Hierarchical height and collaboration of TFs reveal the multiple regulatory patterns in Psph 1448A. a, Close-up representation of 262 TFs hierarchy in Psph 1448A (eight TFs showed no hierarchical characteristic). Nodes depict TFs. Colors of edges represent source-bases. b, Enrichment of different collaborating (direct interaction, indirect interaction and cooperativity) TF-pairs at top (T), middle (M) and bottom (B) levels. We defined indirect interaction if two TFs co-associated with one target DNA. Cooperative TF pair was defined if their common target is from a TF. Gray nodes below the graph represent TFs. The dashed orange line indicates the averaged level of collaboration. c, Thirteen Three-node sub-modules with the number of occurrences and an example. Spire loop is the most enriched sub-module. Edges represent the regulatory direction. d, Autoregulations are accompanied by the number of occurrences and 13 auto-regulators as examples.

Bottom TFs share the same binding sequences to coregulate in Psph 1448A. a, The co-association map for 170 TFs in Psph 1448A shows the co-associated scores of binding peaks of these TFs (rows) that overlap each TF peak (columns). The three colored rectangles represent three different TF levels. C1-C4 represent four clusters of TFs according to the co-associated scores. The TFs in corresponding cluster are shown in the circle diagram. Orange nodes represent top TFs. Green nodes represent middle TFs. Purple nodes represent bottom TFs. The colors of edges are the mixture of two source TF colors. b, The heat-map of MexT indicates the associated scores of binding peaks of TFs in C3 (columns) that overlap the binding peaks (rows) of MexT. PSPPH2411, PSPPH3643 and CysI are the top 3 TFs with high associated scores. c, UpSet plot shows the number of genes uniquely targeted TFs or co-targeted by multiple TFs in C3. The vertical black lines represent shared TF-binding sites. The y axis represents the number of overlapped binding sites across the linked TFs. The x axis represents the number of binding sites for each TF. Orange line represents the most enriched gene cysI that is co-targeted by 12 TFs in C3. d, Motifs of MexT and other 10 TFs in C3 which show similar binding sequences.

Virulence hierarchical regulatory network reveals 35 TFs involved in virulence. a,

Virulence hierarchical regulatory network shows the TF hierarchy and the large pool of target genes of multi-TF. Target genes are related with seven key virulence pathways, including biofilm formation, secondary messengers, motility, T3SS, QS, phytotoxin production and siderophore transporter. Orange nodes represent top TFs. Green nodes represent middle TFs. Purple nodes represent bottom TFs. Blue nodes represent target genes. Yellow edges represent downward point. Red edges represent upward point. b, The head-to-head binding motif of PSPPH1951, the validation of the binding sites of PSPPH1951 by EMSA, and the detection of expression of target gene hopAE1 in WT, ΔPSPPH1951 and complementary strain by RT-qPCR. The validated binding sites are from the promoters of the hrpR, hopAE1 and hopAH2. c, The binding motif of PSPPH2193 is head-to-head. EMSA confirms the direct binding of PSPPH2193 to the promoters of fleQ and flhF. RT-qPCR confirms the positive regulation of PSPPH2193 on the expression of fleQ and flhF. Motility assay validates the weaker motility of ΔPSPPH2193 than WT and complementary strain. d, The binding motif of PSPPH3268 is head-to-head. EMSA confirms the direct binding of PSPPH3268 to the promoters of hrpR, alg44 and pilM. RT-qPCR confirms the negative regulation of PSPPH3268 on the expression of alg44, algX and pilM. Crystal violate staining assay and the quantification of biofilm formation validate the negative regulation of PSPPH3268 on the biofilm formation. Congo red assay confirms the negative regulation of PSPPH3268 on colony morphologies and EPS production. Student’s t test. n.s., not significant, *PL≤L0.05, **PL≤L0.01, and ***PL≤L0.001.

Hundreds of TFs are identified to participate in metabolic pathways. a,

Metabolic hierarchical regulatory network shows the TF hierarchy and the large pool of target genes of multi-TF. Target genes are related with eight key metabolic pathways, including biosynthesis of amino acids, DNA replication, ABC transporter, oxidative phosphorylation, TCA cycle, RNA polymerase, phosphonate metabolism and methyl-accepting chemotaxis. Orange nodes represent top TFs. Green nodes represent middle TFs. Purple nodes represent bottom TFs. Blue nodes represent target genes. Yellow edges represent the direct interaction. b, Radar plots show the putative key regulators identified in six different metabolic pathways, including TCA cycle, oxidative phosphorylation, methyl-accepting, biosynthesis of amino acids, RNA polymerase and ABC transporter. Each radiation line represents a key regulator, and the radial length of the thick colored line is the rate of target genes to the associated genes, representing the significance of the enrichment of the TF target genes within each pathway. c, TFs involved in the methyl-accepting pathway bound to the promoters of TFs in the same category. Brown nodes represent the TFs that are responsible for methyl-accepting pathway. The brown arrows point to the targeted TFs. d, TFs involved in the oxidative phosphorylation pathway bind to the promoters of TFs in the methyl-accepting pathway. Red nodes represent the TFs that are responsible for oxidative phosphorylation pathway. Brown nodes represent the TFs that are responsible for methyl-accepting pathway. Blue nodes represent the TFs involved in these two pathways. The arrows point to the targeted TFs and the arrow colors are source-based.

