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Annals of Applied Biology ISSN 0003-4746

RESEARCH ARTICLE

Effect of Dionaea muscipula extract and plumbagin on maceration of potato tissue by Pectobacterium atrosepticum

A. Szpitter 1 , M. Narajczyk 2 , M. Maciag-Dorszynska 3 , G. Wegrzyn 3 , E. Lojkowska 1 & A. Krolicka 1

1 Department of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Gdansk, Poland

2 Laboratory of Electron Microscopy, University of Gdansk, Gdansk, Poland

3 Department of Molecular Biology, University of Gdansk, Gdansk, Poland

Keywords Antimicrobial activity; efflux pumps; pectinolytic bacteria; plant metabolites.

Correspondence A. Krolicka, Department of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Kladki 24, 80-822 Gdansk, Poland. Email: aleksandra.krolicka@biotech.ug.edu.pl

Received: 29 March 2013; revised version accepted: 5 December 2013; published online: 28 February 2014.

doi:10.1111/aab.12110

Abstract

Pectobacterium atrosepticum (Pba) is a plant pathogen that causes major crop losses. Dionaea muscipula extracts and their antibacterial constituent, plumbagin, inhibit Pba growth in vitro. However, this effect is reduced when the extracts are added to bacterial cultures present on potato tubers or suspended in potato tuber filtrate (PF). To explain this, we examined the response mechanism of Pba cells to Dionaea extract and plumbagin and compared it with the effect of

a bactericidal peptide – CAMEL. The addition of the extract and plumbagin to

a Pba1043 culture in stationary phase increased the extracellular pectate lyase

(Pel) activity in the presence of PF. While the addition of the Dionaea extract and plumbagin caused a dramatic reduction in RNA and protein synthesis in Pba1043, it did not result in cellular damage. PF alone increased the expression of Pba genes encoding protein components of cellular efflux pump systems:

ompX , acrA and emrA . Application of both PF and plumbagin resulted in a synergistic stimulation of acrA gene expression. Plumbagin added to potato tubers inoculated with a field isolate Pba5A/1/2005 increased extracellular Pel activity and reduced tissue maceration but did not affect bacterial counts per gram of tissue. These results show that plumbagin in the presence of compounds from potato tuber stimulates Pel production/secretion in Pba cells and increases the expression of the acrA gene. This may be the molecular basis for the less pronounced effects of Dionaea extract on Pba in planta relative to those observed in vitro.

Introduction

Plant pathogenic bacteria are a significant problem in agriculture. Major losses during the production and storage of potato, carrot, cabbage and tomato are caused by pectinolytic Pectobacterium and Dickeya spp. These bacteria are characterised by high genotypic and phenotypic diversity and are capable of causing disease symptoms on a wide range of host plants (including many vegetables such as potato and ornamental plants) in different climates (Ma et al., 2007). Additionally, diseases caused by Pectobacterium and Dickeya species cannot be controlled by bactericides, and their control currently depends on the early detection of bacteria and resistant cultivar plantation (P erombelon´ and Kelman,

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1980; P erombelon,´ 2002; Zimnoch-Guzowska et al., 2005; Czajkowski et al., 2011). On the basis of various suppressive effects of plant chemicals on bacterial cells, new antibacterial compounds have been developed including inhibitors of quorum sensing and of multidrug resistance pumps (Hentzer et al., 2002; Ball et al., 2006). In spite of the chemical diversity of plant metabolites so far none of those compounds proved to be sufficiently active in order to be introduced to the market with the intention of preventing bacterial diseases in crop plants. This is due to the fact that bacterial plant pathogens have evolved efficient mechanisms to avoid plant chemical defence and to respond to plant antimicrobials as well as plant cell degradation products. Plant phenolics were found to

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induce the expression of efflux pumps (Ravirala et al., 2007) and the components of type III secretion system in

