CHEMBIOCHEM FULL PAPERS DOI: 10.1002/cbic.201402404
Involvement of Ralfuranone Production in the Virulence of Ralstonia solanacearum OE1-1 Kenji Kai,*[a] Hideyuki Ohnishi,[a] Yuka Mori,[b] Akinori Kiba,[b] Kouhei Ohnishi,[c] and Yasufumi Hikichi[b] Ralstonia solanacearum causes a destructive disease called “bacterial wilt” in numerous plant species. Its virulence is controlled by the transcriptional regulator PhcA, the activity of which is, in turn, regulated in a cell-density dependent manner, termed quorum sensing. We herein described the identification and characterization of ralfuranones J–L, new PhcA-regulated secondary metabolites, and the known derivatives, ralfuranones A and B, from R. solanacearum strain OE1-1.
Their structures were determined by spectroscopic and chemical methods. These ralfuranones were also detected in vascular exudates from host plants infected with OE1-1. Deletion of ralA, which encodes an enzyme for ralfuranone biosynthesis, reduced the virulence of OE1-1 in tomato plants. Virulence was restored by complementation of the ralA gene. The results suggest that ralfuranones play important roles in the virulence of OE1-1.
Introduction The Gram-negative bacterium Ralstonia solanacearum is the causative agent of “bacterial wilt” in more than 200 plant species in tropical, subtropical, and warm temperate regions of the world.[1, 2] Its hosts include agronomically important crops, such as tomato, potato, tobacco, peanut, and banana. The pathogen enters the host through wounded roots or the sites of secondary-root emergence; once inside a host, it quickly colonizes the intercellular spaces, then invades xylem vessels and grows vigorously there. When infection exceeds 109 cfu per gram (stem tissue), R. solanacearum causes the wilting symptoms observed in infected plants.[3] The ability of this pathogen to cause host wilting has been mainly attributed to its production of extracellular polysaccharide (EPS), which occludes vascular tissues and prevents water flow.[1, 2] The bacterium also uses plant cell-wall-degrading enzymes (e.g., polygalacturonase and endoglucanase) to invade and grow in host tissue. The production of these virulence factors is controlled by a complex regulatory network in which PhcA, a LysR-type transcriptional regulator, plays a central role.[1, 2, 4] The activity of PhcA is controlled by the PhcS/PhcR two-component regulatory system, which de[a] Prof. K. Kai, H. Ohnishi Graduate School of Life and Environmental Sciences Osaka Prefecture University 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531 (Japan) E-mail: [email protected] [b] Y. Mori, Prof. A. Kiba, Prof. Y. Hikichi Laboratory of Plant Pathology and Biotechnology Kochi University 200 Otsu, Monobe, Nanko-ku, Kochi 783-8502 (Japan) [c] Prof. K. Ohnishi Research Institute of Molecular Genetics Kochi University 200 Otsu, Monobe, Nanko-ku, Kochi 783-8502 (Japan) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cbic.201402404.
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
tects 3-hydroxypalmitic acid methyl ester (3-OH PAME), a PhcBderived quorum sensing (QS) signal.[5–7] QS generally allows bacteria to coordinate their activities in response to population size.[8] The response regulator PhcR post-transcriptionally inhibits the transcription of phcA; however, when the concentration of 3-OH PAME reaches a threshold level, this signal is received by the sensor kinase PhcS, which phosphorylates PhcR, and the transcription of phcA is then activated.[5–7] Here, we referred to this QS mechanism as “phc QS”. The contribution of secondary metabolites, unlike EPS and extracellular enzymes, to the virulence of R. solanacearum remains unclear. Several plant pathogenic bacteria are known to regulate secondary metabolism by using QS systems.[9] For example, the production of toxoflavin (a phytotoxin) is controlled by QS mediated by N-acylhomoserine lactones in the rice pathogen Burkholderia glumae.[10, 11] 3-Hydroxybenzoic acid, an unusual QS signal, regulates the biosynthesis of xanthomonadin (a yellow pigment) in the black rot pathogen Xanthomonas campestris.[12] This metabolite protects the bacterium from photo-oxidative damage.[13] Crosstalk between secondary metabolism and QS in R. solanacearum has only recently become a research topic, and has led to the novel identification of the small molecules, ralfuranones A and B (Scheme 1), from R. solanacearum GMI1000, as well as the characterization of the furanone synthase RalA, an enzyme involved in the biosynthesis of ralfuranones.[14, 15] A recent study reported that ralfuranone I, a biosynthetic precursor of ralfuranones, functions as a reactive Michael acceptor of biomolecular thiols.[16] Furthermore, phc QS was shown to negatively control the production of the siderophore staphyloferrin B in R. solanacearum strain AW1.[17] However, the QS-dependent secondary metabolites of R. solanacearum have not been examined in detail, and little is known about their contribution to bacterial virulence in host plants. Therefore, the identification of secondary metabolites ChemBioChem 0000, 00, 1 – 9
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Scheme 1. Structures of ralfuranones.
(and their production under the control of QS) and the elucidation of their biological roles has become an important theme in the study of the bacteria–plant interactions. The aim of this study was to identify secondary metabolites under the control of phc QS and elucidate their roles in the virulence of R. solanacearum. We compared the metabolic profiles of R. solanacearum strain OE1-1 (isolated in Japan and pathogenic to solanaceous plants)[18] with a phcA-deficient mutant (DphcA).[19] Five metabolites were markedly decreased in DphcA and were identified as known and novel derivatives of ralfuranones (Scheme 1) by spectroscopic and chemical methods. We also investigated the biological roles of ralfuranones by using assays with synthetic ralfuranones and analyzed the ralfuranone-deficient mutant strain of OE1-1.
Results and Discussion Search for phc QS-dependent secondary metabolites To identify phc QS-regulated secondary metabolites, we first compared the HPLC chromatograms of culture extracts of OE11 and DphcA. Previous studies reported that the expression of genes related to the virulence of R. solanacearum was dependent on growth conditions, especially nutrition.[20–22] The xylem fluid of tomato, a representative host of R. solanacearum, contains minerals, amino acids, and a high amount of sucrose.[23, 24] Jacobs et al. demonstrated that host sucrose was important for the virulence of R. solanacearum, and that this was because the bacterium incorporates host sucrose via a specific transporter and uses it as a carbon source during the development of wilt disease.[25] Thus, to identify the secondary metabolites involved in the pathogenicity and virulence of OE1-1, we chose MGRLS medium (MGRL medium supplemented with 3 % sucrose),[19, 26] which mimics the nutrient conditions in the vascular tissues of host plants. We detected five major peaks (compounds 1–5) in the EtOAc extract from a four-day-old culture of OE1-1, but not in the chromatogram of DphcA (Figure 1); this confirmed that these metabolites were under the 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 1. HPLC metabolic profiles of culture extracts from A) OE1-1 and B) DphcA. The data indicate that the production of compounds 1–5 was under the control of phc QS.
control of phc QS. We also examined the culture extracts of OE1-1 grown in 1/4 M63 medium, a minimal medium used in previous ralfuranone studies:[14, 15] 2, 3, and 5 were not detected in these culture extracts. Structure elucidation of phc QS-dependent secondary metabolites To elucidate the structures of 1–5, an EtOAc extract of the culture broth (20 L) of OE1-1 was prepared and subjected to separation by chromatography. The EtOAc extract was purified by ODS column chromatography eluted with H2O/MeOH. The fractions eluted at 40 and 60 % MeOH were further purified by reversed-phase HPLC with an H2O/MeCN gradient (20 to 95 % MeCN) to give 1 (0.6 mg), 2 (2.3 mg), 3 (0.9 mg), 4 (1.2 mg), and 5 (ca. 0.05 mg). The UV spectra of 1–5 were similar to those of ralfuranones previously identified from R. solanacearum GMI1000.[14, 15] This suggested that the compounds were known and new derivatives of ralfuranones. The ESI-MS spectra of 1 and 4 showed ions at m/z 161 [M+H] + and 265 [M H] , corresponding to ralfuranones A and B, respectively. Their 1H NMR spectra were almost identical to those of ralfuranones A and B,[14, 15] thus we concluded that 1 and 4 were ralfuranones A and B, respectively (Scheme 1). A previous study suggested that ralfuranone B might be a chiral molecule.[15] However, as the chiral HPLC analysis of 4 gave two peaks with a ratio of 1:1 (Figure S1 in the Supporting Information), the compound was revealed to be a racemic mixture of C-5 enantiomers. The ESI-MS of purified 2 showed an [M H] ion at m/z 281, thus indicating that its molecular weight is 16 amu larger than that of 4. Thus, the compound was suggested to be an oxygenated derivative of 4. The 1H NMR spectrum of 2 was similar to that of 4; however, the proton signal of H-5 in 2 showed a lower-field shift (dH = 5.18 and 5.26 to dH = 6.50 ppm) and ChemBioChem 0000, 00, 1 – 9
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Table 1. 1H and 13C NMR data for ralfuranones J (2), K (3), and L (5) No. 13
2 3 4 5 6 7 8 9 10 11 12 13 14
C
172.4 131.4 160.1 99.5 131.9 130.2 129.6 131.3 68.2 142.5 127.3 129.3
1
2[a] H, mult. (J, Hz)
6.50, 1 H; s 7.57, 7.44, 7.43, 5.78,
2 H; 2 H; 1 H; 1 H;
m m m s
7.41, 2 H; m 7.29, 2 H; tt (7.3, 1.5) 128.5 7.22, 1 H; tt (7.3, 1.5)
13
C
168.8 127.9 160.8 98.5 129.9 129.8 129.9 132.5 192.3 136.1 130.2 130.0
1
3[b] H, mult. (J, Hz)
6.72, 1 H; s 7.47, 2 H; m 7.36, 2 H; m 7.43, 1 H; m
7.94, 2 H; m 7.48, 2 H; tt (7.4, 1.2) 135.7 7.65, 1 H; t (7.4)
13 [c]
C
177.5 126.1 160.1 72.6 132.4 128.7 130.2 131.6 30.7 139.3 129.2 129.7
1
5[a] H, mult. (J, Hz)
5.24, 2 H; s 7.48, 7.42, 7.42, 3.84,
2 H; 2 H; 1 H; 2 H;
m m m s
7.17, 2 H; m 7.22, 2 H; m
127.6 7.14, 1 H; m
sion between the C-5 enantiomers, 3 was detected as one peak by chiral HPLC (Figure S1). A similar profiling analysis indicated that 5 was a major metabolite of OE1-1; however, the isolated compound was the smallest among these. This might have been due to the different conditions between the small- and large-scale cultures of OE1-1 rather than instability of 5. The structure of 5 was expected to be a deoxygenated derivative of 4, because the [M+H] + ion of 5 was at m/z 251. The aromatic proton signals of 5 suggested the presence of two one-substituted benzene rings (Table 1). The singlet methylene signals at dH = 3.84 and 5.24 ppm were assigned to H-10 and H-5, respectively. Thus, the structure of 5, named ralfruranone L, was deduced (Scheme 1). However, due to the low quantity of 5, its 13C and 2D NMR data could not be obtained, so the complete identification of this structure depended on subsequent chemical synthesis.
[a] in CD3OD. [b] in CD3CN. [c] Data from synthetic 5.
had fewer hydrogens, from 2 to 1 (Table 1). According to the 13 C NMR spectrum, the carbon signal of C-5 in 2 was at dC = 99.5 ppm, which suggests a hemiacetal carbon. The carbon and proton signals responsible for the two 1-substituted benzenes and one 3,4-substituted furanone were observed in the 1 H and 13C NMR spectra of 2. The HMBC correlations from H-5 to C-2 and C-3, from H-10 to C-2, C-3, C-4, C-11, and C-12, and from H-12 to C-10 revealed that two oxymethines were present in the two aryl-substituted furanone ring backbone (Figure 2). Thus, 2 was determined to be the 5-hydroxy derivative of 4, and was given the name ralfuranone J (Scheme 1).
Figure 2. Key HMBC correlations of natural 2 and 3 and synthetic 5.
