Ralstonin Production by Plant Sugars

Jun 17, 2019 - Plant pathogenic bacteria possess sophisticated mechanisms to detect the presence of host plants by sensing host-derived compounds...
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Articles Cite This: ACS Chem. Biol. 2019, 14, 1546−1555

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Activation of Ralfuranone/Ralstonin Production by Plant Sugars Functions in the Virulence of Ralstonia solanacearum Yoko Ishikawa,†,¶ Yuta Murai,†,¶ Megumi Sakata,† Shoko Mori,‡ Shoma Matsuo,† Wakana Senuma,§ Kouhei Ohnishi,∥ Yasufumi Hikichi,§ and Kenji Kai*,†,¶

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†

Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan ‡ Bioorganic Research Institute, Suntory Foundation for Life Sciences, 8-1-1 Seikadai, Seika-cho, Soraku-gun, Kyoto 619-0284, Japan § Laboratory of Plant Pathology and Biotechnology, Kochi University, 200 Otsu, Monobe, Nanko-ku, Kochi 783-8502, Japan ∥ Research Institute of Molecular Genetics, Kochi University, 200 Otsu, Monobe, Nanko-ku, Kochi 783-8502, Japan S Supporting Information *

ABSTRACT: Plant pathogenic bacteria possess sophisticated mechanisms to detect the presence of host plants by sensing host-derived compounds. Ralstonia solanacearum, the causative agent of bacterial wilt on solanaceous plants, employs quorum sensing to control the production of the secondary metabolite ralfuranones/ralstonins, which have been suggested to be involved in virulence. Here, we report that D-galactose and D-glucose, plant sugars, activate the production of ralfuranones/ralstonins in R. solanacearum. As a result, two new derivatives, ralfuranone M (1) and ralstonin C (2), were found in the culture extracts, and their structures were elucidated by spectroscopic and chemical methods. Ralstonin C (2) is a cyclic lipopeptide containing a unique fatty acid, (2S,3S,Z)-3-amino-2-hydroxyicos-13-enoic acid, whereas ralfuranone M (1) has a common aryl-furanone structure with other ralfuranones. D-Galactose and D-glucose activated the expression of the biosynthetic ralfuranone/ralstonin genes and in part became the biosynthetic source of ralfuranones/ralstonins. Ralfuranones and ralstonins were detected from the xylem fluid of the infected tomato plants, and their production-deficient mutants exhibited reduced virulence on tomato and tobacco plants. Taken together, these results suggest that activation of ralfuranone/ralstonin production by host sugars functions in R. solanacearum virulence.

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Ralstonia solanacearum, a soil β-proteobacterium bacterium, causes “bacterial wilt” on a wide range of host plants, including economically important tomato, tobacco, and potato crops.6,7 The bacterium invades the intercellular spaces of roots through openings, such as wounds, accumulates around the stele, and then breaks into and fills xylem vessels through the action of cellulolytic enzymes on vessel walls. The ability of this pathogen to cause host wilting is considered to be mainly attributed to its production of extracellular polysaccharide (EPS) in xylem because bacterial cells and the accumulated EPS prevent water flow.8,9 The production of EPS is regulated by a quorum sensing (QS) system consisting of phc regulatory elements.10,11 We previously reported that R. solanacearum strains possess the phc QS system mediated by either (R)methyl 3-hydroxymyristate or (R)-methyl 3-hydroxypalmitate.12 Several transcriptome analyses revealed that the phc QS system regulates a variety of bacterial activities related to virulence as well as EPS production.13−15

INTRODUCTION

Understanding the molecular events involved in plant-microbe interactions may provide new ways to save crops from the threat of pathogens. It is generally thought that bacterial pathogens sense certain molecules originating from host plants to detect the close presence of the hosts. When receiving such plant-derived compounds, bacterial pathogens evoke a variety of responses related to pathogenesis. For example, Agrobacterium tumefaciens, the causative agent of crown gall disease, senses a structurally diverse group of plant phenolic compounds from wounded plant tissues and then activates the transcription of virulence (vir) genes necessary for the transfer of T-DNA to host cells.1 Plant phenolic glucosides and sugars activate the production of syringomycin, a lipopeptide phytotoxin produced by Pseudomonas syringae.2−4 Moreover, in rhizobia, plant flavonoids activate nodulation (nod) genes essential for the establishment of symbiosis with host plants.5 These examples demonstrate that proteobacteria, which have a close relationship with plants, have developed sophisticated mechanisms to detect the presence of their hosts by sensing specific plant-derived compounds. © 2019 American Chemical Society

Received: April 16, 2019 Accepted: June 17, 2019 Published: June 17, 2019 1546

DOI: 10.1021/acschembio.9b00301 ACS Chem. Biol. 2019, 14, 1546−1555

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Figure 1. Structures of ralfuranones (A) and ralstonins (B).

