Discovery of plant phenolic compounds that act as type III secretion system inhibitors or inducers of the fire blight pathogen, Erwinia amylovora

Abstract

Erwinia amylovora causes a devastating disease called fire blight in rosaceous plants. The type III secretion system (T3SS) is one of the important virulence factors utilized by E. amylovora in order to successfully infect its hosts. By using a green fluorescent protein (GFP) reporter construct combined with a high-throughput flow cytometry assay, a library of phenolic compounds and their derivatives was studied for their ability to alter the expression of the T3SS. Based on the effectiveness of the compounds on the expression of the T3SS pilus, the T3SS inhibitors 4-methoxy-cinnamic acid (TMCA) and benzoic acid (BA) and one T3SS inducer, trans-2-(4-hydroxyphenyl)-ethenylsulfonate (EHPES), were chosen for further study. Both the T3SS inhibitors (TMCA and BA) and the T3SS inducer (EHPES) were found to alter the expression of T3SS through the HrpS-HrpL pathway. Additionally, TMCA altered T3SS expression through the rsmBEa-RsmAEa system. Finally, we found that TMCA and BA weakened the hypersensitive response (HR) in tobacco by suppressing the T3SS of E. amylovora. In our study, we identified phenolic compounds that specifically targeted the T3SS. The T3SS inhibitor may offer an alternative approach to antimicrobial therapy by targeting virulence factors of bacterial pathogens.

Fig 1

Schematic of the effect of compounds on T3SS regulation in E. amylovora. Solid lines indicate direct regulation (protein-protein interaction or direct binding to the promoter region), and dashed lines indicate indirect regulation or hypothetical regulatory links based on evidence shown in E. amylovora or other plant-pathogenic bacteria. IM and OM, inner and outer membrane, respectively. Based on genetic analysis in E. amylovora and biochemical evidence obtained with P. stewartii (65), HrpX/Y forms a two-component system that activates the transcription of hrpL (14). HrpL activates the expression of the T3SS genes such as hrpA and hrpN (16, 52). HrpS is a σ⁵⁴ enhancer-binding protein that activates the transcription of hrpL, independent of HrpXY (14, 17). Based on the two-component system study in E. amylovora and the evidences in P. syringae (22, 23), it is suggested that in E. amylovora, the GacAS two-component system may activate transcription of hrpS. Also, based on previous work in P. carotovorum (54) and the evidence of the functional homolog of rsmB found in E. amylovora E9 (20), the rsmB-RsmA system may regulate the T3SS by affecting the stability of hrpL mRNA in E. amylovora. In this study, we observed that TMCA, BA, and EHPES significantly alter the hrpA promoter activity through HrpS-HrpL pathway. In addition, TMCA alters the T3SS expression through the rsmB_Ea-RsmA_Ea pathway.

Fig 2

Effectiveness of selected compounds that inhibit E. amylovora hrpA promoter activity. A promoter-probe reporter fusion plasmid (phrpA) was examined by measuring GFP in the presence of the selected inhibitory compounds at various concentrations. Dashed lines show the IC₅₀ of these compounds, i.e., the concentrations required for the inhibition of 50% of the promoter activity of hrpA compared to the DMSO control.

Fig 3

Northern blot analysis of hrpA transcripts of E. amylovora 273. RNA was isolated from cells harvested after 6 h of bacterial growth in HIM supplemented with DMSO and 100 μM BA, TMCA, and EHPES. 16S rRNA was used as an RNA loading control. The experiment was repeated three times with similar results. The image was spliced to conserve space.

Fig 4

Northern blot analysis of hrpL, rsmB_Ea, and rsmA_Ea transcripts of E. amylovora 273. RNA was isolated from cells harvested after 6 h of bacterial growth in HIM supplemented with DMSO control and 100 μM TMCA, BA, and EHPES. 16S rRNA was used as an RNA loading control. The experiment was repeated three times with similar results. The image was spliced to conserve space.

Fig 5

mRNA levels of hrpX, hrpY, hrpS, and rpoN E. amylovora 273 in HIM supplemented with 100 μM TMCA, BA, and EHPES compared to levels in HIM with DMSO, as determined by qPCR. RNA was collected at 6 h of bacterial growth. There is no significant difference between HIM supplemented with DMSO and HIM supplemented with TMCA, BA, or EHPES for hrpX, hrpY, and rpoN. Levels of gene expression of hrpS are significantly different between HIM supplemented with DMSO and HIM supplemented with 100 μM TMCA, BA, and EHPES (⁎, P < 0.001). Five replicates were used in this experiment. The P value was calculated with the Relative Expression software tool (44). The experiment was repeated three times with similar results.

Fig 6

Alteration of the T3SS effector hrpN by T3SS inhibitors and inducers in E. amylovora. (A) Northern blot analysis of hrpN. RNA was isolated from cells harvested after 6 h of bacterial growth in HIM supplemented with DMSO control and 100 μM TMCA, BA, and EHPES. 16S rRNA was used as RNA loading control. The experiment was repeated twice with similar results. (B and C) E. amylovora cells were grown in HIM supplemented with DMSO (all blots) or 100 μM BA, TMCA, or EHPES. Western blot analyses of total cell fractions and supernatant fractions were performed using an anti-HrpN polyclonal antibody. Numbers below the blots are relative intensities of secreted HrpN provided by ImageJ. The experiment was repeated three times with similar results. The image in panel C was spliced to conserve space.

Fig 7

(A) Effect on HR development when BA and TMCA were coinfiltrated with E. amylovora 273 cells in Nicotiana tabacum cv. Xanthi leaves. (B) Effect on HR development in tobacco leaves transiently expressing hopQ1. TMCA and BA (100 μM) were injected at different time points after the tobacco leaves had been infiltrated with Agrobacterium tumefaciens GV3101 carrying the effector gene hopQ1 of P. syringae DC3000. In this particular picture, the compounds were injected at 6 h after the infiltration of Agrobacterium. The infiltrated areas are outlined. Both the experiments were repeated three times with similar results.