From the IACR-Long Ashton Research Station,
Department of Agricultural Sciences, University of Bristol, Long
Ashton, Bristol, BS41 9AF, United Kingdom, the ¶ University of
Bristol, Department of Biochemistry, School of Medical Sciences,
University Walk, Bristol BS8 1TD, United Kingdom, the
School of
Biological Sciences, University of Sussex, Falmer, Brighton, BN1 9QG,
United Kingdom, and ** Monsanto, St. Louis, Missouri 63198
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ABSTRACT |
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This article describes the first detailed
analysis of mitochondrial electron transfer and oxidative
phosphorylation in the pathogenic filamentous fungus,
Gaeumannomyces graminis var. tritici. While
oxygen consumption was cyanide insensitive, inhibition occurred following treatment with complex III inhibitors and the alternative oxidase inhibitor, salicylhydroxamic acid (SHAM). Similarly,
maintenance of a across the mitochondrial inner membrane was
unaffected by cyanide but sensitive to antimycin A and SHAM when
succinate was added as the respiratory substrate. As a result, ATP
synthesis through complex V was demonstrated to be sensitive to these
two inhibitors but not to cyanide. Analysis of the cytochrome content of mitochondria indicated the presence of those cytochromes normally associated with electron transport in eukaryotic mitochondria together
with a third, b-type heme, exhibiting a dithionite-reduced absorbance maxima at 560 nm and not associated with complex III. Antibodies raised to plant alternative oxidase detected the presence of
both the monomeric and dimeric forms of this oxidase. Overall this
study demonstrates that a novel respiratory chain utilizing the
terminal oxidases, cytochrome c oxidase and alternative
oxidase, are present and constitutively active in electron transfer in G. graminis tritici. These results are discussed in
relation to current understanding of fungal electron transfer and to
the possible contribution of alternative redox centers in ATP
synthesis.
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INTRODUCTION |
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Under aerobic conditions, respiration of carbon metabolites in animal, plant, and fungal cells occurs in a tightly regulated manner to produce carbon dioxide and water. Transfer of the electron pairs associated with the respiration of carbon metabolites is indirect and complex, involving the reduction of the coenzymes NAD+ and FAD at two sites within glycolysis and four sites within the citric acid cycle. The electrons (associated with NADH and FADH2) are subsequently transferred, via at least four distinct sites, into the electron-transport chain, where a series of reduction and oxidation events occur in a sequential manner at approximately 10 different redox centers. In the classical scheme, these redox centers are composed of a series of cytochromes and iron-sulfur complexes. About half the energy generated is lost as heat. The remaining energy generated by the electron flow is utilized in the translocation of hydrogen ions from the mitochondrial matrix to the inter mitochondrial membrane space. The free energy is thus stored in the proton gradient (proton motive force) and is subsequently used to drive the synthesis of ATP.
While in mammalian systems the components and sequence of events associated with electron transport appear tightly conserved, those of plants and fungi appear more complex and diverse in nature, often involving alternative redox centers and pathways. Significant research has been conducted in the area of plant respiration, leading to the characterization of these alternative systems, but research into fungal respiration has been limited, and this is especially true for the pathogenic filamentous fungi. With the development and recent launch of a new antifungal chemistry (the methoxyacrylates) renewed interest is developing in this field (1, 2).
Associated with the respiratory chain in most higher plants, some fungi
and protozoa are the alternative oxidase, which is reduced by electrons
from the ubiquinol pool (the only conserved element of all
characterized respiratory chains). While biochemically consistent with
a terminal oxidase, as it reduces O2 to water, this oxidase
is distinguished by its insensitivity to cyanide, azide, and carbon
monoxide. Plant alternative oxidase has been the focus of interest
since it was first described (3) but real progress was hampered until
Elthon et al. (4) raised antibodies to the alternative
oxidase of Sauromatum guttatum resulting in the eventual
isolation of its cDNA clone (5). Subsequent analyses of plant
alternative oxidases have shown that the protein has a molecular mass
of between 32 and 40 kDa and exists as a membrane bound protein dimer.