Dickeya dadantii cells (Yang et al., 2008). Bacterial response mechanisms identified so far also include upregulation of plant cell degrading enzymes. The presence of ferulic acid, a plant cell wall degradation product, stimulates the production of feruloyl esterase and pectate lyases in D. dadantii cells (Hassan & Hugouvieux-Cotte-Pattat, 2011). Moreover, the expression of proline iminopeptidase gene in Xanthomonas campestris pv. campestri was shown to be upregulated in the presence of an unknown plant metabolite (Zhang et al., 2007). Knowledge of the molecular pathways underlying bacterial response to plant compounds is important for understanding complex plant–pathogen interactions and also for the development of new strategies for managing bacterial infections in plants. It was previously shown that the carnivorous plant Dionaea muscipula ( Droseraceae ) possesses strong antibacterial properties (Juniper et al., 1989; Finnie & van Staden, 1993; Krolicka et al., 2008). In this work, we examine the response of Pba to treatment with D. muscipula extract, its major antimicrobial constituent, plumbagin, and a reference antimicrobial peptide CAMEL, in order to gain insight into the mechanism by which bacterial pathogens avoid

a plant’s chemical defences. Here, we show that the

difference in Pectobacterium atrosepticum (Pba) resistance

to plant antimicrobials observed in in vitro tests and in potato tubers can be at least partially explained by the stimulation of Pba efflux pump expression and extracellular pectate lyase (Pel) activity by the combined presence of metabolites from potato tuber tissue and plumbagin.

Materials and methods

Bacterial strains and media

Pectobacterium atrosepticum (Pba) strain SCRI 1043 and

a field isolate Pba 5A/1/2005 were from the collection

of Department of Plant Protection and Biotechnology,

University of Gdansk, Poland. In this work, we used only one field-isolated strain of Pba as preliminary tests showed similar susceptibility among different Pba isolates

to tested antimicrobials (data not shown). Bacteria were

grown in standard Luria broth (LB) (Sambrook et al., 1989) and in M63 buffer (Miller, 1972) with 0.2% polygalacturonic acid (Sigma, Munich, Germany) and 0.2% glycerol (referred to as MPG medium) at 28° C. The M63 buffer consisted of 2 g L 1 (NH 4 ) 2 SO 4 , 13.6 g L 1

KH 2 PO 4 , 0.5 mg L 1 FeSO 4 · 7H 2 O and 0.247 mg L 1 MgSO 4 · 7H 2 O. The pH was adjusted to 7.0 with KOH. Where indicated, media were supplemented with 20%

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(final concentration) potato tuber tissue filtrate (PF) (the supplemented media are referred to as LB + PF and MPG + PF, respectively). PF was prepared by adding 0.02% diethyldithiocarbamic acid (Sigma) to a juice made from fresh potato tubers and then centrifuging and filter sterilising the resulting solution. Methanol potato tuber extract (PE) was prepared by adding 50 m L 1 of methanol and 0.01% HCl to 50 g of homogenised fresh potato tuber tissue and incubating the mixture on a rotary shaker (100 rpm) for 2 h in 20 ° C. The extract was then passed through Whatman no. 1 filter paper and lyophilised, and the dry residue was dissolved in methanol and again filtered. The obtained PE was applied to bacterial suspensions at 100–1500 μ g mL 1 .

Dionaea muscipula extract and chemicals

Dionaea muscipula plants were obtained and grown as described by Krolicka et al. (2008). Whole plants were extracted with chloroform and a Soxhlet apparatus. Dried extract was dissolved in methanol to the concentration of 30 mg dry weight (DW) per mL and filtered. In this work, we have also used plumbagin – a major antibacterial constituent present in D. muscipula extract as shown by our preliminary studies of antibacterial activity of High Performance Liquid Chromatography (HPLC) fractions of extract (data not shown). Extract and plumbagin (Sigma, Cat. no P7262) were added to the wells of 48-well plates as methanol solutions, and the organic solvent was allowed to evaporate at room temperature before medium was added. Final concentrations of extract and plumbagin were ranged from 0.025 to 400 μ g mL 1 . In some experiments, we used the antimicrobial, synthetic peptide CAMEL (KWKLFKKIGAVLKVL), which is active against Pba strains and was previously shown to be more effective against Pectobacterium spp. than streptomycin (Kamysz et al., 2005) to determine whether the effects of plant extract and plumbagin were specific. CAMEL was synthesised as previously described (Kamysz et al., 2005) and dissolved in distilled water, filter sterilised and applied at 0.025–16 μ g mL 1 .

