A pattern-triggered immunity-related phenolic, acetosyringone, boosts rapid inhibition of a diverse set of plant pathogenic bacteria

Abstract

Background: Acetosyringone (3,5-dimethoxy-4-hydroxyacetophenone, AS) is a syringyl-type phenolic compound rarely found in plants in free form. It has been shown earlier to inhibit the growth of Pseudomonas bacteria in the presence of hydrogen peroxide and peroxidase (AS mix).

Results: We detected elevated levels of free AS in Nicotiana tabacum and N. benthamiana plants after inducing pattern-triggered immunity (PTI) by injecting bacterial elicitor flg22, or pathogenicity-mutant Pseudomonas syringae pv. syringae 61 hrcC- bacteria; but not after inoculations with compatible or incompatible pathogens at the time of PTI onset. In this study, we demonstrate that the antibacterial effect of the AS mix is general, as growth of several Gram-negative and -positive phytopathogenic bacteria was characteristically inhibited. The inhibition of bacterial metabolism by the AS mix was rapid, shown by the immediate drop of luminescence intensity of P. syringae pv. tomato DC3000 lx strain after addition of AS mix. The mechanism of the bacteriostatic effect was investigated using fluorescent reporter dye assays. SYTOX Green experiments supported others' previous findings that the AS mix does not result in membrane permeabilization. Moreover, we observed that the mode of action could be depolarization of the bacterial cell membrane, as shown by assays carried out with the voltage sensitive dye DIBAC₄(3).

Conclusions: Level of free acetosyringone is elevated during plant PTI responses in tobacco leaves (N. tabacum and N. benthamiana). When combined with hydrogen peroxide and peroxidase (AS mix), components of the mix act synergistically to inhibit bacterial metabolism and proliferation rapidly in a wide range of plant pathogens. This effect is related to depolarization rather than to permeabilization of the bacterial cell membrane. Similar AS mixture to the in vivo model might form locally at sites of invading bacterial attachment to the plant cells and the presence of acetosyringone might have an important role in the inhibition of bacterial proliferation during PTI.

Keywords: Acetosyringone; Antibacterial; Elicitor; Oxidative burst; Pattern-triggered immunity; Pseudomonas syringae.

Fig. 1

Identification of PTI-related phenolic compound acetosyringone by HPLC-DAD-MS using analytical standard in Nicotiana benthamiana. a HPLC-DAD analysis of phenolic compounds from PTI-induced and water-treated control N. benthamiana leaves detected at 298 nm. PTI was induced by P. syringae pv. syringae hrcC- bacteria, water was injected as control. Samples were taken at 2, 4, 6 hpi. b MS spectra of AS had the expected m/z values in both positive and negative modes, identical to that of the standard. c) Identity of acetosyringone was supported by the similar UV spectra of the analytical standard and the unknown peak. Abbreviations: AS: acetosyringone, ESI+, ESI-: electrospray ionization positive and negative modes, Std: standard, W: water

Fig. 2

Relative accumulation of acetosyringone in N. benthamiana leaves. a Response to treatments with P. syringae pv. syringae hrcC- bacteria at 2, 6 and 24 hpi. b Relative accumulation of AS in N. benthamiana leaves in response to treatments with different bacteria at 6 hpi. Error bars indicate standard deviations. Asterisks indicate significant difference from corresponding water-treated controls according to student’s T-test (*p < 0.1; **p < 0.05). Red downward arrows indicate zero values. W: water-treated control; P. s.: Pseudomonas syringae pathovars; flg: flg22 peptide elicitor

Fig. 3

Antimicrobial effect of oxidized form of acetosyringone on different plant pathogenic bacteria. Bacteria (10⁵ CFU/ml) were added to reaction mixtures containing 50 μM AS, 50 μM H₂O₂, and 0.72 U/ml horseradish peroxidase, and various control mixtures from which one or two components were omitted. Serial dilutions were plated following 3 h of co-incubation for CFU determination. a Typical representative examples of serial dilutions plated on Kings’s B agar plates. Right half of each plate: no dilution (0×), left side of each plate: 10× dilution (indicated with blue lettering). Numbering of treatment combinations applied to the bacterial suspensions: 1. non-treated control; 2. AS; 3. H₂O₂; 4. H₂O₂ + POX; 5. AS + H₂O₂ + POX; 6. HK. b Diagram showing quantification of the results. Error bars indicate standard deviations. Asterisks indicate significant difference from corresponding water-treated controls according to student’s T-test (*p < 0.1; **p < 0.05). Downward arrows indicate zero values. Abbreviations: Bact: bacterium, AS: acetosyringone, HK: heat-killed, POX: horseradish peroxidase