Each monomer consists of two membrane spanning -helicies with
hydrophilic domains flanking the membrane anchoring helix which then
extend into the matrix (6). Monomers are separated by a
sulfhydryl-disulfide system, which plays a key regulatory role in the
activity of the oxidase, with increased activity associated with the
reduced state (-SH-HS-). Regulation of the activity has also been
linked with the induction of gene expression (7), the redox state of
the ubiquinol pool (8), levels of
-keto acids (9), and intermediates
of the tricarboxylic acid cycle (10, 11).
The alternative oxidase is of considerable interest in plants due to its nonphosphorylating nature. Although evidence in support of possible roles exist, a precise function remains unclear. Early reports by Bahr and Bonner (12) indicated that the alternative oxidase could be activated following the inhibition or saturation of the cytochrome pathway and, therefore, lead to the suggestion that the alternative oxidase in plants provided an "energy overflow" capability. Subsequent work has challenged this, on the basis that the alternative pathway may compete directly with the main pathway for electrons. A refined function has consequently been proposed whereby the alternative oxidase in plant mitochondria may provide a route for the maintenance of the high rate of electron transfer required under conditions of cold stress or during the operation of the photorespiratory cycle in photosynthesizing tissues.
In fungi, and particularly in filamentous fungal species, the presence, function, and regulation of the alternative oxidase remains controversial. A recent report, based on whole cell sensitivity studies to the alternative oxidase inhibitor, salicylhydroxamic acid (SHAM),1 inferred that the alternative oxidase may be more extensively distributed among pathogenic fungal species than had previously been reported (13), but remains to be conclusively demonstrated.
Induction of the alternative pathway in filamentous fungi following inhibition of cytochrome b by methoxyacrylates and antimycin A, again defined by the development of pathogen sensitivity to SHAM, varies in extent dependent on which cytochrome b inhibitor was employed. This may be explained by the different target sites of the inhibitors within cytochrome b and the resultant electron "leakiness" observed in relation to electron flow through either the Q cycle (14), or the alternative cycle hypothesis of Matsuno-Yagi and Hatefi (15). A common observation for all pathogenic fungi utilizing the alternative respiratory pathway was a fitness penalty, presumably due to the decreased ATP generating capacity of this pathway (2).
Induction and regulation of the plant alternative oxidase by compounds such as salicylic acid has been well documented. However, induction of the fungal alternative oxidase appears more limited, and has only been demonstrated to occur following inhibition of the respiratory chain (16), and as a stimulatory response to the presence of nucleotides AMP, ADP, dAMP, and GMP (17).
In the present study, we present evidence that in the phytopathogenic fungus, Gaeumannomyces graminis var. tritici, the cytochrome and alternative oxidase respiratory pathways are constitutively expressed and active. In contrast to the plant alternative respiratory pathway, the alternative oxidase of G. graminis tritici provides a mechanism for the generation and maintenance of a proton motive force (and hence ATP biosynthesis). Since its reliance on the alternative oxidase apparently carries no discernible fitness penalty, we anticipate that G. graminis tritici provides a model system for future studies to probe the function, regulation, and electron partitioning between cytochrome and alternative pathways in filamentous fungi.
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EXPERIMENTAL PROCEDURES |
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Materials-- All chemicals, unless otherwise stated, were purchased from Sigma, Poole, UK, and were of the highest purity. Carboxin was supplied by Uniroyal Chemicals, Evesham, UK. Stock solutions of dinitrophenol (54 mM), antimycin A (19 mM based on an average formula weight 527.6 as purchased as a mixture of antimycin A1, A2, A3, and A4), oligomycin (13 mM, based on an average formula weight 790.4 as purchased as a mixture of oligomycins A, B, and C), and carboxin (0.01 M) were prepared in ethanol. Potassium cyanide was dissolved in dH2O (0.5 M) and SHAM (0.2 M) was dissolved in dimethyl sulfoxide. All inhibitors were prepared immediately prior to use and the solvent concentration never exceeded 1% (v/v) in any assay. Rhodamine 123 and the luciferin:luciferase ATP determination kit were supplied by Molecular Probes, Inc., Europe.
