Article Figures & Data
Figures
- FIG 1
Growth of Erwinia amylovora is affected by DksA and ppGpp. The graphs show the growth of the E. amylovora WT strain, the relA, spoT, and dksA single mutants, and their corresponding complementation strains in LB (A) and MBMA (B) media and the growth of the E. amylovora WT strain, the relA/spoT (ppGpp0) and relA/dksA double mutants, and their corresponding complementation strains in LB (C) and MBMA (D) media. The experiments were repeated at least two times with similar results.
- FIG 2
ppGpp measurement. Intracellular ppGpp levels in Erwinia amylovora WT and relA, spoT, dksA, relA/spoT, and relA/dksA mutant strains were quantified in HMM by using the fluorescent chemosensor PyDPA as reported previously (44, 45). One-way analysis of variance (ANOVA) and Student's t test (P = 0.05) were used to analyze the data. Values marked with the same letter were not significantly different (P < 0.05). NI, not included in statistical analysis. This experiment was performed twice.
- FIG 3
ppGpp controls cell size in Erwinia amylovora. (A) Epifluorescence microscopy images of E. amylovora WT and relA, spoT, and relA/spoT (ppGpp0) mutant strains constitutively expressing GFP and grown in LB medium or HMM for 4 h. Magnification, ×200. (B) Distributions of sizes and average cell lengths of WT and relA, spoT, and relA/spoT (ppGpp0) mutant strains constitutively expressing GFP and grown in LB medium or HMM. The experiments were repeated at least two times with similar results.
- FIG 4
Pathogenicity and HR assays. (A and B) Symptoms caused by the WT strain, the relA, spoT, dksA, relA/spoT (ppGpp0), and relA/dksA mutants (A), and their complementation strains (B) on immature pear fruits. Immature pears (cv. ‘Bartlett’) were surface sterilized, pricked with a sterile needle, and inoculated with 2 μl of bacterial suspension. Symptoms were recorded and photos were taken at 4 and 8 dpi. (C and D) HR assay on tobacco leaves. The E. amylovora WT strain, the relA, spoT, dksA, relA/dksA, and ppGpp0 mutants (C), and their complementation strains (D) were allowed to infiltrate into 8-week-old tobacco leaves at a concentration of 108 CFU ml−1. PBS was used as a negative control. Photographs were taken at 24 h postinfiltration.
- FIG 5
Both DksA and ppGpp are required for bacterial growth in planta. (A) Growth of the E. amylovora WT strain, the relA, spoT, and dksA single mutants, and their corresponding complementation strains in immature pears. (B) Growth of the E. amylovora WT strain, the relA/dksA and relA/spoT (ppGpp0) double mutants, and their corresponding complementation strains in immature pears. Immature pears (cv. ‘Bartlett’) were surface sterilized, pricked with a sterile needle, and inoculated with 2 μl of bacterial suspension. Tissue surrounding the inoculation site was excised with a no. 4 cork borer and homogenized in 1 ml of 0.5× PBS. Bacterial growth within the pear tissue was monitored at 1, 2, and 3 days postinoculation by dilution plating on LB medium with appropriate antibiotics. d, day.
- FIG 6
Both DksA and ppGpp activate T3SS gene expression in Erwinia amylovora. (A) Expression of T3SS regulatory and effector genes (hrpL, hrpA, hrpN, and dspE) in the relA, spoT, dksA, relA/dksA, and relA/spoT (ppGpp0) mutant strains compared to the WT strain in HMM, as determined by qRT-PCR. (B) Expression of T3SS regulatory and effector genes (hrpL, hrpA, hrpN, and dspE) in the relA, spoT, dksA, relA/dksA, and ppGpp0 mutant strains compared to the WT on immature pear fruits. Relative gene expression of selected T3SS genes was calculated by the 2−ΔΔCT method, utilizing the rpoD gene as an endogenous control. Fold changes are the means of results for three replicates. Each experiment was performed at least two times with similar results. Error bars indicated standard deviations.
- FIG 7
Accumulation of HrpA protein is controlled by DksA and ppGpp. The HrpA-His6 protein in the WT and relA, spoT, dksA, and relA/spoT (ppGpp0) mutant strains was detected by Western blotting using an anti-histidine protein antibody after growth in HMM at 18°C for 6 h. Relative protein abundances were calculated by using ImageJ software, utilizing the average pixel value of the signals and considering the abundance of the WT sample to be 100%.
- FIG 8
Working model illustrating the role of ppGpp in Erwinia amylovora in response to plant and environmental stimuli. This model is based on findings obtained in this study as well as those reported in previous studies (4, 5, 8, 11). Symbols: ↓, positive effect; ⊥, negative effect; IhfA and -B, integration host factors α and β; RNAP, RNA polymerase; OM, outer membrane; IM, inner membrane.
