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Antimicrobial Agents and Chemotherapy, August 2006, p. 2732-2740, Vol. 50, No. 8
0066-4804/06/$08.00+0 doi:10.1128/AAC.00289-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Dual Effects of Plant Steroidal Alkaloids on Saccharomyces cerevisiae
Veronika Simons,1
John P. Morrissey,2
Maita Latijnhouwers,3
Michael Csukai,4
Adam Cleaver,4
Carol Yarrow,4 and
Anne Osbourn1*
Sainsbury Laboratory, John Innes Centre, Norwich NR4 7UH, United Kingdom,1
Microbiology Department, University College Cork,
Ireland,2
Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, United Kingdom,3
Syngenta, Jealott's Hill International Research Centre, Bracknell, RG42 6EY, United Kingdom4
Received 7 March 2006/
Returned for modification 27 April 2006/
Accepted 2 June 2006
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ABSTRACT
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Many
plant species accumulate sterols and triterpenes as antimicrobial
glycosides. These secondary metabolites (saponins) provide built-in
chemical protection against pest and pathogen attack and can also
influence induced defense responses. In addition, they have a variety
of important pharmacological properties, including anticancer activity.
The biological mechanisms underpinning the varied and diverse effects
of saponins on microbes, plants, and animals are only poorly understood
despite the ecological and pharmaceutical importance of this major
class of plant secondary metabolites. Here we have exploited budding
yeast (Saccharomyces cerevisiae) to investigate the effects of
saponins on eukaryotic cells. The tomato steroidal glycoalkaloid
-tomatine has antifungal activity towards yeast, and this
activity is associated with membrane permeabilization. Removal of a
single sugar from the tetrasaccharide chain of
-tomatine
results in a substantial reduction in antimicrobial activity.
Surprisingly, the complete loss of sugars leads to enhanced antifungal
activity. Experiments with
-tomatine and its aglycone
tomatidine indicate that the mode of action of tomatidine towards yeast
is distinct from that of
-tomatine and does not involve
membrane permeabilization. Investigation of the effects of tomatidine
on yeast by gene expression and sterol analysis indicate that
tomatidine inhibits ergosterol biosynthesis. Tomatidine-treated cells
accumulate zymosterol rather than ergosterol, which is
consistent with inhibition of the sterol C24
methyltransferase Erg6p. However, erg6 and erg3
mutants (but not erg2 mutants) have enhanced resistance to
tomatidine, suggesting a complex interaction of erg mutations,
sterol content, and tomatidine
resistance.
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INTRODUCTION
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Plants produce a vast array of structurally diverse secondary
metabolites. These natural products serve as attractants for agents
that mediate pollination and seed dispersal; they also provide chemical
defenses against pests, pathogens, and invasion by neighboring plants
(47). Small molecules
therefore play key roles in ecological interactions between plants and
other organisms. We exploit the rich reservoir of metabolic diversity
provided to us by diverse plant species in order to find new drugs and
other valuable compounds. The chemical "space," in
terms of the number and variety of molecules produced by plants, is
enormous. Structures of over 100,000 diverse compounds have been
reported so far (11), and
this is inevitably just the tip of the iceberg. However,
with a few well-characterized exceptions, we know very little about the
biological properties of plant secondary metabolites. Characterization
of the biological activities of these compounds will be critical, both
from an ecological perspective and a pharmaceutical
perspective.
The terpenes are one of the largest and most diverse
groups of plant secondary metabolites
(9). They include sterols
and triterpenes, complex compounds that are formed by the cyclization
of 2,3-oxidosqualene. Sterols and triterpenes can accumulate as
glycoside conjugates in substantial quantities in plants. These
glycosides, which include steroidal glycoalkaloids, are commonly
referred to as saponins
(24). Saponins have a
broad range of properties that includes antimicrobial, anti-insect, and
allelopathic activity, and there is good evidence that they contribute
to plant defense (8,
20,
24,
30,
36,
38). They also have a
range of important pharmacological applications
(13,
23,
36). Examples of members
of this family of plant secondary metabolites that are
exploited for drug or medical use include digitonin (used for
cardiovascular treatment), diosgenin (a precursor
for chemical synthesis of steroid hormones), the
Quillaja saponins (adjuvants), and avicins (new and effective
anticancer agents) (13,
19,
21,
22,
24,
36). Some saponins have
negative effects and are detrimental to human health. Steroidal
glycoalkaloids, for example, can be toxic when ingested
(16,
24).