Compound 2 gave only two peaks in chiral HPLC analysis (Figure S1), although the compound has two chiral centers (and thus theoretically should give four peaks) and did not display any diastereomeric proton or carbon signal (Table 1). We concluded that 2 was a rapid equilibrium mixture of C-5 epimers. The two peaks observed in the chiral HPLC analysis might have derived from the C-10 epimers. ESI-MS of purified 3 gave an [M+H] + ion at m/z 281, thus indicating that the compound has two hydrogens fewer than 2. The oxymethine proton signal at C-10 in 2 was absent in the 1 H NMR spectrum of 3. Furthermore, the carbon signal of C-10 in 3 shifted to dC = 192.3 ppm, the carbonyl carbon region. The HMBC correlations from H-5 to C-2 and C-3, from H-7 to C-4, and from H-12 to C-10 showed a backbone composed of the two aryl-substituted furanone ring in 3 (Figure 2). Thus, the structure of 3, named ralfuranone K, was revealed to be a 10oxo derivative of 2 (Scheme 1). Because of the rapid conver 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chemical synthesis of ralfuranones To confirm the structures of ralfuranones and obtain them for our biological studies, we synthesized all ralfuranones (Schemes 2 and 3). Arylfuranones have been the targets of synthetic studies because they exhibit many biological activities; ralfuranone A (1) and candidates for the synthetic precursors of other ralfuranones have been synthesized.[27–31] Compound 1 was prepared from bromobenzene (6) by the method by Verendel et al. (Scheme 2; See the Experimental Section).[27] Luche reduction of 13, which was synthesized from benzoic acid (9) in four steps by the method of Peixoto et al.,[28] gave ralfuranone B (4) in good yield (94 %). Ralfuranones J (2) and K (3) were synthesized by catalytic oxidation with molecular oxygen from 19,[29] which was synthesized from ethyl benzoate (14) in five steps by the methods of Basavaiah et al. and Clive et al. (Scheme 3).[30, 31] Although the yields of 2 and 3 were very low, this demonstrated the potential of catalytic oxidation in the synthesis of highly oxidized arylfuranones. Ralfuranone L (5) was synthesized analogously to ralfuranone A (1). Condensation of 7 with 2-benzylacrylic acid yielded ester 20 in 72 % yield. Ring closure of 20 was carried out by using olefin metathesis with Grubbs catalyst second generation (Grubbs 2nd) to give 5 in 41 % yield. Therefore, we accomplished the first total synthesis of ralfuranones B (4), J (2), K (3), and L (5). The ESI-MS data and HPLC retention times of synthetic ralfuranones were identical to those of the natural compounds. Furthermore, the 1H and 13C NMR data of synthetic and natural ralfuranones were almost identical (Figure S2). The complete assignments of the proton and carbon signals of 5 were based on HMQC and HMBC data of the synthetic compound (Table 1; Figure 2). We also confirmed that synthetic ralfuranones B (4), J (2), and K (3), which were obtained as stereoisomeric mixtures of C-5/C-10, had the same retention times as those of the natural compounds in chiral HPLC analysis (Figure S1). Therefore, these ralfuranones have the structures described in Scheme 1. ChemBioChem 0000, 00, 1 – 9
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Scheme 2. Synthesis of ralfuranones A (1) and B (4). a) i: Mg, Et2O, reflux; ii: propargyl alcohol, CuI, Et2O, RT to reflux, 80 %; b) acryloyl chloride, Et3N, CH2Cl2, RT, 83 %; c) Grubbs 2nd, CH2Cl2, reflux, 45 %; d) i: (COCl)2, CH2Cl2, RT; ii: N,O-dimethylhydroxylamine, Et3N, CH2Cl2, RT, quant.; e) tert-butyl acetate, LDA, THF, 78 8C, quant.; f) acetone, Ac2O, H2SO4, RT, 85 %; g) 2-hydroxyacetophenone, toluene, Et3N, MS 4 A, 110 8C, 94 %; h) NaBH4, CeCl3, MeOH, 0 8C, 94 %.
www.chembiochem.org were synthesized by the actions of enzymes encoded by the ral gene cluster.[15] The aminotransferase RalD converts phenylalanine to phenylpyruvic acid, which is then loaded to the furanone synthase RalA, composed of a tridomain nonribosomal peptide synthetase (NRPS)-like enzyme. Two phenylpyruvic acids condense to form the intermediate, ralfuranone I.[16] The compound is further modified by enzymatic or non-enzymatic reactions to afford ralfuranones A (1) and B (4). Based on the structures of ralfuranones, the following metabolic routes were proposed: 1) 5 is synthesized from ralfuranone I and could be a precursor of other ralfuranones; and 2) 4 is oxidized to form ralfuranones J (2) and K (3). To investigate these possibilities, we synthesized deuterium-labeled ralfuranone L ([D5]5) and ralfuranone B ([D5]4), and conducted incorporation studies with the labeled compounds. Incorporation of the [D5]5 deuterium into other ralfuranones was not observed in the LC-MS analysis of the culture extract of OE1-1, which was grown in the presence of [D5]5 (10 mm; Figure S3). Thus, 5 might not be a precursor of other ralfuranones in OE1-1. In contrast, [D5]4 deuterium incorporation into 2 and 3 was confirmed by LC-MS analysis (Figure S4). Based on the areas of the peaks, these incorporation rates were calculated to be 13 and 29 %, respectively. Therefore, 2 and 3 were postulated as the C-5 oxidized products of 4. Production of ralfuranones by OE1-1 in host plants We investigated whether OE1-1 produced ralfuranones in the xylem of host plants. We inoculated OE1-1 into tomato and tobacco plants by the wounded-petiole inoculation method,[32] 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 MilliQ water. Water was extracted with EtOAc, and analyzed by HPLC: all the ralfuranones were detected in the vascular exudates of both tomato and tobacco plants (Figure 3 A and B). Their identities were confirmed by UV spectra and LC-MS analysis. Thus, we confirmed that OE1-1 also produced ralfuranones A (1), B (4), J (2), K (3), and L (5) in host plants. Based on standard curves, we estimated the concentrations of ralfuranones as 80 (4), 730 (2), and 610 (5) pmol per plant in tomato, and 29 (4), 73 (2), and 200 (5) pmol per plant in tobacco; no data means were below the quantification limit. Considering the apoplastic spaces of xylem vessels in plants, the production levels appear to be high. The results suggest that ralfuranones, which are actively produced in hosts, might be important for the optimal virulence of OE1-1.
Scheme 3. Synthesis of ralfuranones J (2), K (3), and L (5). a) methyl acrylate, 1,4-diazabicyclo[2.2.2]octane, RT, 47 %; b) benzene, CH3SO3H, reflux, 66 %; c) LiAlH4, Et2O, 20 8C to RT, 45 %; d) PCC, CH2Cl2, RT, 66 %; e) NaClO2, NaH2PO4, aq EtOH, RT, 58 %; f) O2, N-hydroxyphthalimide, Co(OAc)2, MeCN, 40 8C, 1.3 % for 2, 5.2 % for 3; g) 2-benzylacrylic acid, EDC, DMAP, Et3N, CH2Cl2, RT, 72 %; h) Grubbs 2nd, CH2Cl2, reflux, 41 %.
Feeding study of deuterium-labeled ralfuranones How these novel ralfuranones are biosynthesized in R. solanacearum is unknown. Wackler et al. reported that ralfuranones 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Evaluation of phytotoxic effects of ralfuranones Several secondary metabolites of plant pathogens are known to play roles as phytotoxins against host plants.[33] If the ralfuranones exhibited toxicity against host plants, they would be recognized as new virulence factors of R. solanacearum. To examine this, we investigated the toxic effects of ralfuranones on tobacco plants. After inoculation of ralfuranone (1, 10, and 50 mm; 50 mL) into the leaves,[34] no significant changes were observed between treated and control regions (Figure S5). BacChemBioChem 0000, 00, 1 – 9
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www.chembiochem.org ulence of DralA in tomato plants was significantly less than that of OE1-1 (Figure 4 B). Transformation of DralA with a plasmid containing ralA from OE1-1 (ralA-comp) restored the production of ralfuranones (Figure 4 A) and its virulence in tomato plants (Figure 4 B), thus suggesting the involvement of ralA in OE1-1 virulence. Seven days after inoculation, plants of these strains clearly showed that the degree of host wilting was dependent on the presence of ralA (Figure 4 C). Deletion of ralA led not only to the loss of ralfuranone biosynthesis, but also to a reduction in the virulence of R. solanacearum. Taken together, our results implicate the production of ralfuranones in the full virulence of OE1-1 in host plants.
Conclusions
Figure 3. HPLC analysis of crude extracts of vascular exudates of A) tomato and B) tobacco plants infected with OE1-1. Peaks of 1–5 were assigned from their UV and ESI-MS data.
terial phytotoxins also exhibit antimicrobial activity;[33, 35] therefore, we examined the activity of ralfuranones against Escherichia coli, Bacillus subtilis, and Aspergillus aculeatus by using the disc diffusion assay.[34] The growth of these microbes was not inhibited by ralfuranones A, B, J, or L, although ralfuranone K exhibited weak inhibitory activity against B. subtilis (diameter of inhibition zone: 8 mm, at 0.5 mmol per disc). These results suggest that ralfuranones do not possess strong antimicrobial activity; we assumed that ralfuranones are not phytotoxins.
We have identified the novel ralfuranones J (2), K (3), and L (5), as phc QS-dependent metabolites from OE1-1 and synthesized these compounds, as well as the known derivatives, ralfuranones A (1) and B (4). We confirmed the production of ralfuranones by OE1-1 in host plants and the reduced virulence of DralA in tomato plants. These results strongly suggest the involvement of ralfuranone production in R. solanacearum virulence. However, these ralfuranones did not exhibit apparent toxicity in tobacco plants. Therefore, these ralfuranones might cause changes in the bacterium but not in the host. It remains unknown as to how ralfuranones contributed to the virulence of OE1-1. Therefore, further analysis of DralA and identification of the targets of ralfuranones are needed. As ablation of ralfuranone production did not result in complete loss of bacterial
Virulence assay of OE1-1 and ralfuranone-deficient mutant in tomato plants Although ralfuranones did not appear to be active in host plants, this does not exclude ralfuranone involvement in the virulence of R. solanacearum. To determine whether ralfuranones were involved in the virulence of OE1-1, we created a ralA-deficient mutant of OE1-1 (DralA). The mutant strain did not produce any ralfuranones (Figure 4 A). Combined chromatograms were constructed to compare the HPLC chromatograms of OE1-1 and its mutant easily (Figure S6). Using wounded-petiole inoculation, we assessed the ability of DralA to cause wilt disease in tomato plants. The vir-
Figure 4. A) HPLC analysis of culture extracts from DralA and ralA-comp. B) Disease progress curves of OE1-1 (*), DralA (&), and ralA-comp (&). Eight-week-old tomato plants were inoculated by stabbing pipette tips containing bacterial cell suspensions into the third leaf axil from the plant apex. Plants were rated on a zero-to-five disease index scale. Error bars indicate standard deviations (n = 3). * denotes P < 0.05 (Student’s t-test). C) Phenotypes of tomato plants seven days after inoculation of OE1-1, DralA, and ralA-comp.
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CHEMBIOCHEM FULL PAPERS virulence, these compounds are not essential for wilting. Therefore, the results indicate that ralfuranones might be key molecules that enhance bacterial activity in order to induce wilt disease in host plants.