Strains-- G. graminis tritici strains UK22A-1 and DK22A were isolated from the field in 1995 and strain T7 was a gift from Dr. Paul Bowyer, IACR-Long Ashton Research Station. The strain MT3 was isolated at Long Ashton. All cultures were maintained at 18 °C on Czapek Dox Agar (Oxoid) supplemented with D-biotin (0.2 ppm) and thiamine (0.2 ppm). Cultures for analysis were prepared routinely as described. Five, 5-mm agar plugs removed from the outer zone of 2-week-old cultures were inoculated onto 20 ml of Czapek Dox liquid medium (Oxoid) supplemented with D-biotin (0.2 ppm) and thiamine (0.2 ppm) and allowed to grow again. After 3 days at 18 °C, mycelia were disrupted with a hand-held, all glass, homogenizer (30 ml), and the resulting cellular suspension transferred to either 50 or 100 ml of Czapek Dox liquid medium in 100- or 200-ml conical flasks and maintained at 100 rpm, at 18 °C for 72 h.
Toxicity Assays-- Sensitivity to respiratory and oxidative phosphorylation inhibitors was determined in 10 ml of liquid Czapek Dox medium maintained at 100 rpm at 18 °C, containing approximately 5 mg wet weight mycelia. Inhibition of growth was evaluated after 72 and 96 h, by dry cell weights. All assays were conducted in triplicate.
Measurements of Oxygen Consumption in Whole Cells--
Oxygen
consumption by 10 mg ml1 wet mycelia was measured at
18 °C with a Clark-type oxygen electrode in 2 ml of buffer I (50 mM potassium phosphate (pH 7.4)). Inhibitors were added at
the concentrations indicated with subsequent oxygen consumption
recorded over 15 min unless otherwise indicated.
Spectroscopic Measurements of Mitochondrial Cytochromes--
The
mitochondrial fraction was prepared as follows. Three-day-old mycelia
were washed three times in 20 ml of citrate phosphate buffer containing
50 mM citrate, 50 mM disodium phosphate (pH 5.6), 1.2 M sorbitol, and 20 mM
2-mercaptoethanol. Protoplasts were generated by cell wall digestion
for 50-75 min with 3 mg/ml Novozyme 234 (Interspex Products, Inc.,
Foster City, CA) at 18 °C. Protoplast formation was monitored
microscopically and once 90% of mycelia were protoplasted the Novozyme
was removed by three washes with Buffer II (30 mM Tris-HCl,
15 mM sucrose, 5 mM KCl, 1 mM
K2HPO4, 0.5 mM MgCl2,
and 0.2 mM Na2EDTA (pH 7.4)) at 2,000 × g for 8 min at 0 °C. Protoplast disruption was performed
at 0 °C in a Bead-Beater with 40 × g of acid-washed
glass beads (150-212 µm diameter), 30 ml of protoplasts in buffer
II, and 250 µl of fungal protease inhibitor mixture (Sigma P 8215).
Glass beads and large cellular debris were removed by filtering the
homogenate through Mira cloth (0 °C) and large cellular particulates
were removed by centrifugation at 6,000 × g, for 10 min at 4 °C. The mitochondrial fraction was obtained following
centrifugation at 20,000 × g for 25 min at 4 °C.
The resultant mitochondrial pellet was resuspended in Buffer III (1 ml:
0.1 mM KH2PO4, 0.1 mM
K2HPO4, 20% (v/v) glycerol, 1 mM
EDTA, and 1 mM reduced glutathione). Ten µl of this
suspension was removed for the determination of protein concentration
and the remainder immediately frozen in liquid nitrogen. All
mitochondrial samples were stored at 80 °C. These preparations
remained active for at least 6 months but were typically used within 1 month. Cytochrome difference spectra of G. graminis tritici
strains were obtained from 100 µg of isolated mitochondrial protein
using either a Unicam UV2 100 UV/VIS Spectrometer v4.15 or a
spectrometer developed "in-house" by Prof. Peter Rich at the Glynn
Laboratory, UCL (Gower Street, London, United Kingdom). Prior to
analysis, mitochondria were washed in 2 ml of buffer III (pH 7.4) + 0.6 M KCl to remove any pigment adsorbed to the mitochondrial
membrane during preparation.
