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First published online May 8, 2008; 10.1104/pp.108.118372 Plant Physiology 147:1062-1071 (2008) © 2008 American Society of Plant Biologists A Pathogenic Fungi Diphenyl Ether Phytotoxin Targets Plant Enoyl (Acyl Carrier Protein) Reductase[W]Natural Products Utilization Research Unit, U.S. Department of Agriculture, Agricultural Research Services, University, Mississippi 38677 (F.E.D., Z.P.); Department of Pharmacognosy (D.F., Y.-H.W.), and National Center for Natural Products Research, Research Institute of Pharmaceutical Science (D.A.F., I.A.K.), School of Pharmacy, The University of Mississippi, University, Mississippi 38677; and Department of Entomology and Plant Pathology, Auburn University, Auburn, Alabama 36849 (J.A.M.)
Cyperin is a natural diphenyl ether phytotoxin produced by several fungal plant pathogens. At high concentrations, this metabolite inhibits protoporphyrinogen oxidase, a key enzyme in porphyrin synthesis. However, unlike its herbicide structural analogs, the mode of action of cyperin is not light dependent, causing loss of membrane integrity in the dark. We report that this natural diphenyl ether inhibits Arabidopsis (Arabidopsis thaliana) enoyl (acyl carrier protein) reductase (ENR). This enzyme is also sensitive to triclosan, a synthetic antimicrobial diphenyl ether. Whereas cyperin was much less potent than triclosan on this target site, their ability to cause light-independent disruption of membrane integrity and inhibition of ENR is similar at their respective phytotoxic concentrations. The sequence of ENR is highly conserved within higher plants and a homology model of Arabidopsis ENR was derived from the crystal structure of the protein from Brassica napus. Cyperin mimicked the binding of triclosan in the binding pocket of ENR. Both molecules were stabilized by the  - stacking interaction between one of their phenyl rings and the nicotinamide ring of the NAD+. Furthermore, the side chain of tyrosine is involved in hydrogen bonding with a phenolic hydroxy group of cyperin. Therefore, cyperin may contribute to the virulence of the pathogens by inhibiting ENR and destabilizing the membrane integrity of the cells surrounding the point of infection.
Interactions between pathogenic fungi and their host plants involve complex physical and chemical communications that lead to a series of biochemical actions and reactions by both the infecting microorganisms and the plants being infected (Vera-Estrella et al., 1994  ; Fray et al., 1999; Pedras et al., 2001). The virulence of an organism is sometimes enhanced by its ability to produce phytotoxins that kill cells in the tissue surrounding the point of infection (Baker et al., 1997). Fungi and other microorganisms that produce such biochemical compounds are being studied as potential biocontrol agents for weed management (Duke et al., 1996). These studies have led to the discovery that many pathogenic organisms, such as Preussia fleischhakii, Phoma sorghina, and Ascochyta cypericola, produce cyperin (Fig. 1
; Weber and Gloer, 1988; Stierle et al., 1992; Venkatasubbaiah et al., 1992). These natural toxins are thought to play important roles in inhibiting the physiological processes in the plant cells surrounding the point of infection, enabling the spread of the disease (Feys and Parker, 2000; Staskawicz et al., 2001).
Cyperin is a natural diphenyl ether toxin with structural similarities with diphenyl ether herbicides (i.e. acifluorfen and oxyfluorfen), but it is not a strong inhibitor of the molecular target site of this chemical class, the enzyme protoporphyrinogen oxidase (Harrington et al., 1995 ; Dayan and Allen, 2000). Moreover, the mechanism of action of cyperin appears to be light independent (Harrington et al., 1995), whereas diphenyl ether herbicides require light to cause their phytotoxic effect (Dayan and Duke, 1997). These observations suggest that cyperin acts by another mechanism than that associated with protoporphyrinogen oxidase-inhibiting herbicides.