Experimental Section General: All purchased chemicals were used without further purification. Silica gel column chromatography was performed on Wakogel C-200 gel (Wako Pure Chemical Industries, Osaka, Japan). Synthetic reactions were conducted under a nitrogen atmosphere with dry solvents (Wako Pure Chemical Industries) unless otherwise indicated. The yields of synthetic reactions were not optimized. 1H and 13C NMR spectra were recorded on a JNM-AL400 spectrometer (JEOL, Tokyo, Japan). Chemical shifts are reported as d values (ppm); tetramethylsilane (TMS; dH = 0, dC = 0) or solvents (CDCl3 : dH = 7.26, dC = 77.0; CD3CN: dH = 1.93, dC = 118.2; CD3OD: dH = 3.30, dC = 49.0) were used as internal references. High-resolution mass spectra (HRMS) were obtained with a SYNAPT G2 HDMS mass spectrometer (Waters, Milford, MA). HPLC experiments were performed with a LaChrom Elite HPLC system (Hitachi High-Technologies, Tokyo, Japan) and a Prominence HPLC system (Shimadzu, Kyoto, Japan). Solvents for HPLC were purchased from Kanto Chemical (Tokyo, Japan). LC-MS data were obtained with an LCMS2020 spectrometer (Shimadzu, Kyoto, Japan). Microbial cultivation and extraction: Seed cultures of R. solanacearum strain OE1-1[18] were grown in B medium at 30 8C overnight.[19] These were centrifuged to remove medium, and the bacterial cells were then suspended in the same volume of MGRLS medium (MGRL medium[19, 26] supplemented with 3% sucrose). The suspensions were diluted with MGRLS medium in Erlenmeyer flasks and incubated at 30 8C with rotation (130 rpm) 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. 1/4 M63 medium[14, 15] was also used instead of MGRLS medium. Profiling analysis of secondary metabolites: OE1-1 and DphcA[19] were grown in MGRLS medium (100 mL) in 300 mL Erlenmeyer flasks at 30 8C with rotation (130 rpm) 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 mL) and subjected to HPLC analysis: column, Inertsil ODS-3 (250 4.6 mm, 5 mm; GL Sciences, Tokyo, Japan); column oven, 40 8C; flow rate, 1 mL min 1; eluent, MeCN/H2O (20– 95 % linear gradient in 40 min, then 95 % MeCN for 10 min); injection volume, 10 mL. Purification and structure elucidation of ralfuranones: The extraction of the 20 L MGRLS culture of OE1-1 (100 500 mL Erlenmeyer flasks, each containing 200 mL medium) gave a crude EtOAc extract (112 mg). The extract was chromatographed on Chromatorex ODS (Fuji Silysia Chemical, Aichi, Japan) and eluted with a stepwise gradient of H2O/MeOH (0–100 % MeOH). The 40 and 60 % MeOH eluates (14.1 mg) were combined and subjected to HPLC with an Inertsil ODS-3 column (250 10 mm, 5 mm) and an H2O/MeCN gradient (20–95 % MeCN) to yield 1 (0.6 mg), 2 (2.3 mg), 3 (0.9 mg), 4 (1.2 mg), and 5 (ca. 0.05 mg). Ralfuranone A (1): colorless amorphous solid; 1H NMR (CD3OD): d = 5.37 (d, J = 1.7 Hz, 2 H; H-5), 6.53 (t, J = 1.7 Hz, 1 H; H-3), 7.44–7.57 (m, 3 H; H-8, H-9), 7.68 ppm (dd, J = 1.7, 7.8 Hz, 2 H; H-7); UV (MeCN/H2O): lmax = 202, 212, 271; ESI-MS: m/z 161 [M+H] + . 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org Ralfuranone B (4): colorless amorphous solid; 1H NMR (CD3OD): d = 5.18 (d, J = 17.6 Hz, 1 H; H-5a), 5.26 (d, J = 17.6 Hz, 1 H; H-5b), 5.85 (s, 1 H; H-10), 7.20 (tt, J = 1.5, 7.3 Hz, 1 H; H-14), 7.28 (tt, J = 1.5, 7.3 Hz, 2 H; H-13), 7.40 (m, 2 H; H-12), 7.44 (m, 2 H; H-8), 7.45 (m, 2 H; H-9), 7.57 ppm (m, 2 H; H-7); UV (MeCN/H2O): lmax = 264; ESIMS: m/z 264 [M H] . Ralfuranone J (2): colorless amorphous solid; NMR (CD3OD): Table 1; ESI-MS: m/z 281 [M H] ; UV (MeCN/H2O): lmax = 268; HR-ESI-MS: m/z 305.0784 [M+Na] + (calcd for C17H14O4Na, 305.0790). Ralfuranone K (3): colorless amorphous solid, UV (MeCN/H2O): lmax = 260; NMR (CD3CN): Table 1; ESI-MS: m/z 281 [M+H] + ; HR-ESIMS: m/z 303.0628 [M+Na] + (calcd for C17H12O4Na, 303.0633). Ralfuranone L (5): colorless amorphous solid, UV (MeCN-H2O) lmax = 267; 1H NMR (400 MHz, CD3OD): Table 1; ESI-MS: m/z 251 [M+H] + ; HR-ESI-MS: m/z 273.0886 [M+Na] + (calcd for C17H14O2Na, 273.0891). Synthesis of ralfuranone A: The synthesis was performed as described previously.[27] Grignard regent prepared from bromobenzene (6) and magnesium in Et2O was reacted with propargyl alcohol to afford 7 in 80 % yield. Condensation of 7 with acryloyl chloride in CH2Cl2 gave 8 in 83 % yield. Olefin metathesis of 8 with Grubbs 2nd in CH2Cl2 yielded 1 in 45 % yield. 1H NMR (400 MHz, CD3OD): d = 5.37 (d, J = 1.7 Hz, 2 H; H-5), 6.53 (t, J = 1.7 Hz, 1 H; H3), 7.44–7.57 (m, 3 H; H-8, H-9), 7.68 ppm (dd, J = 1.7, 7.8 Hz, 2 H; H7); 13C NMR (100 MHz, CD3OD): d = 72.9 (C-5), 113.3 (C-3), 128.0 (C7), 130.3 (C-8), 131.1 (C-9), 132.9 (C-6), 167.1 (C-4), 176.7 ppm (C-2); ESI-MS: m/z 161 [M+H] + . Synthesis of ralfuranone B: The synthesis of 13 was performed as described previously.[28] The lithium enolate of tert-butyl acetate was prepared by using lithium diisopropylamide (LDA) in THF. Weinreb amide 10 prepared from benzoic acid (9) was added to the solution to afford 11 and its enol quantitatively. Reaction of 11 with acetone in the presence of Ac2O and H2SO4 yielded dioxinone 12 in 85 % yield. Heating 12 in toluene gave a b-acylketene intermediate, which was reacted with 2-hydroxyacetophenone, and then the conjugate was cyclized by an intramolecular Knoevenagel reaction to afford 13 in 94 % yield. NaBH4 (53.3 mg, 1.41 mmol) was added to 13 (339 mg, 1.28 mmol) and CeCl3·7 H2O (716 mg, 1.92 mmol) in MeOH (20 mL) at 0 8C. After stirring at 0 8C for 30 min, the reaction was quenched with HCl (1 m, 60 mL), and the mixture was extracted three times with an equal volume of EtOAc. The combined EtOAc layer was washed with brine and dried over Na2SO4. The concentrate was purified by silica gel chromatography with hexane/EtOAc (9:1) as the eluent to give 4 (320 mg, colorless amorphous solid, yield 94 %). 1H NMR (400 MHz, CD3OD): d = 5.18 (d, J = 17.6 Hz, 1 H; H-5a), 5.26 (1 H; d, J = 17.6 Hz, H-5b), 5.85 (s, 1 H; H-10), 7.20 (1 H; tt, J = 1.5, 7.3 Hz, H14), 7.28 (2 H; tt, J = 1.5, 7.3 Hz, H-13), 7.40 (m, 2 H; H-12), 7.44 (m, 3 H; H-8, H-9), 7.57 ppm (m, 2 H; H-7); 13C NMR (100 MHz, CD3OD): d = 68.2 (C-10), 72.6 (C-5), 127.2 (C-12), 128.4 (C-14), 128.9 (C-3), 129.2 (C-13), 129.4 (C-7), 129.9 (C-8), 131.7 (C-9), 132.0 (C-6), 142.6 (C-11), 161.8 (C-4), 175.6 ppm (C-2); ESI-MS: m/z 265 [M H] . Synthesis of ralfuranones J and K: The synthesis of 19 was performed as described previously.[30, 31] A Morita-Baylis–Hillman reaction of ethyl 2-oxo-2-phenylacetate (14) with methyl acrylate in the presence of 1,4-diazabicyclo[2.2.2]octane provided the Morita– Baylis–Hillman adduct 15 in 47 % yield. Friedel–Crafts reaction of 15 with benzene in the presence of CH3SO3H yielded furandione 16 in 66 % yield. Reductive cleavage of 16 with LiAlH4 in Et2O gave diol 17 in 45 % yield. Oxidation of 17 with PCC in CH2Cl2 afforded ChemBioChem 0000, 00, 1 – 9
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furan 18 in 66 % yield. Conversion of 18 into g-hydroxybutenolide 19 (58 % yield) was performed by treatment with NaClO2 in EtOH containing NaH2PO4.
H2O (for 4), 25 % MeCN in H2O (for 2), and 30 % MeCN in H2O (for 3). Other LC-MS conditions: column oven, 40 8C; flow rate, 200 mL min 1; injection volume, 5 mL.
A solution of 19 (179 mg, 0.672 mmol), N-hydroxyphthalimide (65.8 mg, 0.403 mmol), and Co(OAc)2 (3.57 mg, 20.2 mmol) in MeCN (5 mL) was stirred at 40 8C for three days under an O2 atmosphere.[29] The mixture was diluted with H2O (40 mL) and extracted three times with an equal volume of EtOAc. The combined EtOAc layer was dried over Na2SO4 and evaporated to dryness. The residue was purified by silica gel chromatography with hexane/EtOAc (8:2) as the eluent to give a mixture of 2 and 3. The mixture was further purified by HPLC (Inertsil ODS-3 250 10 mm) with MeCN/ H2O (6:4) as the eluent to afford 2 (2.5 mg, colorless amorphous solid, yield 1.3 %) and 3 (9.9 mg, colorless amorphous solid, yield 5.2 %). This reaction was repeated to obtain sufficient amounts of 2 and 3 for biological studies. Spectroscopic data of 2: 1H NMR (400 MHz, CD3OD): d = 5.78 (s, 1 H; H-10), 6.49 (s, 1 H; H-5), 7.22 (tt, J = 1.5, 7.3 Hz, 1 H; H-14), 7.29 (tt, J = 1.5, 7.3 Hz, 2 H; H-13), 7.41 (m, 2 H; H-12), 7.43 (m, 1 H; H-9), 7.44 (m, 2 H; H-8), 7.57 (m, 2 H; H-7); 13 C NMR (100 MHz, CD3OD): d = 68.3 (C-10), 99.5 (C-5), 127.3 (C-12), 128.5 (C-14), 129.3 (C-13), 129.6 (C-8), 130.2 (C-7), 131.3 (C-9), 131.4 (C-3), 131.9 (C-6), 142.5 (C-11), 160.1 (C-4), 172.4 ppm (C-2); ESI-MS: m/z 281 [M H] . Spectroscopic data of 3: 1H NMR (400 MHz, CD3CN): d = 6.72 (s, 1 H; H-5), 7.36 (m, 2 H; H-8), 7.43 (m, 2 H; H-9), 7.47 (m, 2 H; H-7), 7.48 (m, 2 H; H-13), 7.65 (tt, J = 1.2, 7.4 Hz, 1 H; H-14), 7.94 ppm (m, 2 H; H-12); 13C NMR (100 MHz, CD3CN): d = 98.6 (C-5), 128.0 (C-3), 129.8 (C-7), 129.9 (C-6, C-8), 130.0 (C-13), 130.2 (C-12), 132.5 (C-9), 135.7 (C-14), 136.1 (C-11), 160.8 (C-4), 168.8 (C2), 192.3 ppm (C-10); ESI-MS: m/z 281 [M+H] + .