Antibacterial activity of Dionaea muscipula extract, plumbagin and CAMEL

Minimal bactericidal concentrations (MBCs) of D. muscip- ula extract, plumbagin and CAMEL were determined in LB medium as described (Thornsberry, 1991). For deter- mination of bacterial generation times, Pba cultures were grown overnight in LB, centrifuged and suspended in LB, LB + PF, MPG or MPG + PF growth media at an OD 600 of 0.1; the cultures were grown at 28 ° C with shaking in 24- well plates for 24 h. Chloroform extract from D. muscipula,

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plumbagin and CAMEL was added to the cultures at 0.1 and 0.25 MBC, and the absorbance was measured at 60- min intervals with a plate reader (Wallac Perkin Elmer, Turku, Finland). Generation times were calculated on the basis of OD 600 values measured during the exponen- tial phase of growth as previously described (Ulanowska et al., 2007).

Extracellular pectate lyase activity in cell-free culture supernatants as affected by plumbagin and CAMEL

Pectate lyase activity was measured in cell-free super- natants from cultures treated or untreated with D. mus- cipula extract or plumbagin at 0.1 and 0.25 MBC and with or without PF. The extract and plumbagin were added at the start of culture or at the beginning of the stationary phase of growth. Extracellular Pel activity was measured with a reaction buffer containing polygalactur- onic acid as described previously (Keen et al., 1984). One unit (U) of Pel activity was defined as the amount of enzyme releasing an amount of oligogalacturonides that increased Abs 235 by 1 unit during a 1-minute reaction. Pel activity was expressed as U g 1 of bacterial DW.

Plant tissue maceration assay and associated extracellular Pel activity as affected by plumbagin and CAMEL

A maceration assay with potato tubers was performed as

described previously (Keen et al., 1984) with some mod- ifications. Briefly, an overnight culture of Pba5A/1/2005

in LB was centrifuged and washed with M63 buffer, and

the cells were suspended at an OD 600 of 0.075. Potato tubers were washed and surface sterilised with 5% cal- cium hypochlorite. Just before tuber inoculation, the bacterial suspension was mixed 1:1 (v/v) with twofold concentrated solutions of plumbagin or CAMEL in M63

buffer. Five sterile tips containing 50 μL of bacterial sus- pension with plumbagin or CAMEL (at 1.0, 4.0 or 8.0 MBC), suspension without these compounds, or M63 buffer alone were inserted into each potato tuber. After 48 h at 28 ° C, the decayed tissue surrounding each tip was weighed, homogenised and diluted 1:4 (v/v) in M63 buffer. Pba colony-forming unit (CFU) were counted as previously described (Jett et al., 1997). Extracellular Pel activity was measured in diluted tissue homogenate as described in the previous section and was expressed as

U g 1 of macerated potato tissue.

Inhibition of DNA, RNA and protein synthesis by Dionaea muscipula extract and plumbagin

The influence of plumbagin, methanol and chloroform extracts from D. muscipula, and CAMEL on the synthesis

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of DNA, RNA and proteins by Pba1043 was estimated by measuring the incorporation of radioactive precursors ([ 3 H]thymidine, [ 3 H]uridine or [ 3 H]leucine, respectively) according to a previously described procedure (Wegrzyn et al., 1991). Briefly, overnight cultures of Pba1043 were diluted 1:100 in fresh LB medium and cultivated to an OD 600 of 0.1 at 28° C. [ 3 H]thymidine, [ 3 H]uridine or [ 3 H]leucine was added to the cultures to final concen- trations of 5 μCi mL 1 of [ 3 H]thymidine or [ 3 H]uridine or 2 μCi mL 1 of [ 3 H]leucine together with plumbagin, extract or CAMEL at 0.1 and 0.25 MBC. Control cultures were supplemented only with radioactive precursors. For measurement of the radioactive compounds, 50 μL samples of the bacterial suspensions were placed on Whatman no. 3 filter paper after 15, 30, 60 or 90 min of incubation, and were then transferred immediately to an ice-cold 10% trichloroacetic acid (TCA) for 10 min. Following washing once in 5% TCA and then twice in 96% ethanol, the filters were dried, and radioactivity was measured in a scintillation counter (Beckman LS3133P, Fullerton, CA, USA) (Ulanowska et al., 2006). Each treatment was examined in a separate experiment with three biological replicates.

