Fig. 3. Transcriptional regulation in the T3SS pathway.
a Bar chart shows the distribution on the number of the putative binding sites per TF. Note that most of TFs target fewer than 200 genes with five TFs exceptional bind more than 500 sites in the genome. b Network illustrates the regulatory relationship between TFs and their target genes in the T3SS pathway. Circles indicate TF proteins, hexagons indicate target genes, and the triangle represents the TF with auto-regulatory activity. TFs that can regulate more than two target genes are highlighted by the red edges. Red arrows show the putative binding sites located in the promoters of the target genes, while gray arrows show the putative binding sites located in the target gene bodies. The TFs without names are named with their locus tag omitting PSPPH_. c Radar plot shows the putative master regulators identified in the T3SS pathway. Each radiation line represents a TF, and the radial length of thick colored line is −log10 (P value), representing the significance of the enrichment of the TF targets within T3SS-associated genes. The names of master regulators are marked in red-type face. d The head-to-head binding motif of PSPPH_3618 (upper) and the validation of the putative binding sites of PSPPH_3618 by EMSA (lower). The validated binding sites were from the promoters of the rhpPC operon and avrB2. The promoter of hopAN1 was negative control. e, f The expression of target gene avrB2 was measured in the WT and ∆PSPPH_3618 strains by RT-qPCR in KB and MM, respectively. P value is 1.460E-04 (e). P value is 1.373E-06 (f). g, h The expression of target gene rhpP was measured in the WT and ∆PSPPH_3618 strains by RT-qPCR in KB and MM, respectively. P value is 3.38E-04 (g). P value is 1.001E-05 (h). i Disease symptoms caused by either WT or ∆PSPPH_3618 strain, 105 CFU/ml bacteria of which were infiltrated into the primary leaves of the bean. Disease symptoms were photographed 6 days after inoculation. j Bacterial numbers were quantified 6 days after inoculation. P value is 4.650E-05 (j). Three independent replicates were performed. Statistic P values by two-tailed Student’s t test are shown. **P < 0.01 and ***P < 0.001 (g, h, j). Error bars show standard deviations. TF transcription factor.