Detection of Alternative Oxidase-- Resolution of proteins by SDS-polyacrylamide gel electrophoresis was essentially as described by Laemmli (18) with equal amounts of mitochondrial protein from Arum maculatum and G. graminis tritici strain, T7 (approximately 20 µg) electrophoresed on a 10% SDS-polyacrylamide electrophoresis gel.
Electroblotting to Nitrocellulose Membrane-- Electrophoresed proteins were transferred to a nitrocellulose membrane (Schleicher and Schuell, 0.45 µm pore size) according to the method of Towbin et al. (19). The transfer buffer contained 12.6 mM Tris buffer (pH 7.5), 192 mM glycine, and 10% methanol. The proteins were transferred at 100 V (constant voltage) for 1 h.
Western Blotting-- Filters were initially incubated with 3% (w/v) bovine serum albumin and 2% (w/v) milk powder in phosphate-buffered saline (145 mM NaCl (pH 7.2), 12.5 mM Na2HPO4, 2.5 mM NaH2PO4) in order to block unbound protein reactive sites. The filters were then washed in 0.6% (w/v) milk powder, 0.1% (v/v) Tween 20 buffer in phosphate-buffered saline. Transferred proteins were probed with the alternative oxidase antibody (4) at a dilution of 1 in 1000 in 3% (w/v) bovine serum albumin and phosphate-buffered saline buffer. The filters were washed as described above, prior to incubation with a secondary anti-mouse antibody linked to horseradish-peroxidase enzyme and used at a concentration of 1 in 1000 as for the primary antibody. Filters were washed in blot rinse buffer (10 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.1% (v/v) Tween 20) and bound antibody was detected using an enhanced chemiluminescence kit (Amersham International plc, Aylesbury, Bucks, United Kingdom).
Inhibitor Effects on the Proton Motive Force across the Inner Mitochondrial Membrane-- Isolated mitochondrial fraction (300 µg) was loaded with 1 µM potentiometric dye, rhodamine 123, in 1 ml of buffer II (pH 7.4) and maintained at 4 °C prior to analysis. The effect of the various inhibitors on rhodamine 123 fluorescence was monitored with a Zeiss Axiophot microscope equipped for epifluorescence with excitation filter BP546, dichroic mirror FT580, and barrier filter LP590.
ATP Quantitation--
Cultures of G. graminis tritici
strains in 100 ml of liquid Czapek Dox medium were maintained at
18 °C for the duration of the assays. At each time point, 15 ml of
culture was removed, rapidly filtered through Mira cloth, and washed
with 50 ml of deionized water. The mycelia were immediately frozen in
liquid nitrogen to prevent further ATP formation or hydrolysis and
stored at 80 °C prior to analysis. ATP was extracted from mycelia
by grinding a known biomass under liquid nitrogen, in a pestle and mortar until a fine powder was obtained. The debris was resuspended in
500 µl of deionized water (2 °C) and immediately refrozen in liquid nitrogen. A 10-µl aliquot was removed for protein estimation. Isolation of ATP was achieved by vortex mixing the mycelial debris in
phenol:chloroform (1:1) for 30 s and centrifuging in a precooled Biofuge 17RS centrifuge (at 4 °C) at 10,000 × g for
10 min to obtain a clear aqueous phase. The aqueous phase was removed
and stored at
80 °C prior to analysis. ATP levels remained
constant over 7 days when stored under these conditions.
Protein Estimations--
Protein levels were measured using the
BCA protein assay reagent system (Pierce) adapted for a 96-well
microtiter plate assay. Absorbance values (560 nm) were converted to
micrograms of protein ml1 with reference to a bovine
serum albumin standard curve.
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RESULTS |
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Toxicity Assays-- To determine the active involvement of the cytochrome and alternative respiratory pathways, in G. graminis tritici strains T7, UK22A-1, DK22A, and MT3, sensitivities to cyanide, antimycin A, carboxin, SHAM, and oligomycin B were determined. Minimum inhibitory concentration (MIC) required for 100% growth inhibition (MIC's) for each inhibitor were obtained and are presented in Table I.