It was recently reported that enoyl (acyl carrier protein) reductases (ENRs) of the type II fatty acid synthase (plants and prokaryotes) were sensitive to the diphenyl ether triclosan (Fig. 1; McMurry et al., 1998
Phytotoxicity of Cyperin and Triclosan Cyperin was phytotoxic to Arabidopsis (Arabidopsis thaliana) and inhibited growth in a dose-dependent manner (Fig. 2 ), with an I50 of 38.4 µM (Table I ). The effect was most evident on root length, but the leaves were also small and chlorotic at the higher concentrations. Triclosan was much more active on Arabidopsis than cyperin, with an I50 of 0.08 µM. Both cyperin and triclosan caused loss of membrane integrity of cucumber (Cucumis sativus) cotyledons in the absence of light, resulting in electrolyte leakage measured as increased conductivity of the medium (Fig. 3 ). The rate of electrolyte leakage was similar for the two compounds, when tested at 333 and 10 µM for cyperin and triclosan, respectively. After 8 h of incubation in the dark, the level of electrolyte leakage was equivalent to approximately 75% of the total leakage measured after boiling the leaf discs (Table I), suggesting a significant membranotropic effect of these compounds. Similar results were obtained when the experiment was carried out in the presence of light (300 µmol m–2 s–1 of light), indicating that the mode of action of these compounds was not modulated by photon flux or light-dependent physiological or biochemical processes (data not shown).
Levels of C18:3 and C18:2 fatty acids were significantly lower in Arabidopsis exposed to sublethal concentrations of cyperin and triclosan (33 and 0.03 µM, respectively) than in control treatments (Table II ). These phenotypic responses suggest that both of these phytotoxic diphenyl ethers share a similar mode of action.
Biochemical Characterization of Arabidopsis ENR Inhibition Arabidopsis ENR was overexpressed with a poly-His tag in Escherichia coli and purified using a nickel-activated column. The activity of ENR was measured spectrophotometrically by monitoring oxidation of NADH in the presence of crotonyl-CoA. Both cyperin and triclosan inhibited ENR catalytic activity (Fig. 4 ). However, as observed in vivo and with the electrolyte leakage experiment, triclosan was much more active than cyperin on this enzyme, with I50 values of 0.046 and 89.0 µM, respectively (Table I).
Examination of the reaction curves of the time-dependent assays revealed a clear difference between the inhibition of ENR activity by triclosan and cyperin (Fig. 5 ). The initial inhibition of ENR activity in the presence of 8.5 nM triclosan was similar to that observed in the presence of 20 µM cyperin, at 72.7% and 71.7% inhibition relative to control. However, inhibition of ENR activity by triclosan increased over the duration of the assay (88.5% inhibition at the end of the assay), which indicates that triclosan binds irreversibly to the enzyme. On the other hand, inhibition of ENR catalytic activity by cyperin remained constant during the entire assay (74.1% inhibition at the end of the assay), suggesting that this natural product is a reversible inhibitor of ENR.
This difference in binding mechanism to ENR was confirmed by titrating ENR concentration with or without inhibitors (Fig. 6 ). The convergence of the regression lines of the assays performed with and without cyperin is indicative of reversible inhibition of ENR (Fig. 6A). The parallel lines observed in the case of the experiments performed with and without triclosan are typical of slow tight-binding inhibitors. Kinetic analysis determined that ENR had a Km of 288 ± 97 µM for crotonyl-CoA and that the binding of cyperin was noncompetitive with respect to crotonyl-CoA, with a Ki of 13.6 ± 2.2 µM (Fig. 7 ).
Modeling of Arabidopsis ENR and Binding of Cyperin to the Catalytic Site The homology between the amino acid sequence of Arabidopsis ENR and nine other plant ENRs ranged from 63.5% to 94.9% amino acid identity. The sequence homology was significantly less with the ENR sequences of nonplant origin (four protozoal and 12 bacterial species), ranging from 32.0% to 68.2% amino acid identity. However, the catalytic domain of ENR is highly conserved across all species (Table III ).
The homology model of Arabidopsis ENR derived from the crystal structure of Brassica napus was analyzed to assess its structural quality. The geometry and stereochemistry, solvent-accessible surface areas, side chain conformational probabilities, and backbone and side chain conformation were checked. No major discrepancies were found. A Ramachandran plot of the model indicated that, as expected, most  angles are negative and  angles are positive. The few outliers with positive were mostly Gly residues that are not subject to the strong conformational constraints due to a lack of side chain. Analysis of the Ramachandran plot showed 95% of the non-Gly residues in most favored regions. Alignment of the homology model to crystal structure of ENR B. napus has a root mean square distance of 0.872 Å, underlining the high degree of similarity between the two enzymes (Fig. 8A
).