Fig. 4

Bioluminescence changes of indicator bacteria after treatment with oxidized AS. 10⁵ CFU/ml of P. syringae pv. tomato DC3000 lux, a luminescent P. syringae strain was added to reaction mixtures containing 50 μM AS, 50 μM H₂O₂, and 0.72 U/ml POX, and various control mixtures from which one or two components were omitted. Bioluminescence was measured at indicated time points. Different letters above the bars denote different levels of luminescence assigned by Tukey’s Test (P < 0.05). Error bars indicate standard deviations. AS: acetosyringone, HK: heat-killed, P.s. tomato DC3000 lux: Pseudomonas syringae pv. tomato DC3000 luminescent strain, POX: horseradish peroxidase

Fig. 5

Evaluation of applicability of different fluorescent dyes for the detection of membrane permeability and membrane depolarisation in the AS mix system. Different fluorescent dyes were tested for usability to measure membrane permeability: SYTOX Green (a), TO-PRO (b); and membrane depolarisation: DIBAC₄(3) (c). Fluorescence of indicator dyes in reaction mixtures containing 50 μM AS, 50 μM H₂O₂, and 0.72 U/ml POX, and various control mixtures from which one or two components were omitted was measured. Fluorescent dyes were added either immediately (0 h) or 3 h after preparation of the mixtures to see if bleaching occurs. HK P. syringae pv. tabaci suspension was also added 3 h after mixture preparation to 5 × 10⁶ CFU/ml final density. Fluorescence was measured 4 h after preparation of the mixtures. Error bars indicate standard deviations. Asterisks indicate significant difference from corresponding controls according to student’s T-test (*p < 0.1; **p < 0.05). Abbreviations: CONT: untreated bacteria, AS: acetosyringone, HK: heat-killed, POX: horseradish peroxidase, AU: arbitrary unit

Fig. 6

Dependence of bacterial membrane depolarisation on bacterial density after treatment with oxidized AS. Suspensions of 5 × 10⁷ (a) and (b) 5 × 10⁶ CFU/ml of P. syringae pv. tabaci, P. syringae pv. tomato DC3000 or A. tumefaciens bacteria were added to reaction mixtures containing 50 μM AS, 50 μM H₂O₂, and 0.72 U/ml POX, and various control mixtures from which one or two components were omitted. Bacterial membrane polarity was tested with the voltage sensitive DIBAC₄(3) stain added 3 h after treatments of bacteria. Error bars indicate standard deviations. Different letters above the bars denote different levels of DIBAC₄(3) fluorescence assigned by Tukey’s Test (p < 0.05). Abbreviations: Cont: non-treated control bacteria, AS: acetosyringone, HK: heat killed, POX: horseradish peroxidase, Blank: buffer only

Fig. 7

Membrane depolarization and permeability testing of bacteria after treatment with oxidized AS as a function of bacterial concentration. Dilution series of bacteria from 5 × 10⁷ to 0.625 × 10⁷ CFU/ml of P. syringae pv. tabaci and A. tumefaciens C58C1 bacteria was added to reaction mixtures containing 50 μM AS, 50 μM H₂O₂, and 0.72 U/ml POX. Untreated bacteria were used as negative, and heat killed bacteria as positive controls. Bacterial membrane polarity was tested with the voltage sensitive DIBAC₄(3) stain, and membrane permeability with SYTOX Green stain, both added 3 h after treatment of bacteria. Error bars indicate standard deviations. Asterisks indicate significant difference from corresponding controls according to student’s T-test (*p < 0.1; **p < 0.05). Abbreviations: BACT: bacterium, AS: acetosyringone, HK: heat-killed, POX: horseradish peroxidase, AU: arbitrary unit

Fig. 8

Fluorescent microscopic images of P. syringae pv. tabaci bacteria treated with an AS mix, and stained to evaluate changes in membrane permeability and polarity. P. syringae pv. tabaci (10⁵ CFU/ml) was treated with a mixture of AS + H₂O₂ + POX. After 3 h of shaking, (a) DIBAC₄(3), an indicator dye of membrane depolarization or (b) SYTOX Green, an indicator of membrane permeability, and DAPI as counterstain were added and co-incubated for 1 h. After concentration of the bacteria, microscopic slides were prepared and photographed within 1–2 h. Bar = 100 μm. Abbreviations: Control: untreated bacteria AS: acetosyringone, HK: heat-killed, POX: horseradish peroxidase

Fig. 9

Proliferation of compatible P. syringae pv. tabaci (10⁶ CFU/ml inoculum) bacteria in tobacco (Nicotiana tabacum). Bacteria were suspended in 0.01 M potassium phosphate buffer pH 6. Mixture of AS+H₂O₂ + POX was added to the bacteria. N. tabacum leaves were infiltrated either immediately or after 3 h of incubation. Samples for bacterial counts were taken at 0, 2, 7 dpi. Error bars indicate standard deviations. Significant difference from untreated bacterial control according to student’s T-test is marked by a * (p < 0.1). Abbreviations: AS mix: acetosyringone combined with hydrogen peroxide and horseradish peroxidase, HK: heat killed, POX: horseradish peroxidase