Morphology of Pectobacterium atrosepticum cells as affected by plumbagin and CAMEL

In order to examine morphology of Pba1043 cells treated with antimicrobials bacteria were grown overnight in LB in 28° C with shaking before CAMEL at 1.0 MBC or plumbagin at 2.0 MBC was added. After 1 h at 28 ° C with shaking, bacteria were adsorbed onto carbon- coated grids (Sigma), stained with 1.5% uranyl acetate (Laboratory Reagent BDH, Chemicals LTD, Netherfield, UK), and immediately examined with a Philips CM100 electron microscope (EM, FEI Company, Eindhoven, the Netherlands).

Construction of Pectobacterium atrosepticum reporter plasmids

Sequences (about 500 bp long) preceding transcription start sites of acrA , ompX and emrA genes from Pba1043 (Bell et al., 2004) were amplified using primers listed in Table 1. Each amplified sequence contained one of the following restriction sites: BamHI, Hin dIII, Sal I or EcoRI. The obtained fragments and a pPROBE-GT Gm r pVS1/p15a plasmid containing promoterless gfp gene preceded by a multicloning site (Miller et al., 2000) were digested with appropriate endonucleases (Fermentas) and ligated using T4 ligase (Fermentas, GMBH, St. Leon- Rot, Germany). The resulting plasmids (pPROBE-GT P acrA , pPROBE-GT P emrA and pPROBE-GT P ompX ) were

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Table 1 Primers used in this study a

Pectobacterium response to plant antimicrobial compounds

Primer name

Primer sequence 5 3

Restriction site

Product length (bp)

PacrA_F

GTGAGT GTCG AC AACGGTTAAACG ATTAAGCCGCCA GAATTCATCAGA ACGCGA AAGCTTCAGTGACAGGCG TGCGGCGTCTGT GG AT CCTGATTT GATAGA AAGCT TCGAGATAATGAT ACCAGCAAATGCG GA TCCTGCGCC

SalI

542

PacrA_R

EcoRI

PemrA_F

HindIII

522

PemrA_R

BamHI

PompX_F

HindIII

520

PompX_R

BamHI

a Restriction sites are underlined, and non-complementary bases are shown in bold.

electroporated into Pba 5A/1/2005 cells using Gene Pulser Xcell (BioRad, Hercules, CA, USA), and bacterial clones carrying plasmids were selected on LB medium with gentamycin (50 μ g mL 1 ).

Efflux pump expression as affected by tested compounds

Plumbagin, CAMEL, PF and PE were added to the wells of 96-well plates. For methanol solutions of plumbagin and PE, the solvent was evaporated. The liquid in each well was then increased to 100 μL by addition of LB. Final concentrations of compounds and extracts in the wells were: for plumbagin 0.025–0.5 μ g mL 1 , CAMEL 0.025–1 μ g mL 1 , PE 100–1500 μ g mL 1 and PF 2–20%. Cultures of Pba 5A/1/2005 containing different reporter plasmids were grown overnight at 28 ° C on LB medium

supplemented with 50 μ g mL 1 gentamycin. The cultures were diluted 1:100 in the same medium and grown to an OD 600 of 0.5. Cells were washed once with LB and resus- pended in the same medium to an OD 600 of 0.5. A 100 μ L volume of suspension was added to the plates containing the tested compounds and extracts. Fluorescence of the suspension in each well was measured immediately ( t = 0) and after 4 h at 28° C ( t = 4) with a plate reader at λ ex = 405 nm and λ em = 535 nm. The change in fluorescence is a measure of transcriptional activity of a corresponding promoter: PacrA , P emrA or P ompX .

Statistical analysis

All data were analysed by a Student’s t -test. Differences were considered significant for P < 0.05. All experiments were performed at least twice with similar results and

Table 2 In vitro growth of two Pba strains as affected by Dionaea muscipula extract, plumbagin and CAMEL.