Tables
- TABLE 1
Bacterial strains and plasmids used in this study
Strain or plasmid Description Reference or source Strains E. amylovora strains Ea1189 Wild type; isolated from apple 72 ΔrelA mutant relA::Cm; Cmr insertional mutant of relA of Ea1189; Cmr This study ΔspoT mutant spoT::Cm; Cmr insertional mutant of spoT of Ea1189; Cmr This study ΔdksA mutant dksA::Cm; Cmr insertional mutant of dksA of Ea1189; Cmr This study ΔrelA/spoT mutant relA::Cm spoT::Km; Kmr insertional mutant of spoT into ΔrelA mutant This study ΔrelA/dksA mutant relA::Cm dksA::Km; Kmr insertional mutant of dksA into ΔrelA mutant This study E. coli DH10B F− mcrA Δ(mrr-hsdRMS-mcrBC) ϕ80dlacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ(ara-leu)7697 galU galK rpsL nupG λ Invitrogen, CA Plasmids pKD46 Apr PBAD gam bet exo pSC101 oriTS 42 pKD32 Cmr FRT cat FRT tL3 oriR6Kγ bla rgnB 42 pkD13 Kmr FRT kan FRT tL3 oriR6Kγ bla rgnB 42 pWSK29 Apr; cloning vector; low copy number 73 pRelA 3.0-kb SacI-KpnI fragment including the relA gene in pWSK29 This study pSpoT 2.8-kb SacI-KpnI fragment including the spoT gene in pWSK29 This study pDksA 1.0-kb SacI-KpnI fragment including the dksA gene in pWSK29 This study pFPV25 Apr; GFP-based promoter trap vector containing promoterless gfpmut3a gene 74 pZW2(HrpL) 608-bp KpnI-XbaI DNA fragment containing promoter sequence of hrpL gene of Ea1189 in pFPV25 75 pHrpA-GFP 708-bp EcoRI-BamHI DNA fragment containing promoter sequence of hrpA gene in pFPV25 40 pHrpA-His6 803-bp DNA fragment containing promoter sequence of hrpA gene and C-terminal His tag coding sequence in pWSK29 This study pKH91 ori15A gfpuv bla Apr tet Tcr 76 - TABLE 2
Comparison of disease severities with Erwinia amylovora strain Ea1189, ppGpp mutants, and complementation strains
Strain No. of shoots infected/no. of shoots inoculated Length of necrosis (cm) (mean ± SD)a Ea1189 7/7 30.2 ± 5.6a ΔrelA mutant 2/7 10 ± 2.8c ΔrelA(pRelA) mutant 7/7 30.8 ± 3.2a ΔspoT mutant 6/7 19 ± 1.6b ΔspoT(pSpoT) mutant 6/7 29 ± 3.1a ΔdksA mutant 0/7 —NI ΔdksA(pDksA) mutant 7/7 32.2 ± 5.08a ΔrelA/spoT mutant 0/7 —NI ΔrelA/spoT(pSpoT) mutant 3/7 1.16 ± 0.28d ΔrelA/dksA mutant 0/7 —NI ΔrelA/dksA(pRelA) mutant 0/7 —NI ΔrelA/dksA(pDksA) mutant 2/8 3.25 ± 1.06c,d ↵a Average necrosis length for 7 or 8 inoculated apple shoots (cv. ‘Gala’) at 7 days postinoculation. —, no disease detected. The experiment was repeated with similar results. One-way ANOVA and Student's t test (P = 0.05) were used to analyze the data. Values marked with the same letter were not significantly different (P < 0.05). NI, not included in statistical analysis.
- TABLE 3
Promoter activities of hrpL and hrpA genes in Erwinia amylovora WT and ppGpp mutant strains
Strain Plasmid (gene)a GFP intensity (geometric mean ± SD)b HMM HMM + 0.1 mM SHX HMM + 0.5% αMG Ea1189 pFPV25 1.34 ± 0.021e 1.35 ± 0.007e 1.36 ± 0.014d pZW2 (hrpL) 1.58 ± 0.035c,d 1.54 ± 0.007c,d 1.52 ± 0.014c ΔrelA mutant pZW2 (hrpL) 1.47 ± 0.007c,d 1.42 ± 0.021d 1.41 ± 0.022c ΔspoT mutant pZW2 (hrpL) 2.3 ± 0.028b 2.64 ± 0.233b 5.15 ± 0.234b Ea1189 pHrpA-GFP 1.72 ± 0.049c 1.93 ± 0.035c 1.98 ± 0.036c ΔrelA mutant pHrpA-GFP 1.42 ± 0.021d 1.49 ± 0.14c,d 1.44 ± 0.15c ΔspoT mutant pHrpA-GFP 3.55 ± 0.355a 5.98 ± 0.63a 13.94 ± 0.96a ↵a Promoter-GFP fusion plasmid.
↵b Bacteria were grown in HMM for 18 h, with or without addition of serine hydroxamate (SHX) or α-methylglucoside (αMG). One-way ANOVA and Student's t test (P = 0.05) were used to analyze the data. GFP intensity values within a treatment marked with the same letter were not significantly different (P < 0.05).