Here, we report that several plant sugars, especially Dgalactose and D-glucose, activate the production of ralfuranones/ralstonins in R. solanacearum strain OE1-1. As a result of this activation, ralfuranone M (1) and ralstonin C (2) (Figure 1) were accumulated in the bacterial culture. The ralfuranone- and ralstonin-deficient mutants were still virulent on tomato and tobacco plants, but their virulence was significantly reduced compared with the wild-type strain. This study clarified the nature of plant compounds that activate the secondary metabolism in R. solanacearum and supports the importance of ralfuranones/ralstonins for virulence.

Ralfuranones A−L are the aryl-furanone metabolites derived from the common intermediate ralfuranone I (Figure 1A), which is synthesized via the action of the aminotransferase RalD and the furanone synthase RalA in R. solanacearum strains GMI1000 and OE1-1.16−20 The expression of these enzymes is regulated by the phc QS system. In our previous studies, ralfuranones were suggested to enhance bacterial activities related to virulence by activating the phc QS circuit.14,21 Ralstonins A and B (originally called ralsolamycins) (Figure 1B) are unique cyclic lipopeptides synthesized by the hybrid polyketide synthase-nonribosomal peptide synthetase RmyA/RmyB.22,23 The expression of these enzymes is also under the control of the phc QS system.22,24 Although rmyAdeficient mutants were still virulent in tomato plants, ralstonins had moderate phytotoxicity against tobacco leaves.23,24 Therefore, these secondary metabolites have been suggested to be important in the virulence of R. solanacearum. Plant-derived compounds may be important for the expression of R. solanacearum virulence. For example, the outer-membrane receptor PrhA was suggested to sense nondiffusible plant signal(s) and activate the Prh pathway, which is involved in the expression of the type III secretion system (TTSS).25 Subsequently, the presence of diffusible plant signal(s) triggering the expression of hrpG, a gene encoding a regulatory protein of TTSS was also suggested.26 However, the nature of these signals has not been elucidated. Previously, we observed that the production of ralfuranones by R. solanacearum were more active in planta than in several artificial media.18 This implied that the secondary metabolism of this plant pathogen is activated by certain plant compound(s).

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RESULTS Sugars Activate Ralfuranone Production in R. solanacearum. The apoplast and xylem fluids of tomato plants are known to contain sucrose and several monosaccharides.26,27 We previously reported that the production of ralfuranones by R. solanacearum strain OE1-1 was more active in planta than in MGRLS (MGRL media supplemented with 3% sucrose) or BG media.18 Furthermore, Mori et al. observed that fructose, mannose, and arabinose induced biofilm formation of strain OE1-1 in the minimal 1/4 M63 media and that the exogenous ralfuranones rescued the reduced biofilm formation in ΔralA, a ralfuranone-deficient mutant of OE1-1.21,28 Thus, we hypothesized that R. solanacearum cells sense host sugars to gauge their success in invading plant tissues. We investigated whether plant sugars (D-glucose, Dmannose, D-fructose, and D-galactose)26 activate the production of ralfuranones in strain OE1-1. The addition of 0.1% Dgalactose, D-glucose, and D-fructose to the bacterial cultures 1547

DOI: 10.1021/acschembio.9b00301 ACS Chem. Biol. 2019, 14, 1546−1555

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Furthermore, the levels of ralfuranone A produced by strain OE1-1 cultures also increased in response to the sugars applied (Figure 3B). The inducing activity of D-galactose was slightly higher than that of D-glucose. Time-course experiments demonstrated that the time of highest ralA expression was faster than that of the high accumulation of ralfuranone A in OE1-1 cultures (Figure S3). Taken together, these experiments indicated that D-galactose/D-glucose activate ralfuranone production by increasing ralA expression in strain OE1-1. Ralfuranones are synthesized from L-phenylalanine, which is derived from monosaccharides through glycolysis and the subsequent shikimate pathways in R. solanacearum.29 This suggested that D-galactose/D-glucose can be the biosynthetic source of ralfuranones. Next, we examined whether these sugars are converted to ralfuranones in strain OE1-1 by feeding experiments with [13C6]-D-galactose or [13C6]-D-glucose. As a result, the addition of [13C6]-D-galactose/[13C6]-D-glucose (0.1%) to the bacterial cultures activated the production of ralfuranones, and the partial incorporation of 13C-label into ralfuranones was observed (Figures 3C and S4). Then, we confirmed that the addition of D-galactose/D-glucose did not significantly affect the levels of L-phenylalanine produced. Thus, D-galactose/D-glucose can be converted to these secondary metabolites in strain OE1-1; however, the main effect of these sugars on ralfuranone production may originate from the activation of biosynthetic gene expression. Isolation and Structure Elucidation of a New Ralfuranone Derivative. The addition of D-galactose at 1% to OE1-1 cultures (MGRLS media) induced the high production of ralfuranones, including an unknown metabolite (named ralfuranone M (1)) (Figure 4A). The UV spectrum of 1 exhibited an absorption maximum at 259 nm, and the absorption profile was similar to that of ralfuranone K (Figure 4B). For the elucidation of its structure, we purified 1 from the EtOAc extract of OE1-1 culture by ODS column chromatography and reversed-phase HPLC. The isolated compound gave an [M + H]+ ion at m/z 265, which was 16 Da smaller than that of ralfuranone K. The molecular formula of 1 was deduced as C17H12O3 by HRESIMS with m/z 265.0860 [M + H]+ (calcd for C17H13O3+, 265.0859). The 1H NMR spectrum of 1 revealed the presence of two benzenes (δH 7.33−7.48 ppm, 7H; δH 7.58 ppm, 1H; δH 7.91−7.95 ppm, 2H) and an uncoupled methylene (δH 5.36 ppm, 2H) (Figure 4C). These data suggested that 1 is a 5-deoxy derivative of ralfuranone K. To identify its structure, the authentic standard was synthesized from benzoic acid in accordance with the previous report.18 Natural 1 gave an identical 1H NMR spectrum and HPLC retention time to those of synthetic 1 (Figure 4C). Therefore, the structure of ralfuranone M (1) was determined, as shown in Figure 1A. D-Galactose/D-Glucose Activate Ralstonin Production in R. solanacearum. The lipopeptide ralstonins A and B are synthesized by RmyA/RmyB in strains GMI1000 and OE11,22,24 and they exhibit moderate phytotoxic activity against tomato plants.22 We therefore examined whether D-galactose/ D-glucose also activate the production of ralstonins in strain OE1-1. D-Galactose/D-glucose (0.1−0.5% in the rich B media) significantly activated the production of ralstonins A and B, as determined by LC/MS analysis of the culture extracts (Figure 5A). Time-course experiments also indicated that, although OE1-1 growth was activated by D-galactose/D-glucose, the biomass increase was not the main source of the activation of ralstonin production (Figure S5). To examine the effects of D-

