Genome-wide identification of P. syringae DNA methylation.

(A) The circle map displays the distribution of 6mA, 5mC, and 5mC in Psph WT. (B) The Venn plot reveals overlapped genes within three types of DNA methylation of Psph WT. (C) The circle map displays the distribution of 6mA, 5mC, and 5mC in Pst WT. (D) The Venn plot reveals overlapped genes within three types of DNA methylation of Pst WT. (E) The circle map displays the distribution of 6mA, 5mC, and 5mC in Pss WT. (F) The Venn plot reveals overlapped genes within three types of DNA methylation of Pss WT.

Functional enrichment analysis of methylation sites in three P. syringae strains.

(A) Repartition of the total pool of modified genes among strains. (B) Proportion of methylated genes detected in one, two, or three genomes for all P. syringae strains and conserved DNA methylation sites with detected genes. (C) The dot plot reveals the functional annotation enrichment of genes within different modified bases among three strains in GO and KEGG terms. The size of the dots indicates the number of related genes.

DNA methylation motifs in P. syringae.

(A) 6mA methylation motifs found in Psph using SMRT-seq. The black arrows indicate the site of adenine methylation. (B) Bar plot shows the abundance of methylated numbers throughout all motif sites in Psph. (C) 6mA and 4mC methylation motifs found in Pst using SMRT-seq. The black arrows indicate the site of base methylation. (D) Bar plot shows the abundance of methylated numbers throughout all motif sites in Pst.

Transcriptional changes profiling of hsdMSR mutant in Psph.

(A) The volcano plot reveals DEGs between Psph WT and ΔhsdMSR. The DEGs were (|log2 FC| > 1 and adjusted P value < 0.05) in blue (Ribosomal protein), pink (Alginate biosynthesis), and yellow (T3SS). Each dot represents one gene. (B) Bubble plot shows enriched GO (Red) and KEGG (Blue) terms based on the DEGs between Psph WT and ΔhsdMSR. The x-axis shows the significance of functional annotation terms (−log10 adjusted P-value), and the y-axis indicates the Z-score of terms. The bubble size represents the gene number of terms. (C) COG classification and distribution of up-regulated DEGs. (D) COG classification and distribution of down-regulated DEGs. COG terms are highlighted in different colors. (E) Venn plot reveals the overlapped genes between DEGs and genes within the HsdMSR motif.

Influence of HsdMSR on virulence and metabolism in Psph.

(A) HsdMSR negatively regulated T3SS-related genes. Data are shown as means ± SD (n = 3). (B) Disease symptoms caused by Psph WT, ΔhsdMSR, and the complemented strains, photographing 6 days after inoculation of 105 CFU/mL bacteria. (C) HsdMSR negatively regulated alginate biosynthesis-related genes. Data are shown as means ± SD (n = 3). (D) The quantification of biofilm production in the Psph WT, ΔhsdMSR, and the complemented strains using a crystal violate staining assay. (E) HsdMSR positively regulated ribosomal protein-related genes. Data are shown as means ± SD (n = 3). (F) The scatterplot shows the TE and mRNA change between Psph WT and ΔhsdMSR. The x-axis presents the log2FC of the mRNA level, and the y-axis shows the log2FC of the TE. The greater TE is represented in red, whereas the lesser TE is displayed in blue.

6mA methylation regulates gene transcription based on fully methylated.

(A) Upstream region sequence of hrpF carrying the HsdMSR motif. Adenine methylation is highlighted in red. (B) Constantly lux activity detection of the hrpF between Psph WT and ΔhsdMSR. (C) Lux activity of hrpF between Psph WT and ΔhsdMSR at the time point 24 hours. (D) Lux activity of hrpF between Psph WT and ΔhsdMSR with point mutation. In the HsdMSR motif, an “A” was replaced by a “C”, highlighted in bold and underlined. (E) Lux activity of hrpF between Psph WT and ΔhsdMSR with double point mutations. In the HsdMSR motif, two “A” was replaced by a “C”, highlighted in bold and underlined.