Epigenetic Modulating Chemicals Significantly Affect the Virulence and Genetic Characteristics of the Bacterial Plant Pathogen Xanthomonas campestris pv. campestris

Abstract

Epigenetics is the study of heritable alterations in phenotypes that are not caused by changes in DNA sequence. In the present study, we characterized the genetic and phenotypic alterations of the bacterial plant pathogen Xanthomonas campestris pv. campestris (Xcc) under different treatments with several epigenetic modulating chemicals. The use of DNA demethylating chemicals unambiguously caused a durable decrease in Xcc bacterial virulence, even after its reisolation from infected plants. The first-time use of chemicals to modify the activity of sirtuins also showed some noticeable results in terms of increasing bacterial virulence, but this effect was not typically stable. Changes in treated strains were also confirmed by using methylation sensitive amplification (MSAP), but with respect to registered SNPs induction, it was necessary to consider their contribution to the observed polymorphism. The molecular basis of the altered virulence was deciphered by using dualRNA-seq analysis of treated Xcc strains infecting Brassica rapa plants. The results of the present study should promote more intensive research in the generally understudied field of bacterial epigenetics, where artificially induced modification by epigenetic modulating chemicals can significantly increase the diversity of bacterial properties and potentially contribute to the further development of the fields, such as bacterial ecology and adaptation.

Keywords: DNA methylation; Xanthomonas campestris; bacterial epigenetics; dual RNA-seq; virulence.

Figure 1

Scale used to evaluate plant reactions to the pathogen. 1—No symptoms. 2—Lesions on 25% of the leaves. 3—Lesions on 25–50% of the leaves. 4—Lesions on 50–75% of the leaves. 5—Lesions on more than 75% of the leaves.

Figure 2

Impact of individual epigenetic modulating chemical treatments on the virulence of ETSs 15 days after the inoculation of B. rapa plants. Bright columns of the respective color represent virulence of the strains used immediately after treatment. Dark columns of the respective color represent virulence of the strains reisolated from the plants 20 days after the first round of inoculation. Means of three independent repetitions are presented, and the standard deviation is indicated as error bars. UPC = untreated positive control (inoculation by untreated Xcc strain); NC = negative control (no Xcc was applied on plants); * = unconfirmed presence of Xcc in the CAM-derived ETS after the second round of inoculation.

Figure 3

Dendrogram depicting similarities between DNA methylation-sensitive profiles of individual strains treated by respective epigenetic modulating chemicals. Numbered samples represent biological replicates of individual variants to control their internal variability.

Figure 4

Characterization of differentially expressed contigs within the analyzed Xcc WHRI 1279A variants. (A) PCA of normalized altered transcriptomes of positive (deplPOS), deplCAY- and deplLOM-treated Xcc variants during their infection of B. rapa plants (B) Selection process for contigs from the first (deplPOS + deplCAY vs. deplLOM Xcc strains) and the second (deplCAY vs. deplLOM + deplPOS) eigenvector at the level of 5%. (C) Enriched KEGG pathways identified as having the highest impact on the differences between the deplLOM and (deplCAY + deplPOS) transcriptomes.

Figure 5

Characterization of differentially expressed contigs in the transcriptomes of the analyzed variants of B. rapa after infection by individual Xcc WHRI 1279A variants. (A) PCA of the normalized altered transcriptomes of B. rapa plants infected by individual Xcc variants (1st and 2nd eigenvectors). (B) PCA of normalized altered transcriptomes of B. rapa plants infected by individual Xcc variants (2nd and 3rd eigenvectors). (C) Selection process of the contigs from the first eigenvector (distinguishing the negative sample deplNEG from the rest) at the level of 1% and respective enriched gene ontology analysis. (D) Selection process of the contigs from the second eigenvector (distinguishing the positive sample deplPOS from the rest) at the level of 1% and respective enriched gene ontology analysis. (E) Selection process of the contigs from the third eigenvector (distinguishing the deplCAY sample from the rest) at the level of 1% and respective enriched gene ontology analysis.

Figure 6

Results of SNPs calling within potentially methylatable motifs by using dual RNA-seq data. * = motif usually associated with m4C methylation; ** = motif usually associated with m5C methylation; respective complementary sequences are also included in depicted frequencies.