(MGRLS media) significantly activated the production of ralfuranones, as confirmed by HPLC analysis of the culture extracts (Figure 2A). The inducing activity of D-galactose was

Figure 2. Activation of ralfuranone production by sugars in R. solanacearum strain OE1-1. (A) Ralfuranones A−L produced by strain OE1-1, which was grown for 4 days in MGRLS media containing 0.1% monosaccharides. (B) Ralfuranone I produced by strain OE1-1, which was grown in MGRLS media containing 0.1% D-galactose or Dglucose. Glc: D-glucose; Man: D-mannose; Fru: D-fructose; Gal: Dgalactose. Error bars mean ± SEM (n = 3). *p < 0.05 versus control (Dunnett’s test).

the highest among the monosaccharides tested. D-Galactose and D-glucose also activated the production of ralfuranone I, a common biosynthetic intermediate of ralfuranones (Figure 2B). The growth of strain OE1-1 was slightly activated by the addition of these sugars (Figure S1). However, time-course experiments (OD600, ralfuranone A, and ralfuranone A/OD600) revealed that the high activation of ralfuranone production by D-galactose/D-glucose was not a result of increased biomass (Figure S2). Therefore, plant monosaccharides, especially Dgalactose and D-glucose, activated the production of ralfuranones in strain OE1-1. D-Galactose and D-Glucose Induce ralA Expression. Allen and colleagues reported that D-galactose is the major compound depleted in the xylem sap of tomato plants infected with R. solanacearum strain GMI1000.27 Similarly, Valls and colleagues reported that strain GMI1000 consumed plant sugars, including D-galactose and D-glucose, when grown in apoplast or xylem sap of tomato plants.26 These previous reports and the above results suggested the importance of these sugars for the activation of ralfuranone production, as well as for the in planta growth of R. solanacearum. Therefore, we analyzed the detailed effects of D-galactose/D-glucose on ralfuranone biosynthesis. To assess the effects of D-galactose/D-glucose on the expression of the biosynthetic gene ralA in strain OE1-1, we constructed the reporter strain RK8343, which has the GFP gene under the control of the ralA promoter. The addition of these sugars in RK8343 cultures activated the expression of the GFP reporter gene in a dose-dependent manner (Figure 3A). 1548

DOI: 10.1021/acschembio.9b00301 ACS Chem. Biol. 2019, 14, 1546−1555

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Figure 3. Effects of D-galactose/D-glucose on ralfuranone production. (A) Activation of pralA-gfp expression by D-galactose (left) and D-glucose (right) in strain RK8343 (MGRLS media, 2 days). The RFU values of bacterial cells were normalized according to the OD600 values. (B) Activation of ralfuranone A production by D-galactose (left) and D-glucose (right) in strain OE1-1. The LC/MS peak areas were normalized according to the OD600 values. Error bars are the mean ± SEM (n = 3). *p < 0.05 versus control (Dunnett’s test). (C) ESI-MS data for ralfuranones A−L prepared from control culture (left) and culture supplemented with [13C6]-D-galactose (right).

