum strains has identified both unique and shared genetic traits influencing their evolution and ability to colonize plant hosts. Previous research has shown that DNA methylation can drive speciation and modulate virulence in bacteria, but the impact of epigenetic modifications on the diversification and pathogenesis of R. solanacearum is unknown. Sequencing of R. solanacearum strains GMI1000 and UY031 using Single Molecule Real-Time technology allowed us to perform a comparative analysis of R. solanacearum methylomes. Our analysis identified a novel methylation motif associated with a DNA methylase that is conserved in all complete Ralstonia spp. genomes and across the Burkholderiaceae, as well as a methylation motif associated to a phage-borne methylase unique to R. solanacearum UY031. Comparative analysis of the conserved methylation motif revealed that it is most prevalent in gene promoter regions, where it displays a high degree of conservation detectable through phylogenetic footprinting. Analysis of hyper- and hypo-methylated loci identified several genes involved in global and virulence regulatory functions whose expression may be modulated by DNA methylation. Analysis of genome-wide modification patterns identified a significant correlation between DNA modification and transposase genes in R. solanacearum UY031, driven by the presence of a high copy number of ISrso3 insertion sequences in this genome and pointing to a novel mechanism for regulation of transposition. These results set a firm foundation for experimental investigations into the role of DNA methylation in R. solanacearum evolution and its adaptation to different plants.

This work was funded by the Spanish Ministry of Economy and Competitiveness projects AGL2013-46898-R and AGL2016-78002-R to MV and by a U.S. National Science Foundation (MCB-1158056) award to IE. We also acknowledge financial support from the CERCA Program of the Catalan Government (Generalitat de Catalunya), the University of Maryland, Baltimore County Office of Research, the “Severo Ochoa Program for Centers of Excellence in R&D” 2016–2019 (SEV-2015-0533) of the Spanish Ministry of Economy and Competitiveness and the COST Action SUSTAIN (FA1208) from the European Union. RG is the recipient of a doctoral fellowship [grant 2012/15197-1, São Paulo Research Foundation (FAPESP)]. JS has a researcher fellowship from CNPq (304881/2015-5). MP holds an APIF doctoral fellowship from Universitat de Barcelona. This work was also performed in collaboration with the GeT core facility, Toulouse, France (http://get.genotoul.fr), and was supported by France Génomique National infrastructure, funded as part of “Investissement d'avenir” program managed by Agence Nationale pour la Recherche (contract ANR-10-INBS-09).

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