Feeding study of deuterium-labeled ralfuranones: Deuteriumlabeled [7,7,8,8’,9-2H5]ralfuranone L ([D5]5) and [12,12’,13,13’,142 H5]ralfuranone B ([D5]4) were synthesized from [D5]bromobenzene ( 98 atom % D, Wako Pure Chemical Industries) and [D5]benzoic acid ( 98 atom % D, Cambridge Isotope Laboratories, MA) by the methods described for the synthesis of the corresponding non-labeled ralfuranones. Spectroscopic data of [D5]5: 1H NMR (400 MHz, CD3OD): d = 3.88 (s, 2 H; H-10), 5.27 (s, 2 H; H-5), 7.17 (m, 1 H; H-14), 7.20 (m, 2 H; H-12), 7.25 (tt, J = 1.2, 7.2 Hz, 2 H; H-13); 13C NMR (100 MHz, CD3OD): d = 30.7 (C-10), 72.6 (C-5), 126.1 (C-3), 127.6 (C14), 129.2 (C-12), 129.7 (C-13), 132.2 (C-6), 139.3 (C-11), 160.0 (C-4), 177.5 ppm (C-2); ESI-MS: m/z 256 [M+H] + . Spectroscopic data of [D5]4: 1H NMR (400 MHz, CD3OD): d = 5.17 (d, J = 17.6 Hz, 1 H; H5a), 5.25 (d, J = 17.6 Hz, 1 H; H-5b), 5.85 (s, 1 H; H-10), 7.44 (m, 3 H; H-8, H-9), 7.57 ppm (m, 2 H; H-7); 13C NMR (100 MHz, CD3OD): d = 68.1 (C-10), 72.6 (C-5), 128.9 (C-3), 129.4 (C-7), 129.9 (C-8), 131.7 (C9), 132.0 (C-6), 142.5 (C-11), 161.7 (C-4), 175.6 ppm (C-2); ESI-MS: m/z 270 [M H] . Deuterium-labeled ralfuranone solutions (100 mL, 10 mm in EtOH) were added to 2-day-old cultures of OE1-1 in MGRLS (100 mL), and the cultures were grown for a further 2 days at 30 8C. Analytical samples were prepared as described above and subjected to LC-MS analysis: column, InertSustain C18 (150 2.1 mm, 3 mm; GL Sciences); column oven, 40 8C; flow rate, 200 mL min 1; eluent system, MeOH in H2O (20–95 % linear gradient in 24 min, then 95 % MeOH in H2O for 6 min); injection volume, 1 mL.
Synthesis of ralfuranone L: A CH2Cl2 solution (15 mL) of 7 (1.23 g, 9.17 mmol), 2-benzylacrylic acid (1.78 g, 11.0 mmol), EDC·HCl (2.64 g, 13.8 mmol), DMAP (224 mg, 1.83 mmol), and Et3N (3.86 mL, 27.5 mmol) was stirred at RT for 16 h. The mixture was diluted with CHCl3 (200 mL) and quenched with HCl (1 m, 200 mL). The organic layer was washed with saturated NaHCO3 and brine, and dried over Na2SO4. After evaporation, the residue was purified by silica gel chromatography with hexane/EtOAc (95:5) as the eluent to give 20 (1.84 g, yellowish oil, yield 72 %). 1H NMR (400 MHz, CDCl3): d = 3.62 (s, 2 H), 5.04 (br s, 2 H), 5.31 (dd, J = 1.2, 2.2 Hz, 1 H), 5.47 (dd, J = 1.5, 2.9 Hz, 1 H), 5.54 (br s, 1 H), 6.23 (m, 1 H), 7.14–7.22 (3 H), 7.24–7.37 (5 H), 7.38–7.43 ppm (2 H); 13C NMR (100 MHz, CDCl3): d = 38.0, 66.0, 115.1, 126.0 (2), 126.3, 126.8, 128.0, 128.4 (2), 1285.5 (2), 129.0 (2), 138.0, 138.5, 139.9, 142.4, 166.6 ppm.
Collection of vascular exudates: Tomato plants (Solanum esculemtum cv. Ohgata-Fukuju) and tobacco plants (Nicotiana benthamiana) were grown in pots containing commercial soil (Hanagokorobaiyoudo; Hanagokoro, Aichi, Japan) in a growth incubator at 25 8C under 10 000 lux for 16 h per day. Three- to four-week-old plants were inoculated by stabbing with 200 mL pipette tips containing bacterial cell suspensions (100 mL, 1.0 107 cfu mL 1) into the third leaf axil from the plant apex.[32] Pipette tips were removed after the entire bacterial suspension had entered the stems. The inoculated plants were grown in an incubator at 25 8C under 10 000 lux for 16 h per day. After 14 days, the stems of five wilting plants were cut with a surgical knife and immersed in MilliQ water (10 mL) for 3 h. The water containing vascular exudates and bacterial cells was extracted with an equal volume of EtOAc three times. The extracts were dried over Na2SO4 and evaporated. The residues were dissolved in MeOH (tomato: 100 mL; tobacco: 20 mL), and the supernatants were analyzed by HPLC as above.
Compound 20 (312 mg, 1.12 mmol) was dissolved in CH2Cl2 (15 mL), and the mixture was heated to reflux. A suspension of Grubbs 2nd (19.0 mg, 22.4 mmol) in CH2Cl2 (5 mL) was added dropwise to the mixture over 1.5 h. After stirring for 10 h, the catalyst suspension was added again to the reaction mixture, and the reaction was continued for an additional 4 h. After filtration to remove the catalyst, the residue was purified by silica gel chromatography with hexane/EtOAc (9:1) as the eluent to give 5 (115 mg, colorless amorphous solid, yield 41 %). 1H NMR (400 MHz, CD3OD): d = 3.88 (s, 2 H; H-10), 5.27 (s, 2 H; H-5), 7.17 (m, 1 H; H-14), 7.20 (m, 2 H; H12), 7.25 (tt, J = 1.2, 7.2 Hz, 2 H; H-13), 7.46 (m, 3 H; H-8, H-9), 7.52 ppm (m, 2 H; H-7); 13C NMR (100 MHz, CD3OD): d = 30.7 (C-10), 72.6 (C-5), 126.1 (C-3), 127.6 (C-14), 128.7 (C-7), 129.2 (C-12), 129.7 (C-13), 130.2 (C-8), 131.6 (C-9), 132.4 (C-6), 139.3 (C-11), 160.1 (C-4), 177.5 ppm (C-2); ESI-MS: m/z 251 [M+H] + . Chiral HPLC analysis: An LCMS-2020 spectrometer was used with a chiral column (CHIRALPAK IA, 250 2.1 mm, 5 mm; Daicel, Osaka, Japan). The following solvent systems were used: 40 % MeCN in 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Phytotoxic assay: Tobacco plants (N. benthamiana) were grown in pots containing commercial soil (Hanagokorobaiyoudo) in a growth incubator at 25 8C under 10 000 lux for 16 h per day. Fiveweek-old plants were used. Solutions of ralfuranones (50 mL; 1, 10, and 50 mm) were inoculated into leaves with a 1 mL syringe.[34] After 1 day, the presence of necrosis and changes to leaves were assessed. Antimicrobial assay: Seed cultures of E. coli and B. subtitles were grown in LB medium (BD) at 30 8C overnight. Cultures (50 mL) was spread on LB-agar medium. The suspension of A. aculeatus spores was spread on potato dextrose agar medium (BD). Advantec paper discs (6 mm; Toyo Roshi Kaisha, Tokyo, Japan) containing an appropriate amount of ralfuranone were placed onto the surface of agar medium. The agar plates were incubated at 30 8C overnight, and the inhibition zones were monitored. ChemBioChem 0000, 00, 1 – 9
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CHEMBIOCHEM FULL PAPERS Deletion and complementation of ralA: An 875 bp DNA fragment (ralA-1) was PCR-amplified from genomic DNA of OE1-1 with primers ral-1-FW (5’-CCGTT GGCGG CGAAT CTTGG-3’) and ral-1-RV (5’GTCCG GCTCA CATCG GATCA TGCCG AATCG-3’). An 875 DNA fragment (ralA-2) was PCR-amplified from genomic DNA of OE1-1 with primers ral-2-FW (5’-TGATC CGATG TGAGC CGGAC GGAGG GGCCG3’) and ral-2-RV (5’-GGGTG TTCCC AGTTT TAGGC G-3’). By using ralA-1 and ralA-2 as templates, a 1,759 bp DNA fragment was PCRamplified with primers ral-1-FW and ral-2-RV, then cloned into TVector pMD20 (Takara Bio, Shiga, Japan) to create pMD20ralA. The pMD20ralA construct was digested with EcoRI and PstI to release a 1.8-kbp fragment, which was ligated into the EcoRI and PstI sites of pK18 mobsacB[36] to create pDralA. This plasmid was electroporated into OE1-1 cells, and kanamycin-resistant and sucrose-sensitive recombinants were selected. Recombinants were incubated in PY medium (polypeptone (0.5 %), yeast extract (0.2 %)) for 6 h, and a kanamycin-sensitive and sucrose-resistant recombinant, DralA, was selected. DNA for sequencing was PCR-amplified with primers ral-SQ-FW3 (5’-TGCGT TAGGC ATGGT TTGG-3’) and ral-SQ-RV3 (5’TTGTA GGCGT TCATG GTCC-3’) to verify the correct substitution of the deleted ralA in OE1-1 (data not shown).
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We are grateful to Dr. Yuichi Masuda (Tohoku University) for the HRMS measurements and Takuya Asai (Kochi University) for his technical assistance. This work was supported by a grant from the Agricultural Chemical Research Foundation to KK and JSPS KAKENHI Grant Number 26660036 to Y.H., K.K., K.O., and A.K.
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Keywords: natural products · quorum sensing · ralfuranones · Ralstonia solanacearum · structure elucidation
Received: July 24, 2014 Published online on && &&, 0000
A 4649 bp DNA fragment containing ralA was PCR-amplified from genomic OE1-1 DNA with primers ralA-C1 (5’-GTCAT GGCGT ACTGC TGCAT G-3’) and ralA-C2 (5’-TCACG CGGCA TCTCC CAACA C-3’). The PCR fragment was cloned into T-Vector pMD20 to create pMD20CralA. The pMD20CralA construct was digested with BamHI and KpnI to release a 4.7-kbp fragment, which was ligated into the BamHI and KpnI sites of pUC18-mini-Tn7-Gm[37] to create pCralA. pCralA was electroporated into DralA to create gentamycin-resistant ralA-comp. DNA sequencing of PCR-amplified DNA fragments (primers ralA-C1 and ralA-C2) was performed to verify the insertion of ralA in ralA-comp (data not shown). Virulence assay: Tomato plants (Solanum esculemtum cv. OhgataFukuju) were grown in pots containing commercial soil (Tsuchitaro; Sumitomo Forestry Landscaping, Tokyo, Japan) in a growth room at 25 8C under 10 000 lux for 16 h per day, and watered with diluted (5 ) Hoagland’s solution.[18] Five-week-old tomato plants were inoculated by stabbing 200 mL pipette tips containing bacterial cell suspensions (50 mL; 1.0 107 cfu mL 1) into the third leaf axil from the plant apex[32] and placed in a 25 8C growth room. Pipette tips were removed after the entire bacterial suspension entered the stems. All inoculated plants were grown at 25 8C under 10 000 lux for 16 h per day. For each trial, five plants of each strain were treated (total: 45 plants). Plants were inspected for wilting symptoms daily for 14 days, and rated on a zero-to-five disease index scale (0 = no wilting; 1 = 1–25 % wilting; 2 = 26–50 % wilting; 3 = 51– 75 % wilting; 4 = 76–100 % wilting; 5 = dead).
Acknowledgements
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FULL PAPERS Caring for tomatoes: We identified new ralfuranones as phc quorum sensing-dependent metabolites from Ralstonia solanacearum OE1-1. Ablation of ralfuranone production resulted in reduced virulence in tomato plants. This is the first study to show that secondary metabolites are involved in the virulence of R. solanacearum.