0.5% was highest (Figure 6A). To elucidate its structure, we prepared the acetone extract of 6000 B agar plates of strain OE1-1. 2 was purified by silica gel column chromatography and the subsequent two-step HPLC separation from the crude extract. Its molecular formula was deduced as C62H100N12O19 by HRESIMS with m/z 1317.7328 [M + H]+ (calcd for C62H101N12O19+, 1317.7300), indicating the presence of 19 degrees of unsaturation in the molecule. Therefore, there may be two more olefinic methines in 2 than in ralstonin A. The 1H NMR spectrum of 2 (in CD3OH) indicated 11 amide protons, one amino proton, one oxymethine proton, ten α-methine/ methylene protons, two exo-methylene protons, para-substituted benzene protons, and overlapping protons (Table S1). In addition, two olefinic methines (δH 5.33 ppm, overlapping) were newly observed. COSY, HSQC, and HMBC indicated the presence of two Gly, Ala, β-Ala, Dha, Val, two Hse, Ser, Thr, β-

glucose/D-galactose on ralstonin biosynthetic genes, we constructed the reporter strain RK8421, which has the LacZ gene under the control of the rmyA promoter. As a result, Dgalactose/D-glucose induced the prmyA-lacZYA expression in a dose-dependent manner (Figures 5B and S6). Feeding of strain OE1-1 with [13C6]-D-galactose/[13C6]-D-glucose (0.1%) demonstrated that these sugars were partially incorporated into ralstonins (probably mainly to the fatty acid moiety of ralstonins) (Figures 5C and S7). Taken together, strain OE1-1 was suggested to utilize D-galactose/D-glucose as chemical inducers and, in part, as a biosynthetic source for ralstonins when grown in the rich B media. Isolation and Structure Elucidation of a New Ralstonin Derivative. The addition of D-galactose to OE11 cultures (B media) induced the production of a new ralstonin derivative (named ralstonin C (2)), and the effect at 1549

DOI: 10.1021/acschembio.9b00301 ACS Chem. Biol. 2019, 14, 1546−1555

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The detailed analysis of the HMBC correlation of 2 suggested that the position of an olefin bond in the C20-fatty acid moiety is present between C-9 and C-14. Recently, olefin cross-metathesis and subsequent LC/MS analysis was applied to determine the double-bond position in long-chain fatty acids.30,31 To identify the position of an olefin bond in the fatty acid moiety of 2, we carried out cross-metathesis reaction of 2 with methyl acrylate using the second-generation Hoveyda− Grubbs catalyst (Figure 6C). The LC/MS analysis of the reaction mixture suggested the formation of compound 3 with m/z 1292 [M + H]+. To confirm the structure, 3 was purified by HPLC and analyzed by LC/HRESIMS. As a result, its molecular formula was confirmed as C58H90N12O21 by m/z 1291.6434 [M + H]+ (calcd for C58H91N12O21+, 1291.6416) (Figure 6C). Therefore, the fatty acid in 2 was revealed to be (Z)-3-amino-2-hydroxyicos-13-enoic acid (Ahie), and the planar structure of 2 was elucidated, as described in Figure 1B. The absolute configurations of the amino acid residues in 2 were determined by Marfey’s derivatization combined with acid hydrolysis and Pronase digestion of 2.22 After acid hydrolysis of 2, the free amino acids in the hydrolysate were derivatized with L-FDAA (1-fluoro-2,4-dinitrophenyl-5-L-alaninamide). An HPLC analysis of the derivatives revealed the presence of L-Ala, L-Val, L- and D-Hse, L-Ser, and L-Thr (Figure S8 and Table S2). Natural β-OH-Tyr was prepared from Pronase digestion of 2 and subsequent HPLC purification. The

Figure 4. Analysis and structure elucidation of ralfuranone M (1). (A) Comparison of HPLC profiles of metabolites produced by strain OE1-1, which was grown for 4 days in control culture (upper) or culture supplemented with 1% D-galactose (lower). (B) UV spectra of ralfuranones K (upper) and M (1) (lower). (C) Comparison of 1H NMR spectra and LC retention times of natural (upper) and synthetic ralfuranone M (1) (lower).

OH-Tyr, and 3-amino-2-hydroxy unsaturated C20-fatty acid (Figure 6B). HMBC and ROESY clarified the sequence of these units, which was identical to that of ralstonin A.

Figure 5. Effects of D-galactose/D-glucose on ralstonin production. (A) Activation of ralstonin production by D-galactose (left) and D-glucose (right) in strain OE1-1 (B media, 3 days). The LC/MS peak areas were normalized according to the OD600 values. (B) Activation of prmyA-lacZYA expression by D-galactose (left) and D-glucose (right) in strain RK8421 (B media, 3 days). The β-galactosidase activities were expressed in Miller units. Error bars are the mean ± SEM (n = 3). *p < 0.05 versus control (Dunnett’s test). (C) ESI-MS data for ralstonins A and B prepared from control culture (left) and culture supplemented with [13C6]-D-galactose (right). 1550

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Figure 6. Structure elucidation of ralstonin C (2). (A) Activation of ralstonin C (2) production by D-galactose in strain OE1-1 (B media, 3 days). The LC/MS peak areas were normalized according to the OD600 values. Error bars are the mean ± SEM (n = 3). *p < 0.05 versus control (Dunnett’s test). LC/MS chromatograms of the extracts from control culture and culture supplemented with 0.5% D-galactose are also shown. (B) Key COSY/TOCSY, HMBC, and ROESY correlations of ralstonins C (2). (C) Olefin cross-metathesis of ralstonin C (2). The HRESIMS spectrum of its product, compound 3, is shown. (D) Comparison of LC/MS retention times for fragment 4 from ralstonin C (2) and 4 from ralstonin A. (E) LC/MS analysis of L-FDAA derivatives of natural and synthetic 5.