Binding of triclosan and cyperin modeled by FlexiDock provided an estimation of the position of the ligands in the catalytic site. Most of the amino acid residues found in the binding site are conserved across all species known (Table III). The amino acid residue Tyr-199 in the catalytic pocket is known to be involved in hydrogen bonding with the phenolic hydroxy group of triclosan. This residue also participated in the stabilization of cyperin. The tetrasubstituted hydroxyphenyl ring of cyperin is also stabilized via - stacking with the nicotinamide ring of the NAD+ (Fig. 8B) in a manner similar to that observed with triclosan. Modeling of cyperin and triclosan showed that these two structures share some structural similarities, such as their diphenyl ether backbone and molecular volumes (Fig. 9, A and B
; Table IV
). However, the presence of several hydroxy groups on cyperin renders it much more hydrophilic than triclosan (Fig. 9, C and D; Table IV).
The ability of a pathogen to infect and invade a compatible host may be facilitated by the production of toxins that induce cell death in the proximity of the invading organism (Baker et al., 1997 ; Dangl and Jones, 2001). Because several microbial disease agents, such as P. fleischhakii, P. sorghina, and A. cypericola, produce cyperin (Weber and Gloer, 1988; Stierle et al., 1992; Venkatasubbaiah et al., 1992), it is possible that this natural diphenyl ether may also play a role in the virulence of these organisms.
Previous work showed that cyperin was an inhibitor of protoporphyrinogen oxidase (Harrington et al., 1995
Triclosan is a relatively small antimicrobial diphenyl ether that is absorbed via diffusion into the bacterial cell wall and causes light-independent disruption of the cellular membranes (Guillén et al., 2004
ENR is an NAD+-dependent enzyme involved in the reduction of a trans-2,3 enoyl moiety to a saturated acyl chain. Functional plant ENR is a homotetramer and each polypeptide chain forms a single domain consisting of a seven-stranded parallel β-sheet surrounded by seven
Subsequent extensive biochemical and structural studies have confirmed that triclosan is a specific inhibitor of ENR in plants and bacteria (Heath et al., 1998
The structural similarity between cyperin and the antimicrobial diphenyl ether triclosan (Fig. 1), and the fact that both diphenyl ethers cause light-independent disruption of membrane integrity (Fig. 3) and similar changes to the fatty acid profile of Arabidopsis (Table II), suggest that these two molecules may share the same molecular target site. Cyperin inhibited purified Arabidopsis ENR. Its potency was much lower than that of triclosan (Fig. 4), but was commensurate with its in vivo activity (Fig. 2). Time-dependent enzyme assays indicated that cyperin inhibited ENR reversibly, whereas triclosan behaved like a slow tight-binding inhibitor (Fig. 5). This difference in binding mechanism was confirmed by enzyme titration assays (Fig. 6). Kinetic analysis determined that cyperin was a reversible inhibitor of ENR and its binding is noncompetitive with respect to crotonyl-CoA, with a Ki of 13.6 ± 2.2 µM (Table I). The lethality of inhibiting plant ENR with natural diphenyl ether cyperin is similar to that observed in the mod1 Arabidopsis mutant line, which is deficient in MOD1, a gene encoding an ENR (Mou et al., 2000
Knowledge-based modeling enabled the construction of a three-dimensional (3-D) model of Arabidopsis ENR. It is established that the root mean square deviation of the C
The orientation of cyperin in the binding pocket of ENR obtained using the FlexiDock method is similar to that of triclosan in the B. napus catalytic site (Fig. 8B). Only minor shifts in the conformation of the amino acid side chains are necessary to accommodate the binding of the inhibitor. Cyperin appears to interact noncovalently with the Arabidopsis protein and the NAD+ cofactor in the substrate binding site. As with triclosan, cyperin appears to mimic the structure of the enoyl moiety of the substrate, and the oxygen atom of the ether bridge of cyperin is positioned where the carbonyl oxygen of the thioester of the substrate is normally localized (Pidugu et al., 2004
The presence of one of the phenolic hydroxy groups on cyperin is in an identical position as in triclosan, which may provide some stabilization of the natural product via hydrogen bonding with Tyr-199 amino acid. Indeed, the presence of the hydroxy group of triclosan is essential for activity. It forms hydrogen bonds with the hydroxy group of Tyr-156 in E. coli that corresponds to Tyr-199 in higher plant ENR (Heath et al., 1999
Whereas cyperin and triclosan have some structural similarity and both caused light-independent loss of membrane integrity (Fig. 3), there was a large difference in their potency (Table I). The absence of chloro groups on cyperin may also contribute to the lower inhibition of ENR and the difference in their binding mechanism. The 5-chloro group of the phenolic moiety of triclosan is known to be very important for tight binding and removal of this group reduced the binding 450,000-fold (Sivaraman et al., 2003
In addition, cyperin is much less lipophilic than triclosan (Fig. 9; Table IV). Consistent with the analysis of several ENR crystal structures (Lee et al., 2007
The biochemical interactions between pathogenic microorganisms and their host plants are very intricate, often resulting from a dynamic and continually evolving process. The ability of certain pathogenic fungi to produce the natural diphenyl ether cyperin in the tissues of infected plants is likely to contribute to the virulence of these disease agents. Whereas the mechanism(s) by which the producing organisms are not affected by cyperin remains to be determined, this article suggests that ENR may be a valid target for the development of new natural product-based herbicides.