Generation time (min) b in four media c

Bacterial strain

Compound added a

LB

LB + PF

MPG

MPG + PF

Pba1043

Control

78 ± 2 307 ± 23 (4) d 1562 ± 442 (20)

79 ± 7 128 ± 7 (1.6) 303 ± 34 (4) 109 ± 9 (1.4) 341 ± 23 (4) 78 ± 2 76 ± 3 73 ± 2 91 ± 5 (1.3) 168 ± 37 (2) 91 ± 5 (1.3) 240 ± 10 (3) 75 ± 4 71 ± 3

117 ± 9 558 ± 261 (5) No growth 752 ± 90 (6) No growth 119 ± 3 115 ± 3 115 ± 2 131 ± 13

67 ± 1 126 ± 7 (2) 351 ± 26 (5) 129 ± 3 (2)

Extract

0.1 MBC

Extract

0.25 MBC

Plumbagin 0.1 MBC

197 ± 41 (3) 2187 ± 442 (28) 87 ± 2 (1.1) 81 ± 2 94 ± 3

Plumbagin

0.25 MBC

316 ± 1

(5)

CAMEL

0.1 MBC

66 ± 0.1 69 ± 2 68 ± 2 83 ± 6 (1.2)

CAMEL

0.25 MBC

Pba 5A/1/2005

Control

Extract

0.1 MBC

147 ± 13 479 ± 85

(1.6)

Extract 0.25 MBC

(5)

475 ± 36 247 ± 39

(4)

197 ± 28 (3) 106 ± 1 (1.6)

Plumbagin

0.1 MBC

282 ± 4 (3) 1127 ± 235 (12) 96 ± 6

(2)

Plumbagin

0.25 MBC

1563 ± 442 (14) 119 ± 5

262 ± 7

(4)

CAMEL

0.1 MBC

69 ± 2 68 ± 1

CAMEL

0.25 MBC

99 ± 0.3

123 ± 2 (1.1)

MBC, minimal bactericidal concentration; Pba, Pectobacterium atrosepticum; PF, potato filtrate. a The MBC were 50 μg mL 1 for D. muscipula chloroform extract (Extract), 50 μg mL 1 for plumbagin and 8 μg mL 1 for CAMEL, for both Pba strains. b Generation times were calculated on the basis of the trend in culture growth recorded for 24 h. Values are means ± SD of three culture replicates. c LB, Luria broth medium; MPG, M63 buffer with 0.2% polygalacturonic acid and 0.2% glycerol; LB + PF/MPG + PF, media supplemented with 20% potato filtrate. d Given in the brackets is the fold increase in generation time in relation to the respective control, presented only for generation times significantly (P < 0.05) different from the control.

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to plant antimicrobial compounds A. Szpitter et al. Figure 1 Specific extracellular pectate lyase (Pel)

Figure 1 Specific extracellular pectate lyase (Pel) activity in Pba1043 culture supernatants as affected by Dionaea muscipula extract, plumbagin and CAMEL. Bacteria were grown in MPG without potato filtrate (PF) (A, C) or with PF (B, D). A chloroform extract from D. muscipula (Extract), plumbagin and CAMEL was added at the beginning of the culture ( t = 0 h) (A, B) or in the early stationary phase (t = 10 h) (C, D) at 0.1 and 0.25 minimal bactericidal concentration (MBC) for the extract and plumbagin and at 0.25 MBC for CAMEL. The results are means ± SD of three culture replicates. Pel activities for control cultures grown for 12, 24 and 48 h on MPG medium were 3.8, 4.3 and 3.8 U g 1 dry weight (DW) of bacteria, respectively, and for those grown on MPG + PF were 1.5, 1.7 and 1.2 U g 1 DW of bacteria, respectively.

only representative results from individual experiments are presented. Standard deviations of the ratios (values relative to the control) were estimated using the total differential method.