Strain OE1-1 also Produces Ralstonins in Planta. We previously confirmed that strain OE1-1 produces ralfuranones in the xylem of tomato and tobacco plants.18 Next, we investigated whether strain OE1-1 also produces ralstonins in planta. We inoculated strain OE1-1 into tomato plants by the wounded-petiole inoculation method and grew these plants until they exhibited severe wilting symptoms. Their vascular exudates and bacterial cells therein were then collected by immersing stem cut ends in Milli-Q water. The water collected was extracted with EtOAc and analyzed by LC/MS. As a result, ralstonins A and B were detected in the EtOAc extract of tomato vascular exudates (Figure 7A), but ralstonin C (2) was not detected. This confirmed that strain OE1-1 also actively produces ralstonins in planta. Many lipopeptides produced by plant pathogens are phytotoxic, and ralstonins cause necrosis in tobacco leaves.22,32 However, it was also reported that ralstonins were not phytotoxic to tomato leaves (cv. Moneymaker).23 Therefore, we examined whether ralstonins are toxic to tomato plants (cv. Ohgata-Fukuju). After the inoculation of ralstonin solutions into tomato leaves, necrosis was observed in these regions but not in control regions (Figure 7B). Only 2.5 mg of ralstonin C (3) was isolated, and most was used for structure analysis;

L-FDLA (l-fluoro-2,4-dinitrophenyl-5-L-leucinamide) derivatives of natural and synthetic β-OH-Tyr were analyzed by LC/MS, revealing that the absolute configuration of β-OH-Tyr residue in 2 was 2S,3S (Figure S9). The above Pronase digestion of 2 also yielded fragment peptide 4 (Figure 6D). This peptide fragment exhibited an identical retention time with that of the same fragment prepared from ralstonin A,22 demonstrating the absolute configurations of these fragments to be identical. Therefore, the Hse′ residue in 4 is D, whereas the remaining Hse residue is L (Figure 1B). The absolute configuration of Ahie was determined by comparing the HPLC retention times of the L-FDAA derivatives of saturated natural and synthetic Ahie (named compound 5). Natural 5 was prepared from the acid hydrolysate of 2 and subsequent methylation with HCl/ MeOH and hydrogenation with Pd/C. We prepared synthetic (2S,3S)- and (2R,3R)-5 (Scheme S1), derivatized them with LFDAA, and compared the retention times of their derivatives with that of the L-FDAA derivative of natural 5 from 2. Natural 5 exhibited an identical retention time with synthetic (2S,3S)-5 (Figure 6E); thus, the absolute configuration of natural 5 was assigned as 2S,3S. The structure of 2, including absolute configuration, was concluded, as shown in Figure 1B.

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Figure 7. Production and biological activity of ralstonins in tomato. (A) LC/MS analysis of crude extracts of vascular exudates collected from tomato plants infected with strain OE1-1. Arrowheads indicate the peaks of ralstonins. (B) Phytotoxic activity of ralstonins A and B in tomato leaves. Induction of necrosis was observed after inoculation of ralstonin solutions.

thus, we were unable to test its phytotoxicity. It was therefore suggested that ralstonins act as phytotoxins and function in R. solanacearum virulence. Deletion of Ralfuranone/Ralstonin Production Resulted in Reduced Virulence on Tomato and Tobacco. The above results suggested that ralstonins/ralfuranones are involved in R. solanacearum virulence. Therefore, we compared the virulence of the rmyA mutant (ralstonin-deficient, Figure S10), ΔralA (ralfuranone-deficient),18 and OE1-1 on tomato seedlings using a root inoculation method recently reported by Ray and colleagues.33 As observed in previous studies,14,18 the virulence of ΔralA was significantly reduced compared with that of strain OE1-1 (Figure 8A). The virulence of rmyA mutant was weaker than those of OE1-1 and ΔralA. We also carried out virulence assay for these strains in tobacco plants and confirmed that rmyA mutant and ΔralA also had reduced virulence on tobacco plants (Figure 8B). These results strongly suggest that ralstonins function in R. solanacearum virulence on tomato and tobacco plants, although they are not essential for pathogenicity.

Figure 8. Virulence data for OE1-1, ΔralA, and rmyA mutant in the inoculated tomato seedlings (A) and tobacco plants (B). The % values were the average of three experiments, and 30 plants were used in each experiment. Error bars are the mean ± SEM. *p < 0.05 versus control (Dunnett’s test). Representative assay images are shown as insets.