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K. Kai,* H. Ohnishi, Y. Mori, A. Kiba, K. Ohnishi, Y. Hikichi && – && Involvement of Ralfuranone Production in the Virulence of Ralstonia solanacearum OE1-1
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Involvement of Ralfuranone Production in the Virulence of Ralstonia solanacearum OE1-1 Kenji Kai,*[a] Hideyuki Ohnishi,[a] Yuka Mori,[b] Akinori Kiba,[b] Kouhei Ohnishi,[c] and Yasufumi Hikichi[b] Ralstonia solanacearum causes a destructive disease called “bacterial wilt” in numerous plant species. Its virulence is controlled by the transcriptional regulator PhcA, the activity of which is, in turn, regulated in a cell-density dependent manner, termed quorum sensing. We herein described the identification and characterization of ralfuranones J–L, new PhcA-regulated secondary metabolites, and the known derivatives, ralfuranones A and B, from R. solanacearum strain OE1-1.
Their structures were determined by spectroscopic and chemical methods. These ralfuranones were also detected in vascular exudates from host plants infected with OE1-1. Deletion of ralA, which encodes an enzyme for ralfuranone biosynthesis, reduced the virulence of OE1-1 in tomato plants. Virulence was restored by complementation of the ralA gene. The results suggest that ralfuranones play important roles in the virulence of OE1-1.
Introduction The Gram-negative bacterium Ralstonia solanacearum is the causative agent of “bacterial wilt” in more than 200 plant species in tropical, subtropical, and warm temperate regions of the world.[1, 2] Its hosts include agronomically important crops, such as tomato, potato, tobacco, peanut, and banana. The pathogen enters the host through wounded roots or the sites of secondary-root emergence; once inside a host, it quickly colonizes the intercellular spaces, then invades xylem vessels and grows vigorously there. When infection exceeds 109 cfu per gram (stem tissue), R. solanacearum causes the wilting symptoms observed in infected plants.[3] The ability of this pathogen to cause host wilting has been mainly attributed to its production of extracellular polysaccharide (EPS), which occludes vascular tissues and prevents water flow.[1, 2] The bacterium also uses plant cell-wall-degrading enzymes (e.g., polygalacturonase and endoglucanase) to invade and grow in host tissue. The production of these virulence factors is controlled by a complex regulatory network in which PhcA, a LysR-type transcriptional regulator, plays a central role.[1, 2, 4] The activity of PhcA is controlled by the PhcS/PhcR two-component regulatory system, which de[a] Prof. K. Kai, H. Ohnishi Graduate School of Life and Environmental Sciences Osaka Prefecture University 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531 (Japan) E-mail: [email protected] [b] Y. Mori, Prof. A. Kiba, Prof. Y. Hikichi Laboratory of Plant Pathology and Biotechnology Kochi University 200 Otsu, Monobe, Nanko-ku, Kochi 783-8502 (Japan) [c] Prof. K. Ohnishi Research Institute of Molecular Genetics Kochi University 200 Otsu, Monobe, Nanko-ku, Kochi 783-8502 (Japan) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cbic.201402404.
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tects 3-hydroxypalmitic acid methyl ester (3-OH PAME), a PhcBderived quorum sensing (QS) signal.[5–7] QS generally allows bacteria to coordinate their activities in response to population size.[8] The response regulator PhcR post-transcriptionally inhibits the transcription of phcA; however, when the concentration of 3-OH PAME reaches a threshold level, this signal is received by the sensor kinase PhcS, which phosphorylates PhcR, and the transcription of phcA is then activated.[5–7] Here, we referred to this QS mechanism as “phc QS”. The contribution of secondary metabolites, unlike EPS and extracellular enzymes, to the virulence of R. solanacearum remains unclear. Several plant pathogenic bacteria are known to regulate secondary metabolism by using QS systems.[9] For example, the production of toxoflavin (a phytotoxin) is controlled by QS mediated by N-acylhomoserine lactones in the rice pathogen Burkholderia glumae.[10, 11] 3-Hydroxybenzoic acid, an unusual QS signal, regulates the biosynthesis of xanthomonadin (a yellow pigment) in the black rot pathogen Xanthomonas campestris.[12] This metabolite protects the bacterium from photo-oxidative damage.[13] Crosstalk between secondary metabolism and QS in R. solanacearum has only recently become a research topic, and has led to the novel identification of the small molecules, ralfuranones A and B (Scheme 1), from R. solanacearum GMI1000, as well as the characterization of the furanone synthase RalA, an enzyme involved in the biosynthesis of ralfuranones.[14, 15] A recent study reported that ralfuranone I, a biosynthetic precursor of ralfuranones, functions as a reactive Michael acceptor of biomolecular thiols.[16] Furthermore, phc QS was shown to negatively control the production of the siderophore staphyloferrin B in R. solanacearum strain AW1.[17] However, the QS-dependent secondary metabolites of R. solanacearum have not been examined in detail, and little is known about their contribution to bacterial virulence in host plants. Therefore, the identification of secondary metabolites ChemBioChem 0000, 00, 1 – 9
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Scheme 1. Structures of ralfuranones.
(and their production under the control of QS) and the elucidation of their biological roles has become an important theme in the study of the bacteria–plant interactions. The aim of this study was to identify secondary metabolites under the control of phc QS and elucidate their roles in the virulence of R. solanacearum. We compared the metabolic profiles of R. solanacearum strain OE1-1 (isolated in Japan and pathogenic to solanaceous plants)[18] with a phcA-deficient mutant (DphcA).[19] Five metabolites were markedly decreased in DphcA and were identified as known and novel derivatives of ralfuranones (Scheme 1) by spectroscopic and chemical methods. We also investigated the biological roles of ralfuranones by using assays with synthetic ralfuranones and analyzed the ralfuranone-deficient mutant strain of OE1-1.
Results and Discussion Search for phc QS-dependent secondary metabolites To identify phc QS-regulated secondary metabolites, we first compared the HPLC chromatograms of culture extracts of OE11 and DphcA. Previous studies reported that the expression of genes related to the virulence of R. solanacearum was dependent on growth conditions, especially nutrition.[20–22] The xylem fluid of tomato, a representative host of R. solanacearum, contains minerals, amino acids, and a high amount of sucrose.[23, 24] Jacobs et al. demonstrated that host sucrose was important for the virulence of R. solanacearum, and that this was because the bacterium incorporates host sucrose via a specific transporter and uses it as a carbon source during the development of wilt disease.[25] Thus, to identify the secondary metabolites involved in the pathogenicity and virulence of OE1-1, we chose MGRLS medium (MGRL medium supplemented with 3 % sucrose),[19, 26] which mimics the nutrient conditions in the vascular tissues of host plants. We detected five major peaks (compounds 1–5) in the EtOAc extract from a four-day-old culture of OE1-1, but not in the chromatogram of DphcA (Figure 1); this confirmed that these metabolites were under the 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 1. HPLC metabolic profiles of culture extracts from A) OE1-1 and B) DphcA. The data indicate that the production of compounds 1–5 was under the control of phc QS.
control of phc QS. We also examined the culture extracts of OE1-1 grown in 1/4 M63 medium, a minimal medium used in previous ralfuranone studies:[14, 15] 2, 3, and 5 were not detected in these culture extracts. Structure elucidation of phc QS-dependent secondary metabolites To elucidate the structures of 1–5, an EtOAc extract of the culture broth (20 L) of OE1-1 was prepared and subjected to separation by chromatography. The EtOAc extract was purified by ODS column chromatography eluted with H2O/MeOH. The fractions eluted at 40 and 60 % MeOH were further purified by reversed-phase HPLC with an H2O/MeCN gradient (20 to 95 % MeCN) to give 1 (0.6 mg), 2 (2.3 mg), 3 (0.9 mg), 4 (1.2 mg), and 5 (ca. 0.05 mg). The UV spectra of 1–5 were similar to those of ralfuranones previously identified from R. solanacearum GMI1000.[14, 15] This suggested that the compounds were known and new derivatives of ralfuranones. The ESI-MS spectra of 1 and 4 showed ions at m/z 161 [M+H] + and 265 [M H] , corresponding to ralfuranones A and B, respectively. Their 1H NMR spectra were almost identical to those of ralfuranones A and B,[14, 15] thus we concluded that 1 and 4 were ralfuranones A and B, respectively (Scheme 1). A previous study suggested that ralfuranone B might be a chiral molecule.[15] However, as the chiral HPLC analysis of 4 gave two peaks with a ratio of 1:1 (Figure S1 in the Supporting Information), the compound was revealed to be a racemic mixture of C-5 enantiomers. The ESI-MS of purified 2 showed an [M H] ion at m/z 281, thus indicating that its molecular weight is 16 amu larger than that of 4. Thus, the compound was suggested to be an oxygenated derivative of 4. The 1H NMR spectrum of 2 was similar to that of 4; however, the proton signal of H-5 in 2 showed a lower-field shift (dH = 5.18 and 5.26 to dH = 6.50 ppm) and ChemBioChem 0000, 00, 1 – 9
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Table 1. 1H and 13C NMR data for ralfuranones J (2), K (3), and L (5) No. 13
2 3 4 5 6 7 8 9 10 11 12 13 14
C
172.4 131.4 160.1 99.5 131.9 130.2 129.6 131.3 68.2 142.5 127.3 129.3
1
2[a] H, mult. (J, Hz)
6.50, 1 H; s 7.57, 7.44, 7.43, 5.78,
2 H; 2 H; 1 H; 1 H;
m m m s
7.41, 2 H; m 7.29, 2 H; tt (7.3, 1.5) 128.5 7.22, 1 H; tt (7.3, 1.5)
13
C
168.8 127.9 160.8 98.5 129.9 129.8 129.9 132.5 192.3 136.1 130.2 130.0
1
3[b] H, mult. (J, Hz)
6.72, 1 H; s 7.47, 2 H; m 7.36, 2 H; m 7.43, 1 H; m
7.94, 2 H; m 7.48, 2 H; tt (7.4, 1.2) 135.7 7.65, 1 H; t (7.4)
13 [c]
C
177.5 126.1 160.1 72.6 132.4 128.7 130.2 131.6 30.7 139.3 129.2 129.7
1
5[a] H, mult. (J, Hz)
5.24, 2 H; s 7.48, 7.42, 7.42, 3.84,
2 H; 2 H; 1 H; 2 H;
m m m s
7.17, 2 H; m 7.22, 2 H; m
127.6 7.14, 1 H; m
sion between the C-5 enantiomers, 3 was detected as one peak by chiral HPLC (Figure S1). A similar profiling analysis indicated that 5 was a major metabolite of OE1-1; however, the isolated compound was the smallest among these. This might have been due to the different conditions between the small- and large-scale cultures of OE1-1 rather than instability of 5. The structure of 5 was expected to be a deoxygenated derivative of 4, because the [M+H] + ion of 5 was at m/z 251. The aromatic proton signals of 5 suggested the presence of two one-substituted benzene rings (Table 1). The singlet methylene signals at dH = 3.84 and 5.24 ppm were assigned to H-10 and H-5, respectively. Thus, the structure of 5, named ralfruranone L, was deduced (Scheme 1). However, due to the low quantity of 5, its 13C and 2D NMR data could not be obtained, so the complete identification of this structure depended on subsequent chemical synthesis.
[a] in CD3OD. [b] in CD3CN. [c] Data from synthetic 5.
had fewer hydrogens, from 2 to 1 (Table 1). According to the 13 C NMR spectrum, the carbon signal of C-5 in 2 was at dC = 99.5 ppm, which suggests a hemiacetal carbon. The carbon and proton signals responsible for the two 1-substituted benzenes and one 3,4-substituted furanone were observed in the 1 H and 13C NMR spectra of 2. The HMBC correlations from H-5 to C-2 and C-3, from H-10 to C-2, C-3, C-4, C-11, and C-12, and from H-12 to C-10 revealed that two oxymethines were present in the two aryl-substituted furanone ring backbone (Figure 2). Thus, 2 was determined to be the 5-hydroxy derivative of 4, and was given the name ralfuranone J (Scheme 1).
Figure 2. Key HMBC correlations of natural 2 and 3 and synthetic 5.