Results

Effect of Dionaea muscipula extract, plumbagin and CAMEL on viability and virulence of Pectobacterium atrosepticum

To assess the effects of D. muscipula extracts and plumbagin on viability of Pba, we have determined MBCs of them. As a positive control, CAMEL – a known antimicrobial pep- tide – was used. The MBC values against both tested Pba

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strains were 100 μ g mL 1 for the D. muscipula chloroform extract, 50 μ g mL 1 for the naphthoquinone plumbagin and 8 μ g mL 1 for CAMEL. At 0.1 and 0.25 MBC, the extract and plumbagin increased Pba generation times by 1.6- to 28-fold in LB medium and by 2- to 14-fold in MPG medium (Table 2). However, for cultures supplemented with extract and plumbagin at 0.25 MBC the addition of potato filtrate (PF) to the medium significantly reduced the observed growth inhibition in comparison to control. The field isolate Pba 5A/1/2005 was more resistant than Pba1043 to D. muscipula extract and plumbagin at 0.25 MBC in all media except for LB + PF (Table 2). CAMEL peptide at 0.1 and 0.25 MBC did not cause significant growth inhibition in the majority of Pba cultures (Table 2).

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Given that PF affects the growth of Pba cells in the presence of plant antimicrobials we also tested the influence of PF on the production of Pel, one of the major virulence determinants in Pba cells. In the determination of extracellular Pel activity in cell-free culture supernatants of Pba1043 cultures, addition of D. muscipula extract and plumbagin at the start of the culture reduced the specific extracellular Pel activity in relation to control ( P < 0.05 after 48 h culture), regardless of the presence of PF in the medium (Fig. 1A, and Fig. 1B). However, when the extract or plumbagin was added in the early stationary phase, an increase in the activity of Pel was observed on MPG medium with PF (Fig. 1D) in comparison to Pel activity on MPG medium alone after 48 h culture (Fig. 1C). Interestingly, CAMEL increased Pel activity in all cases in comparison to control after 48 h culture (Fig. 1A–Fig. 1D). Application of plumbagin at concentrations of 1.0–8.0 MBC to potato tubers tended to reduce tissue maceration caused by Pba 5A/1/2005 (Fig. 2A). Plumbagin at 8.0 MBC significantly increased extracellular Pel activity per gram of macerated tissue (Fig. 2B) and caused only an eightfold reduction in the number of viable bacteria per gram of macerated tissue (data not shown). CAMEL at 4.0 and 8.0 MBC completely inhibited potato tissue maceration (Fig. 2A) and Pel activity (Fig. 2B).

Mechanism of extract and plumbagin effect on Pectobacterium atrosepticum cells

To learn about mechanisms of action of D. muscipula extracts and plumbagin on Pba cells, their effects on crucial biochemical processes proceeding in bacterial cells were monitored. Relative to the control and after 60 min, plumbagin at 0.25 MBC caused a significant 37% reduction in DNA synthesis (Fig. 3A), a 60% reduction in RNA synthesis (Fig. 3B) and a 70% reduction in protein synthesis in Pba1043 cells (Fig. 3C). Chloroform extract from D. muscipula at 0.5 MBC caused a decrease in RNA and protein formation but only protein synthesis was reduced by the methanol extract at 0.5 MBC (Fig. 3D–Fig. 3F). As indicated by EM and relative to the control (Fig. 4A), the morphology of Pba1043 cells was unaffected by incubation for 1 h in LB containing plumbagin at 2.0 MBC (Fig. 4B) but was substantially altered by incubation for 1 h in LB containing CAMEL at 1.0 MBC (Fig. 4C). We examined the influence of the tested compounds on the expression of proteins being a part of efflux pump systems in Pba5A/1/2005 cells by monitoring the expression of gfp in reporter strains with ompX , acrA and emrA gene promoter fusions. Expression of all tested genes increased markedly relative to the control in the

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Pectobacterium response to plant antimicrobial compounds Figure 2 Influence of plumbagin and CAMEL on potato tuber

Figure 2 Influence of plumbagin and CAMEL on potato tuber tissue maceration (A) and extracellular pectate lyase (Pel) activity (B) in tubers infected with Pba5A/1/2005. Each value is the mean + SDfrom at least nine potato tubers. Asterisks indicate values significantly (P < 0.05) different from the respective control.

presence of PE (Fig. 5A) as well as PF (Fig. 5B). Plumbagin alone (Fig. 5C and Fig. 5D) did not significantly stimulate expression of the tested genes but addition of plumbagin with PF resulted in an increase in acrA gene expression, which was significantly greater than elevation caused by PF alone (Fig. 5D). CAMEL at 0.025–1 μ g mL 1 tested alone and in combination with PE or PF failed to affect gene expression (data not shown).