Materials and Synthesis
Cyperin [2-(3-hydroxy-5-methylphenoxy)-5-methoxy-3-methyl phenol; CAS: 33716–82–4; Fig. 1] was synthesized (>95% purity) according to the method reported by Harrington et al. (1995)
Arabidopsis (Arabidopsis thaliana Columbia [Col-0] wild type; Lehle Seeds) seeds were surfaced sterilized in a solution of 2.5% (v/v) sodium hypochlorite with 0.05% (v/v) Tween 20 for 10 min and rinsed four times with sterile deionized water. Twenty seeds per dish were plated onto square culture dishes (L100 x W100 x H15 mm) containing one-half-strength Murashige and Skoog growth medium in agar (2.7 g L–1) with concentrations ranging from 3 to 1,000 µM for cyperin and from 0.01 to 3 µM for triclosan. Controls received equivalent amounts of acetone (0.1% [v/v]). Plates were placed vertically in a CU-32 L plant growth chamber (Percival Scientific) at 21°C with a 16-h day photoperiod (approximately 300 µmol m–2 s–1). Root lengths of 15 seedlings were measured for all treatments after 12 d of growth. The experiment was repeated three times.
Arabidopsis seeds were surfaced sterilized as before and placed in six-well culture plates containing 5 mL of medium (Murashige and Skoog) per well containing either 0.03 µM triclosan or 33 µM cyperin. The control contained a similar amount of acetone (0.1% [v/v]). Plates were cultured for 15 d as described above. Approximately 60 mg of tissue from each well was harvested for extraction. Total fatty acids were extracted according to Sasser (1990)
Tissues were treated with 333 µM cyperin or 10 µM triclosan and their effect on membrane integrity was tested by monitoring electrolyte leakage as described by Kenyon et al. (1985)
Total RNA was extracted from Arabidopsis leaves using TRIzol reagent and converted to first-strand cDNA using Superscript II RNase H– reverse transcriptase according to the manufacturer's instruction (Invitrogen). The open reading frame of fab1 was amplified from the cDNA by using PCR. The PCR reaction was performed using PfuTurbo DNA polymerase (Stratagene) according to the manufacturer's instructions with upstream primer 5'-ctagccATGGCTGAATCCAGTGAAAACAAGGC-3' and downstream primer 5'-ccgctcgagATTCTTGCTGTTAAGGTCTTTGAAC-3'. The PCR product was digested with NcoI and XhoI that were introduced into the primers at the 5' and 3' ends of the primers, respectively, and cloned into similarly prepared pET28b. The clone was sequenced to confirm without errors. This plasmid was named pETFab and transformed BL21(DE3) cells for expression. Cells were collected by centrifugation, resuspended in cold lysing buffer (50 mM Tris-HCl, pH 7.5, 1 M NaCl, 5 mM imidazole, 10% [v/v] glycerol, 1 µg mL–1 leupeptin), and lysed with a French Press (SIM-AMINCO). Benzonase (1 µL mL–1 of 25 units µL–1) and 1 mM phenylmethylsulfonyl fluoride were added immediately to the lysate. After 15-min incubation at room temperature, the lysate was centrifuged in a Sorvall SS34 rotor at 15,000g for 20 min. The supernatant was saved for purification of ENR. A HisTrap HP column (GE Healthcare Bio-Sciences) was activated with 2 mL of 0.1 M NiSO4 and washed column with 10 mL of distilled water. The column was equilibrated with 10 mL of buffer A (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5 mM imidazole). ENR was eluted from the column with elution buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 250 mM imidazole). The ENR-containing fraction was desalted on a PD-10 column equilibrated with cold desalting buffer (10 mM sodium phosphate, pH 7.2, 10 mM dithiothreitol, 10% [v/v] glycerol). Protein concentration was determined using Bio-Rad protein reagent.