to contain millimolar levels of sugars (e.g., D-glucose, Dfructose, sucrose),7,26 this pathogen can produce larger amounts of EPS within host xylems than expected. This implies that R. solanacearum can utilize large carbon sources in planta by exploiting host resources. The degradation of plant cell walls may comprise some part of that carbon source;6,27 however, the possibility that R. solanacearum subverts the host cell metabolism to reorientate metabolic fluxes for its own purpose was also suggested.27 In rice, OsSWEET11, a gene encoding a sugar efflux carrier, is co-opted during infection by Xanthomonas oryzae pv. oryzae and is required for bacterial growth.36 Therefore, we suspect that R. solanacearum has a chance to sense and use the relatively high levels of sugars in planta. What is the mechanism of activation of ralfuranone/ ralstonin production by sugars in R. solanacearum? Several plant pathogenic bacteria are also known to activate the secondary metabolism in response to host metabolites, including sugars.37 However, the detailed mechanism of this activation remains elusive. As observed in this study, the utilization of host sugars as the carbon sources of secondary metabolites contributes, in part, to the activation of their production. However, a specific sensing mechanism, such as lectins and chemosensory proteins,38,39 may be involved in the activation of ralfuranone/ralstonin production in R. solanacearum. Recently, Deng et al. found that the plant pathogen Xanthomonas campestris utilizes host sugars to produce its QS signals.40 If this is also true for R. solanacearum, the activation of a secondary metabolism by plant sugars may be a reasonable response; this is under investigation. Furthermore, Genin et al. reported that host resource allocation drives metabolic versatility in this pathogen.41 Thus, plant sugars may act as “chemical switches” for metabolic adaptation of R. solanacearum to the in planta environment.

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DISCUSSION In this study, we demonstrated that D-galactose/D-glucose activate the production of ralfuranones/ralstonins by increasing the expression of the biosynthetic genes and by partly becoming the biosynthetic source in R. solanacearum strain OE1-1. This activation of ralfuranone/ralstonin production caused the accumulation of their new derivatives, ralfuranone M (1) and ralstonin C (2), in strain OE1-1 culture. This study is the first to report monosaccharides being responsible for the activation of the secondary metabolism in R. solanacearum, and provides an insight into the ability of R. solanacearum to thrive and adapt to the in planta environment. We also demonstrated the importance of ralfuranone/ralstonin production in R. solanacearum virulence. Recently, Natsume and colleagues reported a potential new chemoattractant of R. solanacearum strain MAFF730138 from tomato root exudates.34,35 In their study, strain MAFF730138 also exhibited chemoattractant activity toward D-galactose. Therefore, R. solanacearum might utilize this sugar throughout its life cycle to detect the presence of host plants and the in planta environment. Although tomato xylem saps are expected 1552

DOI: 10.1021/acschembio.9b00301 ACS Chem. Biol. 2019, 14, 1546−1555

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ACS Chemical Biology

(24−30 min), column oven at 40 °C, flow rate of 200 μL/min, detection ESI positive, m/z 1291.7 for ralstonin A, and m/z 1305.7 for ralstonin B. Isolation of Ralfuranone M (1). The extraction of the 10-L MGRLS culture of strain OE1-1 (50 × 500 mL Erlenmeyer flasks, each containing 200 mL of medium) with EtOAc gave a crude extract. The EtOAc extract was chromatographed on Cosmosil 140C18-OPN gel (Nacalai Tesque) and eluted with a stepwise gradient of H2O/ MeOH (0−100% MeOH). The 40 and 60% MeOH eluates were combined and subjected to HPLC with an Inertsil ODS-3 column (250 × 10 mm, 5 μm) and an H2O/MeCN gradient (20−95% MeCN) to yield ralfuranone M (1) (1 mg). Isolation of Ralstonin C (2). OE1-1 cells grown in B medium for 6 h were spread on 6000 B agar plates containing 0.5% (w/v) Dgalactose, and the plates were incubated at 30 °C for 3 days. B agar was cut into small pieces and soaked in acetone (150 L) for 2 h. The acetone extract was collected by filtration, and the agar residue was soaked again in acetone (150 L) for 2 h. The combined extracts were evaporated to remove acetone, extracted with EtOAc three times, and dried over Na2SO4. The EtOAc extracts were concentrated and chromatographed on a silica gel column. They were eluted stepwise with solvents of increasing polarity from n-hexane to EtOAc and then from EtOAc to MeOH. The fractions containing ralstonins (60% and 100% MeOH eluates) were subjected to HPLC with an InertSustain C18 column (250 × 10 mm, 5 μm) at a flow rate of 4 mL/min with 45% MeCN in 0.1% aq TFA to yield ralstonins C (2) with minor impurities. The compound was finally purified by HPLC with a COSMOSIL πNAP (250 × 10 mm, 5 μm, Nacalai Tesque) at a flow rate of 4 mL/min with 40% MeCN in 0.1% aq TFA to yield ralstonin C (2) (2.5 mg). Reporter Gene Assays. RK8343 (pralA-gfp) cells grown in B medium at 30 °C for 4−6 h were diluted to an OD600 of 0.05 with MGRLS medium. Each 2 mL cell suspension was transferred to test tubes and incubated for 48 h at 30 °C with shaking. After incubation, the bacterial cells were collected by centrifugation and suspended in 0.85% (w/v) aq NaCl (2 mL). The cell suspension (200 μL) was used to measure GFP fluorescence (Ex. 492 nm/Em. 530 nm) and OD600. RK8421 (prmyA-lacZYA) cells grown in B medium at 30 °C for 4− 6 h were diluted to an OD600 of 0.05 in B medium. A sample (50 μL) of the cell suspension was pipetted onto B agar plates (control and 0.1−0.5% D-galactose/D-glucose), and the plates were incubated for 3 days at 30 °C. Bacterial cells were collected in Milli-Q water (5 mL), and the OD600 values were measured. β-Galactosidase activity was determined by measuring the rate of hydrolysis of O-nitrophenyl-β-Dgalactopyranoside and expressed in Miller units.