Compound 2 gave only two peaks in chiral HPLC analysis (Figure S1), although the compound has two chiral centers (and thus theoretically should give four peaks) and did not display any diastereomeric proton or carbon signal (Table 1). We concluded that 2 was a rapid equilibrium mixture of C-5 epimers. The two peaks observed in the chiral HPLC analysis might have derived from the C-10 epimers. ESI-MS of purified 3 gave an [M+H] + ion at m/z 281, thus indicating that the compound has two hydrogens fewer than 2. The oxymethine proton signal at C-10 in 2 was absent in the 1 H NMR spectrum of 3. Furthermore, the carbon signal of C-10 in 3 shifted to dC = 192.3 ppm, the carbonyl carbon region. The HMBC correlations from H-5 to C-2 and C-3, from H-7 to C-4, and from H-12 to C-10 showed a backbone composed of the two aryl-substituted furanone ring in 3 (Figure 2). Thus, the structure of 3, named ralfuranone K, was revealed to be a 10oxo derivative of 2 (Scheme 1). Because of the rapid conver 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chemical synthesis of ralfuranones To confirm the structures of ralfuranones and obtain them for our biological studies, we synthesized all ralfuranones (Schemes 2 and 3). Arylfuranones have been the targets of synthetic studies because they exhibit many biological activities; ralfuranone A (1) and candidates for the synthetic precursors of other ralfuranones have been synthesized.[27–31] Compound 1 was prepared from bromobenzene (6) by the method by Verendel et al. (Scheme 2; See the Experimental Section).[27] Luche reduction of 13, which was synthesized from benzoic acid (9) in four steps by the method of Peixoto et al.,[28] gave ralfuranone B (4) in good yield (94 %). Ralfuranones J (2) and K (3) were synthesized by catalytic oxidation with molecular oxygen from 19,[29] which was synthesized from ethyl benzoate (14) in five steps by the methods of Basavaiah et al. and Clive et al. (Scheme 3).[30, 31] Although the yields of 2 and 3 were very low, this demonstrated the potential of catalytic oxidation in the synthesis of highly oxidized arylfuranones. Ralfuranone L (5) was synthesized analogously to ralfuranone A (1). Condensation of 7 with 2-benzylacrylic acid yielded ester 20 in 72 % yield. Ring closure of 20 was carried out by using olefin metathesis with Grubbs catalyst second generation (Grubbs 2nd) to give 5 in 41 % yield. Therefore, we accomplished the first total synthesis of ralfuranones B (4), J (2), K (3), and L (5). The ESI-MS data and HPLC retention times of synthetic ralfuranones were identical to those of the natural compounds. Furthermore, the 1H and 13C NMR data of synthetic and natural ralfuranones were almost identical (Figure S2). The complete assignments of the proton and carbon signals of 5 were based on HMQC and HMBC data of the synthetic compound (Table 1; Figure 2). We also confirmed that synthetic ralfuranones B (4), J (2), and K (3), which were obtained as stereoisomeric mixtures of C-5/C-10, had the same retention times as those of the natural compounds in chiral HPLC analysis (Figure S1). Therefore, these ralfuranones have the structures described in Scheme 1. ChemBioChem 0000, 00, 1 – 9
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Scheme 2. Synthesis of ralfuranones A (1) and B (4). a) i: Mg, Et2O, reflux; ii: propargyl alcohol, CuI, Et2O, RT to reflux, 80 %; b) acryloyl chloride, Et3N, CH2Cl2, RT, 83 %; c) Grubbs 2nd, CH2Cl2, reflux, 45 %; d) i: (COCl)2, CH2Cl2, RT; ii: N,O-dimethylhydroxylamine, Et3N, CH2Cl2, RT, quant.; e) tert-butyl acetate, LDA, THF, 78 8C, quant.; f) acetone, Ac2O, H2SO4, RT, 85 %; g) 2-hydroxyacetophenone, toluene, Et3N, MS 4 A, 110 8C, 94 %; h) NaBH4, CeCl3, MeOH, 0 8C, 94 %.
www.chembiochem.org were synthesized by the actions of enzymes encoded by the ral gene cluster.[15] The aminotransferase RalD converts phenylalanine to phenylpyruvic acid, which is then loaded to the furanone synthase RalA, composed of a tridomain nonribosomal peptide synthetase (NRPS)-like enzyme. Two phenylpyruvic acids condense to form the intermediate, ralfuranone I.[16] The compound is further modified by enzymatic or non-enzymatic reactions to afford ralfuranones A (1) and B (4). Based on the structures of ralfuranones, the following metabolic routes were proposed: 1) 5 is synthesized from ralfuranone I and could be a precursor of other ralfuranones; and 2) 4 is oxidized to form ralfuranones J (2) and K (3). To investigate these possibilities, we synthesized deuterium-labeled ralfuranone L ([D5]5) and ralfuranone B ([D5]4), and conducted incorporation studies with the labeled compounds. Incorporation of the [D5]5 deuterium into other ralfuranones was not observed in the LC-MS analysis of the culture extract of OE1-1, which was grown in the presence of [D5]5 (10 mm; Figure S3). Thus, 5 might not be a precursor of other ralfuranones in OE1-1. In contrast, [D5]4 deuterium incorporation into 2 and 3 was confirmed by LC-MS analysis (Figure S4). Based on the areas of the peaks, these incorporation rates were calculated to be 13 and 29 %, respectively. Therefore, 2 and 3 were postulated as the C-5 oxidized products of 4. Production of ralfuranones by OE1-1 in host plants We investigated whether OE1-1 produced ralfuranones in the xylem of host plants. We inoculated OE1-1 into tomato and tobacco plants by the wounded-petiole inoculation method,[32] 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 MilliQ water. Water was extracted with EtOAc, and analyzed by HPLC: all the ralfuranones were detected in the vascular exudates of both tomato and tobacco plants (Figure 3 A and B). Their identities were confirmed by UV spectra and LC-MS analysis. Thus, we confirmed that OE1-1 also produced ralfuranones A (1), B (4), J (2), K (3), and L (5) in host plants. Based on standard curves, we estimated the concentrations of ralfuranones as 80 (4), 730 (2), and 610 (5) pmol per plant in tomato, and 29 (4), 73 (2), and 200 (5) pmol per plant in tobacco; no data means were below the quantification limit. Considering the apoplastic spaces of xylem vessels in plants, the production levels appear to be high. The results suggest that ralfuranones, which are actively produced in hosts, might be important for the optimal virulence of OE1-1.
Scheme 3. Synthesis of ralfuranones J (2), K (3), and L (5). a) methyl acrylate, 1,4-diazabicyclo[2.2.2]octane, RT, 47 %; b) benzene, CH3SO3H, reflux, 66 %; c) LiAlH4, Et2O, 20 8C to RT, 45 %; d) PCC, CH2Cl2, RT, 66 %; e) NaClO2, NaH2PO4, aq EtOH, RT, 58 %; f) O2, N-hydroxyphthalimide, Co(OAc)2, MeCN, 40 8C, 1.3 % for 2, 5.2 % for 3; g) 2-benzylacrylic acid, EDC, DMAP, Et3N, CH2Cl2, RT, 72 %; h) Grubbs 2nd, CH2Cl2, reflux, 41 %.
Feeding study of deuterium-labeled ralfuranones How these novel ralfuranones are biosynthesized in R. solanacearum is unknown. Wackler et al. reported that ralfuranones 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Evaluation of phytotoxic effects of ralfuranones Several secondary metabolites of plant pathogens are known to play roles as phytotoxins against host plants.[33] If the ralfuranones exhibited toxicity against host plants, they would be recognized as new virulence factors of R. solanacearum. To examine this, we investigated the toxic effects of ralfuranones on tobacco plants. After inoculation of ralfuranone (1, 10, and 50 mm; 50 mL) into the leaves,[34] no significant changes were observed between treated and control regions (Figure S5). BacChemBioChem 0000, 00, 1 – 9
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www.chembiochem.org ulence of DralA in tomato plants was significantly less than that of OE1-1 (Figure 4 B). Transformation of DralA with a plasmid containing ralA from OE1-1 (ralA-comp) restored the production of ralfuranones (Figure 4 A) and its virulence in tomato plants (Figure 4 B), thus suggesting the involvement of ralA in OE1-1 virulence. Seven days after inoculation, plants of these strains clearly showed that the degree of host wilting was dependent on the presence of ralA (Figure 4 C). Deletion of ralA led not only to the loss of ralfuranone biosynthesis, but also to a reduction in the virulence of R. solanacearum. Taken together, our results implicate the production of ralfuranones in the full virulence of OE1-1 in host plants.
Conclusions
Figure 3. HPLC analysis of crude extracts of vascular exudates of A) tomato and B) tobacco plants infected with OE1-1. Peaks of 1–5 were assigned from their UV and ESI-MS data.
terial phytotoxins also exhibit antimicrobial activity;[33, 35] therefore, we examined the activity of ralfuranones against Escherichia coli, Bacillus subtilis, and Aspergillus aculeatus by using the disc diffusion assay.[34] The growth of these microbes was not inhibited by ralfuranones A, B, J, or L, although ralfuranone K exhibited weak inhibitory activity against B. subtilis (diameter of inhibition zone: 8 mm, at 0.5 mmol per disc). These results suggest that ralfuranones do not possess strong antimicrobial activity; we assumed that ralfuranones are not phytotoxins.
We have identified the novel ralfuranones J (2), K (3), and L (5), as phc QS-dependent metabolites from OE1-1 and synthesized these compounds, as well as the known derivatives, ralfuranones A (1) and B (4). We confirmed the production of ralfuranones by OE1-1 in host plants and the reduced virulence of DralA in tomato plants. These results strongly suggest the involvement of ralfuranone production in R. solanacearum virulence. However, these ralfuranones did not exhibit apparent toxicity in tobacco plants. Therefore, these ralfuranones might cause changes in the bacterium but not in the host. It remains unknown as to how ralfuranones contributed to the virulence of OE1-1. Therefore, further analysis of DralA and identification of the targets of ralfuranones are needed. As ablation of ralfuranone production did not result in complete loss of bacterial
Virulence assay of OE1-1 and ralfuranone-deficient mutant in tomato plants Although ralfuranones did not appear to be active in host plants, this does not exclude ralfuranone involvement in the virulence of R. solanacearum. To determine whether ralfuranones were involved in the virulence of OE1-1, we created a ralA-deficient mutant of OE1-1 (DralA). The mutant strain did not produce any ralfuranones (Figure 4 A). Combined chromatograms were constructed to compare the HPLC chromatograms of OE1-1 and its mutant easily (Figure S6). Using wounded-petiole inoculation, we assessed the ability of DralA to cause wilt disease in tomato plants. The vir-
Figure 4. A) HPLC analysis of culture extracts from DralA and ralA-comp. B) Disease progress curves of OE1-1 (*), DralA (&), and ralA-comp (&). Eight-week-old tomato plants were inoculated by stabbing pipette tips containing bacterial cell suspensions into the third leaf axil from the plant apex. Plants were rated on a zero-to-five disease index scale. Error bars indicate standard deviations (n = 3). * denotes P < 0.05 (Student’s t-test). C) Phenotypes of tomato plants seven days after inoculation of OE1-1, DralA, and ralA-comp.
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CHEMBIOCHEM FULL PAPERS virulence, these compounds are not essential for wilting. Therefore, the results indicate that ralfuranones might be key molecules that enhance bacterial activity in order to induce wilt disease in host plants.