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Oxygen Consumption Assays-- To establish that the inhibitor activities observed in the toxicity assays were indicative of inhibition of the components of the respiratory chain, oxygen consumption was measured in whole cells. It was also anticipated that the data would provide information as to the possible induction of the alternative respiratory pathway following inhibition of the primary cytochrome pathway. Inhibitor doses were selected with reference to previous studies with other fungi (2, 13, 20) and reflect concentrations in excess to that required for 100% inhibition of their respective target sites.
Treatment with 1 mM KCN resulted in no discernible reduction in the rate of O2 consumption over the assay period in any G. graminis tritici strain (Table II), indicating either a target site alteration or that KCN was prevented from gaining access to cytochrome aa3. In contrast, treatment with 20 µM antimycin A reduced O2 consumption by between 36 and 48% compared with the control consumption rate in the G. graminis tritici strains. This apparently contradictory finding was reproducible in all G. graminis tritici strains as shown by the O2 consumption rate changes in Table II, and confirms the involvement of cytochrome b in the respiratory pathway of G. graminis tritici.
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Characterization of the Respiratory Components of G. graminis tritici-- To determine whether the inhibitory effects of antimycin A and KCN resulted from inhibition of the cytochrome components of the respiratory chain, we sought evidence for the presence of their respective target cytochromes. Sodium dithionite reduced spectra were obtained by scanning intact mitochondria of T7, DK22A, UK22A-1, and MT3 from 400 to 650 nm at room temperature. A representative reduced versus oxidized difference spectrum is reproduced in Fig. 1A. The pronounced absorbance peak, with maxima at 531 and 560-562 nm, corresponded to cytochrome b. The proportion of this attributed to the b-hemes of complex III was determined from the succinate reduced, antimycin A-treated absorbance spectra (Fig. 1B). As the mitochondrial content of cytochrome bL (antimycin A sensitive) is equivalent to that of cytochrome bH, the proportion of the total cytochrome b reduced absorbance peak derived from the hemes of the bc1 complex can be estimated as the sum of these two absorbances, and was therefore equivalent to 40%. The remainder of the absorbance was attributed to, an as yet uncharacterized, b-heme. Peaks corresponding to cytochromes c, c1, and aa3 with absorbance maxima at 550 nm (+521 nm), 554 nm (+523), and 603 nm, respectively, are as indicated.
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DISCUSSION |
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The present study was conducted in order to evaluate the components of the electron transport chains in the filamentous fungus, G. graminis tritici, and their contributions to cellular respiration and ATP synthesis.
Analysis of the biochemical effects of respiratory chain inhibitors,
both in whole cells and in isolated mitochondria, describe several
novel features associated with the regulation of fungal respiration and
the contribution that the alternative pathway makes to mitochondrial
ATP generation. Treatment with the cytochrome b inhibitor
antimycin A suppressed whole cell oxygen consumption rates, promoted
the rapid disruption of the (and hence proton motive force)
associated with the inner mitochondrial membrane within 1.5 min
following treatment, and significantly decreased cellular levels of
ATP. However, cyanide treatment had no effect on oxygen consumption in
whole cells or on the
and only a brief transitory inhibitory
effect on ATP generation was seen. This indicated that a second,
cyanide-insensitive terminal oxidase was present in G. graminis
tritici, which has a proton pumping capability associated with its
capacity to reduce oxygen.
Spectral analysis of the cytochrome content of G. graminis tritici indicated the presence of the standard cytochrome configuration, but that levels of cytochrome c, c1, and aa3 were significantly lower than that of cytochrome b, as predicted from the dithionite reduced minus oxidized spectra. The relative proportion of b-heme (identified from this spectral trace) associated with the bc1 complex was determined by analysis of the reduced minus oxidized spectra of antimycin A (Fig. 1B) and myxothiazol (data not shown) treated mitochondria (reducing substrate was succinate). The level of b-heme associated with the bc1 complex was estimated to be approximately 40% of the total b-heme in mitochondria and was, therefore, as expected, comparable to the levels of aa3 and c1. Characterization of the non-bc1 associated b-heme, with an absorbance maximum at 560 nm, is the focus of further research.