We isolated and identified ralfuranone M (1) and ralstonin C (2) as new secondary metabolites of strain OE1-1. Ralstonin C (2) is the first lipopeptide containing (2S,3S,Z)-Ahie as a fatty acid moiety. We applied olefin cross-metathesis to determine the double-bond position in the fatty acid moiety of ralstonin C (2) (Figure 6C). This provides a new methodology to elucidate the structures of lipopeptides with an unsaturated fatty acid moiety. The cross-metathesis conjugation of the lipopeptides with a functional tag, such as a fluorescent dye or biotin, may be also useful to study the mode of action in the target organisms.42 Ralfuranone M (1) and ralstonin C (2) may have lower importance for R. solanacearum virulence because their levels in strain OE1-1 cultures were low compared with their major derivatives. However, the identification of new derivatives of ralfuranones/ ralstonins supports that R. solanacearum actively produces ralfuranones/ralstonins in response to plant-derived sugars. rmyA mutant, which cannot produce ralstonins, exhibited reduced virulence on tomato seedlings compared with the wild-type strain, OE1-1 (Figure 8A). This result was inconsistent with the previous report by Nett et al., where ΔrmyA did not exhibit reduced virulence on tomato plants.23 However, we are aware of the importance of inoculation methods (e.g., inoculation through petioles or roots) and plants (e.g., growth stage, cultivars) for the results of virulence assays of R. solanacearum strains. For example, the virulence of ΔralA differed on the basis of the inoculation methods used.14,18 In addition, the reduced virulence of rmyA mutant was also observed using tobacco plants (Figure 8B). We confirmed that ralstonins had moderate phytotoxicity against tomato (Figure 7B) and tobacco.22 Therefore, we suggest that ralstonins function as the virulence factors of R. solanacearum strain OE1-1. In addition, the possibility that ralstonins play other unknown biological roles, such as biosurfactants,32 during pathogenesis remains. To answer such questions, we must continue to analyze the actions of rmyA mutant in planta. In conclusion, as observed in other plant-related proteobacteria, R. solanacearum may also sense host compounds and then activate the production of ralfuranones/ralstonins. We suspect that this pathogen evaluates the environment to shift its cell activity in response to the stage of host invasion. We are also interested in the nature of plant signals that activate the expression of TTSS in R. solanacearum. In the future, we may be able to clarify the entire picture of chemical communication between R. solanacearum and plant cells during invasion and pathogenesis. We believe that these efforts will help to develop methodologies to save crops from bacterial wilt disease.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.9b00301. Supporting methods and NMR data (PDF)

METHODS

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Analysis of Ralfuranones/Ralstonins Produced by Strain OE1-1. OE1-1 cells grown in B medium at 30 °C for 4−6 h were diluted to an OD600 of 1.0 with new medium. The cell suspension (1 mL) was diluted with MGRLS medium (100 mL) in 300 mL Erlenmeyer flasks and incubated at 30 °C with rotation for 4 days. Following growth, the bacterial cultures were extracted three times with an equal volume of EtOAc. The combined EtOAc extracts were dried over Na2SO4 and evaporated to dryness. The residues were dissolved in MeOH (500 μL) and subjected to the following HPLC analysis for ralfuranones: column InertSustain C18 (150 × 4.6 mm, 3 μm, GL Sciences), column oven at 40 °C, flow rate of 1 mL/min, and eluent of 20−95% (v/v) MeCN in H2O (0−24 min) and 95% (v/v) MeCN (24−30 min). The following LC/MS conditions were used for ralstonins: InertSustain C18 column (150 × 2.1 mm, 3 μm), eluent of 20−95% MeCN in 0.1% aq formic acid (0−24 min) and 95% MeCN

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Kenji Kai: 0000-0002-4036-9959 Author Contributions ¶