Experimental Section General: All purchased chemicals were used without further purification. Silica gel column chromatography was performed on Wakogel C-200 gel (Wako Pure Chemical Industries, Osaka, Japan). Synthetic reactions were conducted under a nitrogen atmosphere with dry solvents (Wako Pure Chemical Industries) unless otherwise indicated. The yields of synthetic reactions were not optimized. 1H and 13C NMR spectra were recorded on a JNM-AL400 spectrometer (JEOL, Tokyo, Japan). Chemical shifts are reported as d values (ppm); tetramethylsilane (TMS; dH = 0, dC = 0) or solvents (CDCl3 : dH = 7.26, dC = 77.0; CD3CN: dH = 1.93, dC = 118.2; CD3OD: dH = 3.30, dC = 49.0) were used as internal references. High-resolution mass spectra (HRMS) were obtained with a SYNAPT G2 HDMS mass spectrometer (Waters, Milford, MA). HPLC experiments were performed with a LaChrom Elite HPLC system (Hitachi High-Technologies, Tokyo, Japan) and a Prominence HPLC system (Shimadzu, Kyoto, Japan). Solvents for HPLC were purchased from Kanto Chemical (Tokyo, Japan). LC-MS data were obtained with an LCMS2020 spectrometer (Shimadzu, Kyoto, Japan). Microbial cultivation and extraction: Seed cultures of R. solanacearum strain OE1-1[18] were grown in B medium at 30 8C overnight.[19] These were centrifuged to remove medium, and the bacterial cells were then suspended in the same volume of MGRLS medium (MGRL medium[19, 26] supplemented with 3% sucrose). The suspensions were diluted with MGRLS medium in Erlenmeyer flasks and incubated at 30 8C with rotation (130 rpm) 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. 1/4 M63 medium[14, 15] was also used instead of MGRLS medium. Profiling analysis of secondary metabolites: OE1-1 and DphcA[19] were grown in MGRLS medium (100 mL) in 300 mL Erlenmeyer flasks at 30 8C with rotation (130 rpm) 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 mL) and subjected to HPLC analysis: column, Inertsil ODS-3 (250 4.6 mm, 5 mm; GL Sciences, Tokyo, Japan); column oven, 40 8C; flow rate, 1 mL min 1; eluent, MeCN/H2O (20– 95 % linear gradient in 40 min, then 95 % MeCN for 10 min); injection volume, 10 mL. Purification and structure elucidation of ralfuranones: The extraction of the 20 L MGRLS culture of OE1-1 (100 500 mL Erlenmeyer flasks, each containing 200 mL medium) gave a crude EtOAc extract (112 mg). The extract was chromatographed on Chromatorex ODS (Fuji Silysia Chemical, Aichi, Japan) and eluted with a stepwise gradient of H2O/MeOH (0–100 % MeOH). The 40 and 60 % MeOH eluates (14.1 mg) were combined and subjected to HPLC with an Inertsil ODS-3 column (250 10 mm, 5 mm) and an H2O/MeCN gradient (20–95 % MeCN) to yield 1 (0.6 mg), 2 (2.3 mg), 3 (0.9 mg), 4 (1.2 mg), and 5 (ca. 0.05 mg). Ralfuranone A (1): colorless amorphous solid; 1H NMR (CD3OD): d = 5.37 (d, J = 1.7 Hz, 2 H; H-5), 6.53 (t, J = 1.7 Hz, 1 H; H-3), 7.44–7.57 (m, 3 H; H-8, H-9), 7.68 ppm (dd, J = 1.7, 7.8 Hz, 2 H; H-7); UV (MeCN/H2O): lmax = 202, 212, 271; ESI-MS: m/z 161 [M+H] + . 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org Ralfuranone B (4): colorless amorphous solid; 1H NMR (CD3OD): d = 5.18 (d, J = 17.6 Hz, 1 H; H-5a), 5.26 (d, J = 17.6 Hz, 1 H; H-5b), 5.85 (s, 1 H; H-10), 7.20 (tt, J = 1.5, 7.3 Hz, 1 H; H-14), 7.28 (tt, J = 1.5, 7.3 Hz, 2 H; H-13), 7.40 (m, 2 H; H-12), 7.44 (m, 2 H; H-8), 7.45 (m, 2 H; H-9), 7.57 ppm (m, 2 H; H-7); UV (MeCN/H2O): lmax = 264; ESIMS: m/z 264 [M H] . Ralfuranone J (2): colorless amorphous solid; NMR (CD3OD): Table 1; ESI-MS: m/z 281 [M H] ; UV (MeCN/H2O): lmax = 268; HR-ESI-MS: m/z 305.0784 [M+Na] + (calcd for C17H14O4Na, 305.0790). Ralfuranone K (3): colorless amorphous solid, UV (MeCN/H2O): lmax = 260; NMR (CD3CN): Table 1; ESI-MS: m/z 281 [M+H] + ; HR-ESIMS: m/z 303.0628 [M+Na] + (calcd for C17H12O4Na, 303.0633). Ralfuranone L (5): colorless amorphous solid, UV (MeCN-H2O) lmax = 267; 1H NMR (400 MHz, CD3OD): Table 1; ESI-MS: m/z 251 [M+H] + ; HR-ESI-MS: m/z 273.0886 [M+Na] + (calcd for C17H14O2Na, 273.0891). Synthesis of ralfuranone A: The synthesis was performed as described previously.[27] Grignard regent prepared from bromobenzene (6) and magnesium in Et2O was reacted with propargyl alcohol to afford 7 in 80 % yield. Condensation of 7 with acryloyl chloride in CH2Cl2 gave 8 in 83 % yield. Olefin metathesis of 8 with Grubbs 2nd in CH2Cl2 yielded 1 in 45 % yield. 1H NMR (400 MHz, CD3OD): d = 5.37 (d, J = 1.7 Hz, 2 H; H-5), 6.53 (t, J = 1.7 Hz, 1 H; H3), 7.44–7.57 (m, 3 H; H-8, H-9), 7.68 ppm (dd, J = 1.7, 7.8 Hz, 2 H; H7); 13C NMR (100 MHz, CD3OD): d = 72.9 (C-5), 113.3 (C-3), 128.0 (C7), 130.3 (C-8), 131.1 (C-9), 132.9 (C-6), 167.1 (C-4), 176.7 ppm (C-2); ESI-MS: m/z 161 [M+H] + . Synthesis of ralfuranone B: The synthesis of 13 was performed as described previously.[28] The lithium enolate of tert-butyl acetate was prepared by using lithium diisopropylamide (LDA) in THF. Weinreb amide 10 prepared from benzoic acid (9) was added to the solution to afford 11 and its enol quantitatively. Reaction of 11 with acetone in the presence of Ac2O and H2SO4 yielded dioxinone 12 in 85 % yield. Heating 12 in toluene gave a b-acylketene intermediate, which was reacted with 2-hydroxyacetophenone, and then the conjugate was cyclized by an intramolecular Knoevenagel reaction to afford 13 in 94 % yield. NaBH4 (53.3 mg, 1.41 mmol) was added to 13 (339 mg, 1.28 mmol) and CeCl3·7 H2O (716 mg, 1.92 mmol) in MeOH (20 mL) at 0 8C. After stirring at 0 8C for 30 min, the reaction was quenched with HCl (1 m, 60 mL), and the mixture was extracted three times with an equal volume of EtOAc. The combined EtOAc layer was washed with brine and dried over Na2SO4. The concentrate was purified by silica gel chromatography with hexane/EtOAc (9:1) as the eluent to give 4 (320 mg, colorless amorphous solid, yield 94 %). 1H NMR (400 MHz, CD3OD): d = 5.18 (d, J = 17.6 Hz, 1 H; H-5a), 5.26 (1 H; d, J = 17.6 Hz, H-5b), 5.85 (s, 1 H; H-10), 7.20 (1 H; tt, J = 1.5, 7.3 Hz, H14), 7.28 (2 H; tt, J = 1.5, 7.3 Hz, H-13), 7.40 (m, 2 H; H-12), 7.44 (m, 3 H; H-8, H-9), 7.57 ppm (m, 2 H; H-7); 13C NMR (100 MHz, CD3OD): d = 68.2 (C-10), 72.6 (C-5), 127.2 (C-12), 128.4 (C-14), 128.9 (C-3), 129.2 (C-13), 129.4 (C-7), 129.9 (C-8), 131.7 (C-9), 132.0 (C-6), 142.6 (C-11), 161.8 (C-4), 175.6 ppm (C-2); ESI-MS: m/z 265 [M H] . Synthesis of ralfuranones J and K: The synthesis of 19 was performed as described previously.[30, 31] A Morita-Baylis–Hillman reaction of ethyl 2-oxo-2-phenylacetate (14) with methyl acrylate in the presence of 1,4-diazabicyclo[2.2.2]octane provided the Morita– Baylis–Hillman adduct 15 in 47 % yield. Friedel–Crafts reaction of 15 with benzene in the presence of CH3SO3H yielded furandione 16 in 66 % yield. Reductive cleavage of 16 with LiAlH4 in Et2O gave diol 17 in 45 % yield. Oxidation of 17 with PCC in CH2Cl2 afforded ChemBioChem 0000, 00, 1 – 9
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furan 18 in 66 % yield. Conversion of 18 into g-hydroxybutenolide 19 (58 % yield) was performed by treatment with NaClO2 in EtOH containing NaH2PO4.
H2O (for 4), 25 % MeCN in H2O (for 2), and 30 % MeCN in H2O (for 3). Other LC-MS conditions: column oven, 40 8C; flow rate, 200 mL min 1; injection volume, 5 mL.
A solution of 19 (179 mg, 0.672 mmol), N-hydroxyphthalimide (65.8 mg, 0.403 mmol), and Co(OAc)2 (3.57 mg, 20.2 mmol) in MeCN (5 mL) was stirred at 40 8C for three days under an O2 atmosphere.[29] The mixture was diluted with H2O (40 mL) and extracted three times with an equal volume of EtOAc. The combined EtOAc layer was dried over Na2SO4 and evaporated to dryness. The residue was purified by silica gel chromatography with hexane/EtOAc (8:2) as the eluent to give a mixture of 2 and 3. The mixture was further purified by HPLC (Inertsil ODS-3 250 10 mm) with MeCN/ H2O (6:4) as the eluent to afford 2 (2.5 mg, colorless amorphous solid, yield 1.3 %) and 3 (9.9 mg, colorless amorphous solid, yield 5.2 %). This reaction was repeated to obtain sufficient amounts of 2 and 3 for biological studies. Spectroscopic data of 2: 1H NMR (400 MHz, CD3OD): d = 5.78 (s, 1 H; H-10), 6.49 (s, 1 H; H-5), 7.22 (tt, J = 1.5, 7.3 Hz, 1 H; H-14), 7.29 (tt, J = 1.5, 7.3 Hz, 2 H; H-13), 7.41 (m, 2 H; H-12), 7.43 (m, 1 H; H-9), 7.44 (m, 2 H; H-8), 7.57 (m, 2 H; H-7); 13 C NMR (100 MHz, CD3OD): d = 68.3 (C-10), 99.5 (C-5), 127.3 (C-12), 128.5 (C-14), 129.3 (C-13), 129.6 (C-8), 130.2 (C-7), 131.3 (C-9), 131.4 (C-3), 131.9 (C-6), 142.5 (C-11), 160.1 (C-4), 172.4 ppm (C-2); ESI-MS: m/z 281 [M H] . Spectroscopic data of 3: 1H NMR (400 MHz, CD3CN): d = 6.72 (s, 1 H; H-5), 7.36 (m, 2 H; H-8), 7.43 (m, 2 H; H-9), 7.47 (m, 2 H; H-7), 7.48 (m, 2 H; H-13), 7.65 (tt, J = 1.2, 7.4 Hz, 1 H; H-14), 7.94 ppm (m, 2 H; H-12); 13C NMR (100 MHz, CD3CN): d = 98.6 (C-5), 128.0 (C-3), 129.8 (C-7), 129.9 (C-6, C-8), 130.0 (C-13), 130.2 (C-12), 132.5 (C-9), 135.7 (C-14), 136.1 (C-11), 160.8 (C-4), 168.8 (C2), 192.3 ppm (C-10); ESI-MS: m/z 281 [M+H] + .