Respiration in G. graminis tritici Is a Function of Both the
Cytochrome and Alternative Pathways--
Inhibitor and spectral data
confirmed the presence of an active cytochrome electron transport chain
in G. graminis tritici, similar in composition and function
to that of other eukaryotes. This electron transport chain contained
complex I (mitochondria were sensitive to rotenone, data not shown) and
able to utilize -hydroxybutyrate as a reducing substrate. Complex II
was also present as succinate was a utilizable reducing substrate in
isolated mitochondria and toxicity assays with whole cells demonstrated sensitivity to carboxin. The presence of complexes III and IV were
demonstrated spectrophotometrically with inhibitor studies (antimycin
A, myxothiazol, and cyanide), and CO photolysis of CO bound
aa3 (data not shown).
Contributions of the Cytochrome and Alternative Electron Transport
Chains on ATP Generation--
The function of the alternative oxidase
has been the subject of considerable discussion in relation to a
possible ATP generating system. In fungi, where the presence of the
active form has been indicated, no functional roles have been ascribed.
The present study has demonstrated that the alternative oxidase is
required by actively growing G. graminis tritici for
respiration and ATP formation, because it provides an essential
function in the generation and maintenance of the associated
with ATP synthesis. Indeed, the inhibitory effects of SHAM on
mitochondrial function (O2 consumption, proton accumulation
at the cytosolic side of the inner mitochondrial membrane, and ATP
generation) were similar both in relation to the time of onset of the
inhibitor effects following treatment and in the magnitude of their
effects to those observed following antimycin A treatment. Maximal
inhibitory rates of O2 consumption and ATP synthesis were
only achieved following treatment with both compounds, indicating that
both pathways are essential for ATP synthesis and cell growth.
Site of Electron Partitioning to the Alternative Pathway-- The alternative respiratory pathway of plants withdraws electrons from the ubiquinol pool and complete inhibition of O2 consumption only occurs following inhibition of both cytochrome and alternative pathways. In keeping with the nonphosphorylating nature of the plant alternative oxidase, ATP biosynthesis remains largely unaffected by SHAM, due to the presence of all the proton translocating sites within the cytochrome pathway.
In G. graminis tritici, electron transport appears more complex with the site of electron partitioning between the two pathways less clear. ATP synthesis and O2 consumption were inhibited to a comparable level following treatment with either a cytochrome bc1 or an alternative oxidase inhibitor. Maximal rates of inhibition of these two processes were only obtained following treatment with both inhibitor classes, indicating the presence of two, inter-related pathways, each with a terminal oxidase and a proton translocatory function capable of generating a proton motive force of sufficient magnitude to drive ATP synthesis. The site of electron partitioning between these two pathways is not clear. Sensitivities to antimycin A and SHAM would support the site of electron partitioning being the same as for plants (namely at the ubiqinone pool), whereas the cyanide insensitivity and the ATP generating capacity of the SHAM-sensitive pathway would indicate that the partitioning event may occur downstream of cytochrome b, but prior to cytochrome aa3. We propose, therefore, from our inhibitor studies on both O2 consumption and ATP synthesis that the alternative pathway of electron transport in G. graminis tritici involves two components. The first possesses the proton translocatory capacity associated with the SHAM sensitive alternative pathway and provides the proton motive force for ATP synthesis. Given the presence of a second, antimycin A and myxothiazol insensitive b-type heme, this cytochrome may form a redox center in the alternative system, and analogous to the b-type hemes of the bc1 complex, may possess a proton translocatory ability. The terminal oxidase of the alternative respiratory pathway is the alternative oxidase and we therefore propose that respiration in G. graminis tritici may follow the scheme depicted in Fig. 5.
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ACKNOWLEDGEMENT |
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We thank Prof. Peter R. Rich (University College, London) for advice and help in the analysis of the cytochromes of G. graminis tritici.
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FOOTNOTES |
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* This work was supported in part by the Institute of Arable Crops Research which receives support from the Biotechnology and Biological Sciences Research Council of the United Kingdom.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by a grant from Monsanto during the period of this study. To whom correspondence should be addressed. Tel.: 01275-549323; Fax: 01275-394007; E-mail: TIM.JOSEPH-HORNE{at}bbsrc.ac.uk.
1 The abbreviations used are: SHAM, salicylhydroxamic acid; MIC, minimal inhibitory concentration.
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REFERENCES |
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