Discussion

Although plumbagin, the major naphthoquinone from D. muscipula, exhibited high antimicrobial activity against

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to plant antimicrobial compounds A. Szpitter et al. Figure 3 Inhibition of macromolecule synthesis in Pba1043

Figure 3 Inhibition of macromolecule synthesis in Pba1043 cells by plumbagin (A–C) and Dionaea muscipula extracts (D–F). Incorporation of radioactive precursors: [ 3 H]thymidine, [ 3 H]uridine and [ 3 H]leucine to DNA (A, D), RNA (B, E) and proteins (C, F), respectively. Extracts from D. muscipula were added at 0.5 minimal bactericidal concentration (MBC) (50 μg mL 1 for the chloroform extract and 250 μg mL 1 for the methanol extract). Values are means ± SD of three culture replicates. Asterisks indicate values significantly ( P < 0.05) different from the respective control.

Pba strains in vitro, its inhibitory activity was severely reduced in vivo (i.e. in potato tubers). Similarly, addition of potato filtrate (PF) to the culture medium decreased the antimicrobial activity of plumbagin and D. muscipula extract. Potato filtrate is a complex mixture of plant metabolites including organic acids, sugars, amino acids and phenylpropanoids. Although these metabolites are in a more oxidised state in filtrates than in plant tissue, we suspect that addition of PF to the medium generates some of the conditions experienced by bacteria in the potato tuber. Phenolic compounds were previously reported to increase the resistance of another pectinolytic bacterium, Dickeya chrysanthemi , to antimicrobials by stimulating

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expression of multidrug efflux pump-encoding genes (Ravirala et al., 2007). Researchers have postulated that these bacteria can ‘sense’ plant phenolics (e.g. salicylic acid) and increase their resistance before the plant is able to synthesise antimicrobial compounds (Palumbo et al., 1998; Ravirala et al., 2007). To examine the increased resistance of Pba to plant metabolites in the presence of potato filtrate, we monitored the promoter activities of three genes ( ompX , acrA and emrA ) that encode proteins that are a part of major efflux pump systems in the Enterobacteriaceae . As expected, expression of those genes was induced by PF and PE. Previous research with plant-associated

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A. Szpitter et al. Figure 4 Influence of plumbagin and CAMEL on Pba cell morphology as

Figure 4 Influence of plumbagin and CAMEL on Pba cell morphology as indicated by electron microscopy. (A) Control culture (no addition); (B) plumbagin at 2.0 minimal bactericidal concentration (MBC); (C) CAMEL at 1.0 MBC. Pba1043 cells were incubated for 1 h in Luria broth (LB) medium with the indicated compound. In(C), arrows indicate the abnormal morphology caused by CAMEL. Bars at the lower right corner are 2 μm. The experiment was performed twice and the results obtained in both analyses were similar.

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bacteria demonstrated that efflux pumps are important for establishing a successful plant–bacterium interaction and that they can be induced by plant compounds (Palumbo et al., 1998; Kang and Gross, 2005; Valecillos et al., 2006; Ravirala et al., 2007). The outer membrane protein TolC, which is a part of the multidrug efflux system AcrAB-TolC, was crucial for colonisation of plants by D. chrysanthemi , and tolC mutants were found to be susceptible to a wide range of plant antimicrobials (Barabote et al., 2003). In addition, plant phytoalexins increased expression of the acrAB operon in another bacterial plant pathogen, Erwinia amylovora (Burse et al.,

2004).