Feeding study of deuterium-labeled ralfuranones: Deuteriumlabeled [7,7,8,8’,9-2H5]ralfuranone L ([D5]5) and [12,12’,13,13’,142 H5]ralfuranone B ([D5]4) were synthesized from [D5]bromobenzene ( 98 atom % D, Wako Pure Chemical Industries) and [D5]benzoic acid ( 98 atom % D, Cambridge Isotope Laboratories, MA) by the methods described for the synthesis of the corresponding non-labeled ralfuranones. Spectroscopic data of [D5]5: 1H NMR (400 MHz, CD3OD): d = 3.88 (s, 2 H; H-10), 5.27 (s, 2 H; H-5), 7.17 (m, 1 H; H-14), 7.20 (m, 2 H; H-12), 7.25 (tt, J = 1.2, 7.2 Hz, 2 H; H-13); 13C NMR (100 MHz, CD3OD): d = 30.7 (C-10), 72.6 (C-5), 126.1 (C-3), 127.6 (C14), 129.2 (C-12), 129.7 (C-13), 132.2 (C-6), 139.3 (C-11), 160.0 (C-4), 177.5 ppm (C-2); ESI-MS: m/z 256 [M+H] + . Spectroscopic data of [D5]4: 1H NMR (400 MHz, CD3OD): d = 5.17 (d, J = 17.6 Hz, 1 H; H5a), 5.25 (d, J = 17.6 Hz, 1 H; H-5b), 5.85 (s, 1 H; H-10), 7.44 (m, 3 H; H-8, H-9), 7.57 ppm (m, 2 H; H-7); 13C NMR (100 MHz, CD3OD): d = 68.1 (C-10), 72.6 (C-5), 128.9 (C-3), 129.4 (C-7), 129.9 (C-8), 131.7 (C9), 132.0 (C-6), 142.5 (C-11), 161.7 (C-4), 175.6 ppm (C-2); ESI-MS: m/z 270 [M H] . Deuterium-labeled ralfuranone solutions (100 mL, 10 mm in EtOH) were added to 2-day-old cultures of OE1-1 in MGRLS (100 mL), and the cultures were grown for a further 2 days at 30 8C. Analytical samples were prepared as described above and subjected to LC-MS analysis: column, InertSustain C18 (150 2.1 mm, 3 mm; GL Sciences); column oven, 40 8C; flow rate, 200 mL min 1; eluent system, MeOH in H2O (20–95 % linear gradient in 24 min, then 95 % MeOH in H2O for 6 min); injection volume, 1 mL.
Synthesis of ralfuranone L: A CH2Cl2 solution (15 mL) of 7 (1.23 g, 9.17 mmol), 2-benzylacrylic acid (1.78 g, 11.0 mmol), EDC·HCl (2.64 g, 13.8 mmol), DMAP (224 mg, 1.83 mmol), and Et3N (3.86 mL, 27.5 mmol) was stirred at RT for 16 h. The mixture was diluted with CHCl3 (200 mL) and quenched with HCl (1 m, 200 mL). The organic layer was washed with saturated NaHCO3 and brine, and dried over Na2SO4. After evaporation, the residue was purified by silica gel chromatography with hexane/EtOAc (95:5) as the eluent to give 20 (1.84 g, yellowish oil, yield 72 %). 1H NMR (400 MHz, CDCl3): d = 3.62 (s, 2 H), 5.04 (br s, 2 H), 5.31 (dd, J = 1.2, 2.2 Hz, 1 H), 5.47 (dd, J = 1.5, 2.9 Hz, 1 H), 5.54 (br s, 1 H), 6.23 (m, 1 H), 7.14–7.22 (3 H), 7.24–7.37 (5 H), 7.38–7.43 ppm (2 H); 13C NMR (100 MHz, CDCl3): d = 38.0, 66.0, 115.1, 126.0 (2), 126.3, 126.8, 128.0, 128.4 (2), 1285.5 (2), 129.0 (2), 138.0, 138.5, 139.9, 142.4, 166.6 ppm.
Collection of vascular exudates: Tomato plants (Solanum esculemtum cv. Ohgata-Fukuju) and tobacco plants (Nicotiana benthamiana) were grown in pots containing commercial soil (Hanagokorobaiyoudo; Hanagokoro, Aichi, Japan) in a growth incubator at 25 8C under 10 000 lux for 16 h per day. Three- to four-week-old plants were inoculated by stabbing with 200 mL pipette tips containing bacterial cell suspensions (100 mL, 1.0 107 cfu mL 1) into the third leaf axil from the plant apex.[32] Pipette tips were removed after the entire bacterial suspension had entered the stems. The inoculated plants were grown in an incubator at 25 8C under 10 000 lux for 16 h per day. After 14 days, the stems of five wilting plants were cut with a surgical knife and immersed in MilliQ water (10 mL) for 3 h. The water containing vascular exudates and bacterial cells was extracted with an equal volume of EtOAc three times. The extracts were dried over Na2SO4 and evaporated. The residues were dissolved in MeOH (tomato: 100 mL; tobacco: 20 mL), and the supernatants were analyzed by HPLC as above.
Compound 20 (312 mg, 1.12 mmol) was dissolved in CH2Cl2 (15 mL), and the mixture was heated to reflux. A suspension of Grubbs 2nd (19.0 mg, 22.4 mmol) in CH2Cl2 (5 mL) was added dropwise to the mixture over 1.5 h. After stirring for 10 h, the catalyst suspension was added again to the reaction mixture, and the reaction was continued for an additional 4 h. After filtration to remove the catalyst, the residue was purified by silica gel chromatography with hexane/EtOAc (9:1) as the eluent to give 5 (115 mg, colorless amorphous solid, yield 41 %). 1H NMR (400 MHz, CD3OD): d = 3.88 (s, 2 H; H-10), 5.27 (s, 2 H; H-5), 7.17 (m, 1 H; H-14), 7.20 (m, 2 H; H12), 7.25 (tt, J = 1.2, 7.2 Hz, 2 H; H-13), 7.46 (m, 3 H; H-8, H-9), 7.52 ppm (m, 2 H; H-7); 13C NMR (100 MHz, CD3OD): d = 30.7 (C-10), 72.6 (C-5), 126.1 (C-3), 127.6 (C-14), 128.7 (C-7), 129.2 (C-12), 129.7 (C-13), 130.2 (C-8), 131.6 (C-9), 132.4 (C-6), 139.3 (C-11), 160.1 (C-4), 177.5 ppm (C-2); ESI-MS: m/z 251 [M+H] + . Chiral HPLC analysis: An LCMS-2020 spectrometer was used with a chiral column (CHIRALPAK IA, 250 2.1 mm, 5 mm; Daicel, Osaka, Japan). The following solvent systems were used: 40 % MeCN in 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Phytotoxic assay: Tobacco plants (N. benthamiana) were grown in pots containing commercial soil (Hanagokorobaiyoudo) in a growth incubator at 25 8C under 10 000 lux for 16 h per day. Fiveweek-old plants were used. Solutions of ralfuranones (50 mL; 1, 10, and 50 mm) were inoculated into leaves with a 1 mL syringe.[34] After 1 day, the presence of necrosis and changes to leaves were assessed. Antimicrobial assay: Seed cultures of E. coli and B. subtitles were grown in LB medium (BD) at 30 8C overnight. Cultures (50 mL) was spread on LB-agar medium. The suspension of A. aculeatus spores was spread on potato dextrose agar medium (BD). Advantec paper discs (6 mm; Toyo Roshi Kaisha, Tokyo, Japan) containing an appropriate amount of ralfuranone were placed onto the surface of agar medium. The agar plates were incubated at 30 8C overnight, and the inhibition zones were monitored. ChemBioChem 0000, 00, 1 – 9
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CHEMBIOCHEM FULL PAPERS Deletion and complementation of ralA: An 875 bp DNA fragment (ralA-1) was PCR-amplified from genomic DNA of OE1-1 with primers ral-1-FW (5’-CCGTT GGCGG CGAAT CTTGG-3’) and ral-1-RV (5’GTCCG GCTCA CATCG GATCA TGCCG AATCG-3’). An 875 DNA fragment (ralA-2) was PCR-amplified from genomic DNA of OE1-1 with primers ral-2-FW (5’-TGATC CGATG TGAGC CGGAC GGAGG GGCCG3’) and ral-2-RV (5’-GGGTG TTCCC AGTTT TAGGC G-3’). By using ralA-1 and ralA-2 as templates, a 1,759 bp DNA fragment was PCRamplified with primers ral-1-FW and ral-2-RV, then cloned into TVector pMD20 (Takara Bio, Shiga, Japan) to create pMD20ralA. The pMD20ralA construct was digested with EcoRI and PstI to release a 1.8-kbp fragment, which was ligated into the EcoRI and PstI sites of pK18 mobsacB[36] to create pDralA. This plasmid was electroporated into OE1-1 cells, and kanamycin-resistant and sucrose-sensitive recombinants were selected. Recombinants were incubated in PY medium (polypeptone (0.5 %), yeast extract (0.2 %)) for 6 h, and a kanamycin-sensitive and sucrose-resistant recombinant, DralA, was selected. DNA for sequencing was PCR-amplified with primers ral-SQ-FW3 (5’-TGCGT TAGGC ATGGT TTGG-3’) and ral-SQ-RV3 (5’TTGTA GGCGT TCATG GTCC-3’) to verify the correct substitution of the deleted ralA in OE1-1 (data not shown).
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We are grateful to Dr. Yuichi Masuda (Tohoku University) for the HRMS measurements and Takuya Asai (Kochi University) for his technical assistance. This work was supported by a grant from the Agricultural Chemical Research Foundation to KK and JSPS KAKENHI Grant Number 26660036 to Y.H., K.K., K.O., and A.K.
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Keywords: natural products · quorum sensing · ralfuranones · Ralstonia solanacearum · structure elucidation
Received: July 24, 2014 Published online on && &&, 0000
A 4649 bp DNA fragment containing ralA was PCR-amplified from genomic OE1-1 DNA with primers ralA-C1 (5’-GTCAT GGCGT ACTGC TGCAT G-3’) and ralA-C2 (5’-TCACG CGGCA TCTCC CAACA C-3’). The PCR fragment was cloned into T-Vector pMD20 to create pMD20CralA. The pMD20CralA construct was digested with BamHI and KpnI to release a 4.7-kbp fragment, which was ligated into the BamHI and KpnI sites of pUC18-mini-Tn7-Gm[37] to create pCralA. pCralA was electroporated into DralA to create gentamycin-resistant ralA-comp. DNA sequencing of PCR-amplified DNA fragments (primers ralA-C1 and ralA-C2) was performed to verify the insertion of ralA in ralA-comp (data not shown). Virulence assay: Tomato plants (Solanum esculemtum cv. OhgataFukuju) were grown in pots containing commercial soil (Tsuchitaro; Sumitomo Forestry Landscaping, Tokyo, Japan) in a growth room at 25 8C under 10 000 lux for 16 h per day, and watered with diluted (5 ) Hoagland’s solution.[18] Five-week-old tomato plants were inoculated by stabbing 200 mL pipette tips containing bacterial cell suspensions (50 mL; 1.0 107 cfu mL 1) into the third leaf axil from the plant apex[32] and placed in a 25 8C growth room. Pipette tips were removed after the entire bacterial suspension entered the stems. All inoculated plants were grown at 25 8C under 10 000 lux for 16 h per day. For each trial, five plants of each strain were treated (total: 45 plants). Plants were inspected for wilting symptoms daily for 14 days, and rated on a zero-to-five disease index scale (0 = no wilting; 1 = 1–25 % wilting; 2 = 26–50 % wilting; 3 = 51– 75 % wilting; 4 = 76–100 % wilting; 5 = dead).
Acknowledgements
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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FULL PAPERS Caring for tomatoes: We identified new ralfuranones as phc quorum sensing-dependent metabolites from Ralstonia solanacearum OE1-1. Ablation of ralfuranone production resulted in reduced virulence in tomato plants. This is the first study to show that secondary metabolites are involved in the virulence of R. solanacearum.
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
K. Kai,* H. Ohnishi, Y. Mori, A. Kiba, K. Ohnishi, Y. Hikichi && – && Involvement of Ralfuranone Production in the Virulence of Ralstonia solanacearum OE1-1
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