(Y.I., Y.M., K.K.) These authors contributed equally to this work. Notes

The authors declare no competing financial interest. 1553

DOI: 10.1021/acschembio.9b00301 ACS Chem. Biol. 2019, 14, 1546−1555

Articles

ACS Chemical Biology

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VsrAD and PhcA control secondary metabolism in the plant pathogen Ralstonia solanacearum. ChemBioChem 10, 2730−2732. (17) Pauly, J., Spiteller, D., Linz, J., Jacobs, J., Allen, C., Nett, M., and Hoffmeister, D. (2013) Ralfuranone thioether production by the plant pathogen Ralstonia solanacearum. ChemBioChem 14, 2169−2178. (18) Kai, K., Ohnishi, H., Mori, Y., Kiba, A., Ohnishi, K., and Hikichi, Y. (2014) Involvement of ralfuranone production in the virulence of Ralstonia solanacearum OE1-1. ChemBioChem 15, 2590− 2597. (19) Kai, K., Ohnishi, H., Kiba, A., Ohnishi, K., and Hikichi, Y. (2016) Studies on the biosynthesis of ralfuranones in Ralstonia solanacearum. Biosci., Biotechnol., Biochem. 80, 440−444. (20) Kai, K. (2018) Bacterial quorum sensing in symbiotic and pathogenic relationships with hosts. Biosci., Biotechnol., Biochem. 82, 363−371. (21) Mori, Y., Hosoi, Y., Ishikawa, S., Hayashi, K., Asai, Y., Ohnishi, H., Shimatani, M., Inoue, K., Ikeda, K., Nakayashiki, H., Nishimura, Y., Ohnishi, K., Kiba, A., Kai, K., and Hikichi, Y. (2018) Ralfuranones contribute to mushroom-type biofilm formation by Ralstonia solanacearum strain OE1-1. Mol. Plant Pathol. 19, 975−985. (22) Murai, Y., Mori, S., Konno, H., Hikichi, Y., and Kai, K. (2017) Ralstonins A and B, lipopeptides with chlamydospore-inducing and phytotoxic activities from the plant pathogen Ralstonia solanacearum. Org. Lett. 19, 4175−4178. (23) Baldeweg, F., Kage, H., Schieferdecker, S., Allen, C., Hoffmeister, D., and Nett, M. (2017) Structure of ralsolamycin, the interkingdom morphogen from the crop plant pathogen Ralstonia solanacearum. Org. Lett. 19, 4868−4871. (24) Li, P., Yin, W., Yan, J., Chen, Y., Fu, S., Song, S., Zhou, J., Lyu, M., Deng, Y., and Zhang, L.-H. (2017) Modulation of inter-kingdom communication by PhcBSR quorum sensing system in Ralstonia solanacearum phylotype I strain GMI1000. Front. Microbiol. 8, 1172. (25) Aldon, D., Brito, B., Boucher, C., and Genin, S. (2000) A bacterial sensor of plant cell contact controls the transcriptional induction of Ralstonia solanacearum pathogenicity genes. EMBO J. 19, 2304−2314. (26) Zuluaga, A. P., Puigvert, M., and Valls, M. (2013) Novel plant inputs influencing Ralstonia solanacearum during infection. Front. Microbiol. 4, 349. (27) Lowe-Power, T. M., Hendrich, C. G., von Roepenack-Lahaye, E., Li, B., Wu, D., Mitra, R., Dalsing, B. L., Ricca, P., Naidoo, J., Cook, D., Jancewicz, A., Masson, P., Thomma, B., Lahaye, T., Michael, A. J., and Allen, C. (2018) Metabolomics of tomato xylem sap during bacterial wilt reveals Ralstonia solanacearum produces abundant putrescine, a metabolite that accelerates wilt disease. Environ. Microbiol. 20, 1330−1349. (28) Mori, Y., Inoue, K., Ikeda, K., Nakayashiki, H., Higashimoto, C., Ohnishi, K., Kiba, A., and Hikichi, Y. (2016) The vascular plantpathogenic bacterium Ralstonia solanacearum produces biofilms required for its virulence on the surfaces of tomato cells adjacent to intercellular spaces. Mol. Plant Pathol. 17, 890−902. (29) Wackler, B., Schneider, P., Jacobs, J. M., Pauly, J., Allen, C., Nett, M., and Hoffmeister, D. (2011) Ralfuranone biosynthesis in Ralstonia solanacearum suggests functional divergence in the quinone synthetase family of enzymes. Chem. Biol. 18, 354−360. (30) Kwon, Y., Lee, S., Oh, D.-C., and Kim, S. (2011) Simple determination of double-bond positions in long-chain olefins by crossmetathesis. Angew. Chem., Int. Ed. 50, 8275−8278. (31) Shin, B., Park, S. H., Kim, B.-Y., Jo, S.-I., Lee, S. K., Shin, J., and Oh, D.-C. (2017) Deinococcucins A−D, aminoglycolipids from Deinococcus sp., a gut bacterium of the carpenter ant Camponotus japonicus. J. Nat. Prod. 80, 2910−2916. (32) Raaijmakers, J. M., de Bruijn, I., Nybroe, O., and Ongena, M. (2010) Natural functions of lipopeptides from Bacillus and Pseudomonas: more than surfactants and antibiotics. FEMS Microbiol. Rev. 34, 1037−1062. (33) Singh, N., Phukan, T., Sharma, P. L., Kabyashree, K., Barman, A., Kumar, R., Sonti, R. V., Genin, S., and Ray, S. K. (2018) An

ACKNOWLEDGMENTS We gratefully acknowledge H. Konno (Yamagata University) for providing the synthetic β-OH-Tyr standards. This work was supported by JSPS KAKENHI (Grant No. 17K19244) and the grant from the Project of the NARO Bio-Oriented Technology Research Advancement Institution (Research Program on Development of Innovative Technology, Grant No. 29003A).

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DOI: 10.1021/acschembio.9b00301 ACS Chem. Biol. 2019, 14, 1546−1555