Variable Suites of Non-effector Genes Are Co-regulated in the Type III Secretion Virulence Regulon across the Pseudomonas syringae Phylogeny
Figure 6
A novel HrpL-regulated virulence operon linked to avrD in Pph1448A identified by RNA-seq analysis.
(A) Color coded genomic context of avrD and downstream genes from PSPPH_A0106 to A0112. Grey arrows represent transposases. White arrows represent additional ORFs that are not necessary related. (B) Graphical representation of avrD operon according to IMG/ER conserved neighborhood region search with the 45 Pseudomonas strains presented in Figure 5 and S5. Grey, white, black, and colored arrows as in A. Brackets represent breaks and end of contigs or scaffolds. Not to scale. (C) PCR was performed using primers spanning intragenic regions between PSPPH_A0109 and A0110 (top panels), or PSPPH_A0112 and avrD (middle panels) on cDNA prepared from Pph1448A (Pph) or an isogenic Pph1448AΔhrpL mutant (ΔhrpL) grown in MM media. Total RNA was subject to reverse transcriptase (+RT), or without reverse transcriptase (−RT, as negative control). gDNA, DW indicate respectively that genomic DNA or distilled water were used as template for positive and negative controls of amplification. Equal loading was controlled by monitoring gap-1 amplification across samples (bottom panels). (D) Two week old bean cv. Tendergreen beans were dip inoculated with wild type Pph1448A (Pph), two independent clean deletion avrD mutants (ΔavrD #1, ΔavrD #2), and two independent clean deletion PSPPH_A0107 mutants (ΔPSPPH_A0107 #1, ΔPSPPH_A0107 #2) at OD600 = 0.001. Bacterial growth of each strain was determined after 3.5 dpi. Letters represent significant differences with P<0.05 according to Tukey's highly significant difference and error bars display standard deviation. This experiment was repeated at least twice.