Various conservations are observed in TFs between different P. syringae pathovars. a, Proportion of the TF target genes detected in one, two, three or four tested genomes. c1-c4 represent the conservation of targets in one, two, three and four strains. b-c, Repartition of the total pool of target genes (b) or TF-target interactions (c) in four tested strains. d, Enrichment coverage tracks of ChIP-seq against negative controls for TF Irp with binding sites on the promoter of gidA in all four genomes. e, Enrichment coverage tracks of ChIP-seq against negative controls (gray tracks) for the three TFs (Irp, PSPPH2193 and PSPPH4127) with binding sites on the promoter of rpoD in four tested strains.

Functional modularized regulatory network in P. syringae exhibits the specific functions of both TFs and their target genes. a, The functional categorical regulation network in Psph 1448A analyzed by Gephi (resolution 0.9). The 16 modules (both TFs and their target genes) are labeled in different colors. TF nodes are shown as corresponding sized circles representing their expression level. Their target genes are shown as corresponding-colored dots in the background. TF-target edges are shown as corresponding-colored lines between nodes. b, Graph diagram indicates the connections between TF and their targets in modules. Module nodes are shown as corresponding-colored circles with size proportional to the number of nodes within. Edge colors are source-based, and edge thicknesses represent the connected quantity between modules. c-d, Functional category enrichment analysis of genes in each module, p < 0.05.

Global transcriptional regulatory network in Psph 1448A. The integrated transcriptional regulatory network reflects the interactions between all TFs classified into 39 families from different DNA binding domain types and target genes annotated from pathway annotations. The targets are shown in 12 pathways with various colors. TF nodes are gray as corresponding sizes representing TF number in the family. Edge colors are target-based.

Summary of ChIP-seq results in Psph 1448A. a, Locations of all the 301 annotated TFs in Psph 1448A genome. Blue lines represent the TF loci. b, ChIPed TFs are classified as respective family with different colors. Square size represents the targeted enrichment of each TF. c, The preferential enrichment at different genome loci of each ChIPed TF, including upstream, overlapStart, inside, overlapEnd, downstream and includeFeature regions.

Graph diagram of feed-forward loop in Psph 1448A. TF columns in feed-forward loop (M13, n=696) are classified as families. TF-TF edges are distinguished with separate colors.

Co-association and virulence-related functional category of TFs at top level. a-b,

The co-associated map and functional category of top TFs. The peak number of top TFs are shown at upper corresponding to the TFs in the lower map. Functional enrichment analysis was performed by hypergeometric test (BH-adjusted pl<l0.05).

Co-association and virulence-related functional category of TFs at middle level.

a-b, The co-associated map and functional category of middle TFs. The peak number of middle TFs are shown at upper corresponding to the TFs in the lower map. Functional enrichment analysis was performed by hypergeometric test (BH-adjusted pl<l0.05).

Co-association and virulence-related functional category of TFs at bottom level.

a-b, The co-associated map and functional category of bottom TFs. The peak number of bottom TFs are shown at upper corresponding to the TFs in the lower map. Functional enrichment analysis was performed by hypergeometric test (BH-adjusted pl<l0.05).

The validation of the binding sites of virulence-related TFs in Psph 1448A. a-c, The promoter of rpoD is the negative control of PSPPH1951 and PSPPH2193. The promoter of PSPPH3658 is the negative control of PSPPH3268. d, The validation of binding sites of PSPPH1951. The validated binding sites are from the promoters of pilZ, pilF and pilG. e, The validation of binding sites of PSPPH3798. The validated binding sites are from the promoters of fliK, fliE, fliD and fleQ. The promoter of PSPPH2263 is the negative control. f, A regulatory cascade of PSPPH3504. / means sibling nodes. – means downward regulation. g, Bacterial number (colony-forming units, CFUs) of Δ2193 strain on the bean leaf surface after dip inoculation. ∗P < 0.05. Results were indicated as mean ± SD. All experiments were repeated at least three times.

Metabolic functional category of TFs at three different levels.

a-c, Functional category according to different metabolic pathways of top (a), middle (b) and bottom (c) TFs.

Key TFs in different metabolic pathways. a,

Radar plots show the putative key regulators identified in two different metabolic pathways, including DNA replication, and phosphonate and phosphonate metabolism. Each radiation line represents a key regulator, and the radial length of the thick colored line is the rate of target genes to the associated genes, representing the significance of the enrichment of the TF target genes within each pathway. b, The monomer motif of PSPPH0755 and the validation of the binding sites of PSPPH0755 by EMSA. The validated binding sites are from promoters of PSPPH5210 and PSPPH3109. The promoter of PSPPH4598 is negative control. c, The head-to-head motif of PSPPH3798 and the validation of the binding sites of PSPPH3798 by EMSA. The validated binding sites are from promoters of PSPPH3881 and PSPPH5119. d, The monomer motif of PSPPH4638 and the validation of the binding sites of PSPPH4638 by EMSA. The validated binding sites are from promoters of PSPPH3881 and PSPPH0550. The promoter of PSPPH4598 is the negative control.

Motif and functional enrichment analysis of PSPPH3122 in Psph 1448A, Pss B728a and Pst DC3000 strains. Motifs of PSPPH3122 in Psph 1448A (a) and Pst DC3000 (b) strains. c, GO analysis by hypergeometric test (BH-adjusted pl<l0.05) of PSPPH3122 in Psph 1448A, Pst DC3000 and Pss B728a strains.

Motif and functional enrichment analysis of PSPPH4127 in Psph 1448A, Pss B728a, Pst DC3000 and Psa C48 strains. Motifs of PSPPH4127 in Psph 1448A (a), Pss B728a (b), Pst DC3000 (c) and Psa C48 (d) strains. e, GO analysis by hypergeometric test (BH-adjusted pl<l0.05) of PSPPH4127 in Psph 1448A, Pss B728a, Pst DC3000 and Psa C48 strains.