The assay buffer consisted of 10 mM sodium phosphate, pH 7.2. The I50 values (concentration of inhibitor required for 50% inhibition of activity) of cyperin and triclosan on Arabidopsis ENR were determined by testing the inhibitors at concentrations ranging from 0.1 to 1,000 µM for cyperin and 0.001 to 3 µM for triclosan. All inhibitors were dissolved in acetone and control samples received equivalent amounts of acetone (1% [v/v] final concentration). ENR (24 nM or 1 ng mL–1) was incubated with the compounds in the presence of 100 µM NAD+ for 30 min at room temperature prior to assay. Crotonyl-CoA (100 µM) was added to the mixture and the reaction was initiated by the addition of 100 µM NADH. The time-dependent and enzyme titration experiments were performed with 240 nM ENR (10 ng mL–1) and 200 µM each of crotonyl-CoA, NADH, and NAD+. The kinetic study to determine the Km of crotonyl-CoA and the Ki of cyperin was done with 120 nM ENR (5 ng mL–1) and 100 µM of NADH and NAD+. The experiment was carried out in the presence of 2, 4, 6, and 8 µM cyperin with either 50 or 100 µM crotonyl-CoA using the enzyme assay conditions described by Sivaraman et al. (2004)
The time dependence of ENR inhibition by cyperin and triclosan was determined by conduction enzyme assays over 6 min. The nature of the interaction between cyperin and triclosan and their binding site on ENR was further investigated by modifying the method of Ellis et al. (1995)
The National Center for Biotechnology Information (NCBI) Arabidopsis genomic database was searched using BLAST (http://www.ncbi.nlm.nih.gov/BLAST) with sequence of Brassica napus (GenBank accession no. P80030) as query. Twelve cDNA sequences were identified by this search. Analysis of these sequences with the software MegAlign (DNASTAR) showed that 11 were identical. One sequence (GenBank accession no. NM_126612) was used for this study.
The x-ray crystal coordinates of B. napus ENR/NAD+/triclosan complex at a resolution of 1.9 Å from Roujeinikova et al. (1999) The 3-D structure of triclosan was obtained from its x-ray crystal coordinates found in the 1D7O.pdb file. The structure of cyperin was derived from the coordinate of triclosan. The binding of either cyperin or triclosan was adjusted within the binding site using the FlexiDock module of Sybyl 7.2. The ligands were prepositioned in the binding pocket by using the coordinates of triclosan in the crystal structure of B. napus ENR. The binding pocket was defined by selecting all the residues within a 4 Å boundary around the ligand. Briefly, partial charges were assigned to the atoms. All donor and receptor atoms and rotatable bonds were selected in the binding pocket and the ligands to optimize the interaction. The distances between atoms involved in hydrogen bonding in the ligand-receptor interaction were measured in the resulting models.
Data from dose-response experiments were analyzed with the add-on package for dose-response curves (drc; Ritz and Streibig, 2005 Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers P80030 and NM_126612.
The following materials are available in the online version of this article.
We are grateful for the excellent technical support provided by the staff of the Computer Modeling Laboratory of the National Center for Natural Products Research of the University of Mississippi. We also extend our thanks to Mrs. Susan Watson, Mrs. J'Lynn Howell, and Mrs. Marilyn Ruscoe for their technical assistance in the laboratory. Received February 25, 2008; accepted May 6, 2008; published May 8, 2008.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Franck E. Dayan (franck.dayan{at}ars.usda.gov).
[W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.108.118372 * Corresponding author; e-mail franck.dayan{at}ars.usda.gov.
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ABSTRACT
RESULTS
) and cyperin (
) against the root growth of Arabidopsis. Error bars = ±1 SD. Curves were analyzed with the drc add-on package for dose-response curves in R version 2.2.1 (R Development Core Team, 2005
). Error bars = ±1 SD. The dotted line indicates maximal conductivity in the bathing medium following complete lysis of the membranes by boiling the samples.
-helices (Rafferty et al., 1995
= 6.3 mM–1 cm–1 (Ward et al., 1999