The present work documented a synergistic effect of plumbagin and potato filtrate in the activation of acrA transcription in Pba cells. We hypothesise that this effect could result from the stimulation of the Mar regula- tory network governing bacterial cell resistance to stress, which has been identified in many species of Enter- obacteriaceae. In Escherichia coli , acrAB and tolC promoters contain a marbox sequence recognised by MarA, SoxS and Rob proteins. Expression of marA and soxS is stimulated by phenolic compounds and oxidative stress, respec- tively (Martin et al., 1999; Ravirala et al., 2007). Other signalling pathways and mechanisms were reported to be involved in the response of bacterial pathogens to plant metabolites – GacA/S – RsmA/B (Yang et al., 2008) and regulators of the LuxR family (Zhang et al., 2007). Although plumbagin inhibited potato tissue maceration caused by Pba 5A/1/2005, it also stimulated production and/or secretion of Pel enzymes in potato tissues. In the presence of PF, moreover, addition of D. muscipula chloroform extract and plumbagin to Pba1043 cultures in the stationary phase increased the extracellular Pel activity in culture supernatants in relation to control (Fig. 1). In soft-rotting bacteria, the production of extracellular enzymes, as well as proteins that constitute the type II secretion system taking part in the export of those enzymes out of the bacterial cell, is regulated by a multitude of signalling pathways in response to environmental stimuli such as plant growth regulators or phenolic acids (Liu et al., 2008). The effect of plumbagin seems to be unspecific because CAMEL, an antimicrobial peptide with a completely different mode of action, could also induce production and/or secretion of Pel enzymes in Pba cells. The 15-residue hybrid peptide CAMEL was previously reported to be an effective antimicrobial against a range of Pectobacterium and Dickeya species (Kamysz et al., 2005). Although it has strong bactericidal properties against tested Pba strains, it did not inhibit growth in this study (Table 2) and had no influence on macromolecule synthesis in Pba1043 cells at the concentrations tested (results not shown). This could

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to plant antimicrobial compounds A. Szpitter et al. Figure 5 Influence of PE (A, C) and

Figure 5 Influence of PE (A, C) and PF (B, D) alone and in combination with plumbagin (C, D) on promoter activities of genes encoding efflux pumps (PompX , PacrA, PemrA ) in Pba 5A/1/2005. Each treatment was performed at least in triplicate. Bars represent ± SD. Single asterisks on C and D indicate values significantly (P < 0.05) different from the control, double asterisk indicates significant (P < 0.01) difference between values for cultures supplemented with PF and with PF + plumbagin. The concentrations were: 300 μg mL 1 PE in (C), 2% PF in (D) and 0.2 μg mL 1 plumbagin in (C) and (D). RFU, difference in Gfp fluorescence after 4 h of incubation of reporter strains with tested compounds (Sample) or in Luria broth (LB) medium only (Control). PE, potato extract; PF, potato filtrate.

be due to the fact that most antimicrobial peptides act through interaction with bacterial cell envelopes and cause rapid cell death only if their concentration exceeds a bactericidal threshold (Marcos et al., 2008). Our microscopic observations seem to confirm this hypothesis. We showed that plumbagin, present in the chloroform extract from D. muscipula (Krolicka et al., 2008), inhibits RNA and protein synthesis in Pba cells. Plumbagin is known to induce apoptotic cell death in cancer cells through generation of reactive oxygen species (ROS) and subsequent inhibition of topoisomerase II (Kawiak et al., 2007; Wang et al., 2008). In bacteria, this naphthoquinone probably acts through a similar mechanism that involves oxidative stress-dependent DNA damage (Chen et al., 2006). We have previously shown that ramentaceone, which is a naphthoquinone produced by the carnivorous plant Drosera aliciae, exerts its antimicrobial effect on human bacterial pathogens by inhibiting nucleic acid synthesis, especially DNA replication, but not by inhibiting protein synthesis (Krolicka et al., 2009). The

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antimicrobial effect of ramentaceone was independent of the generation of oxidative stress. In conclusion, the results presented in this report indicate that in response to the plant antimicrobial plumbagin in the presence of potato filtrate, Pba cells increase production and/or secretion of Pel enzymes as well as the expression of the acrA gene encoding efflux pump protein. This increase in virulence and resistance of bacterial cells could explain the observed discrepancy between plumbagin in vitro and in vivo antibacterial activity against Pba. Moreover the effect of plumbagin on Pba cells probably does not rely on cell wall damaging effect. The signalling pathways underlying response of Pba cells to plant antimicrobials and potato metabolites remain to be determined.

Acknowledgements

We thank Prof. Wojciech Kamysz from Medical University of Gdansk for CAMEL peptide. This work was supported by the National Science Centre grant # N N405 165239.

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