Although it is
clear that saponins have a diverse range of biological activities, very
little is known about the mode of action of these compounds. Saponins
form complexes with sterols and cause sterol-dependent membrane
permeabilization (30).
The antifungal activity of saponins is generally attributed to these
membrane-permeabilizing properties. The precise mechanism of membrane
disruption is unknown, but the sugars are critical for activity
(13,
16,
24,
30). For example, the
tomato steroidal glycoalkaloid
-tomatine has a tetrasaccharide
chain attached to carbon 3 (Fig.
1A). A number of fungal pathogens of tomato produce enzymes that hydrolyze
sugars from
-tomatine (collectively known as tomatinases)
(reviewed in reference
30). Some of these remove
just one sugar, while others hydrolyze all four sugars to give the
aglycone tomatidine (Fig.
1A). The removal of sugars
from saponins is traditionally associated with a reduction in
antimicrobial activity
(30). However,
-tomatine hydrolysis products are able to suppress induced
plant defense responses, indicating that they have other as yet
uncharacterized effects on plant cells
(7,
25).
-Tomatine
and its hydrolysis products have also been associated with a variety of
effects on human health, including toxicity, cholesterol lowering,
enhanced immune responses as cancer chemotherapy agents, and protection
against pathogenic fungi and other microorganisms
(16). The biological
mechanisms underpinning the varied and diverse effects of these
compounds on microbes, plants, and animals are not yet
understood.

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FIG. 1. Differential
effects of -tomatine and tomatidine on Saccharomyces
cerevisiae. A. Structure of the tomato leaf saponin
-tomatine. The site of cleavage by fungal
extracellular enzymes to yield the aglycone tomatidine is indicated. B.
The sensitivities of wild-type S. cerevisiae strains INVSc1
and KT1115 to -tomatine and tomatidine were measured in agar
plate assays. The strains were pregrown in YEPD and the cell densities
adjusted to 2 x 107 cells/ml. This cell suspension
and 1:10, 1:100, and 1:1,000 dilutions were replica plated onto YEPD
containing different concentrations of -tomatine or
tomatidine. Growth tests were carried out at a range of pH
values, since the antifungal activity of -tomatine is pH
dependent. C. Electrolyte leakage measurements. Cells of S.
cerevisiae strain INVSc1 were suspended in distilled water (5
x 108 cells/ml) and treated with -tomatine,
tomatidine, a solvent control (DMF), or a lysis control (chloroform).
Conductivity, which is a measure of leakage of electrolytes from the
cell (cell lysis), was measured over time. Mean values for three
independent experiments are presented, with bars indicating standard
error
values.
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Budding yeast (Saccharomyces cerevisiae) is
an established model organism for investigation of the modes of action
of antifungal and therapeutic compounds
(1,
4,
18,
29,
42). It has also been
used to study the biological properties of defensins, another class of
molecule that play a role in plant defense against fungal attack
(43,
44). Here we have
exploited S. cerevisiae to investigate the effects of
-tomatine and tomatidine on eukaryotic
cells.
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MATERIALS AND METHODS
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Yeast strains and media.
The S. cerevisiae strains
used are listed in Table
1.
Reagents.
Stock solutions were made in
dimethylformamide (DMF) or dimethyl sulfoxide
(DMSO) as indicated.
-Tomatine, tomatidine, and nystatin
were purchased from Sigma (Gillingham, Dorset, United
Kingdom) and flutriafol and fenpropimorph from Riedel-de Haën
(Seelzen, Germany).
Assays of antifungal activity.
Yeast strains
were tested for sensitivity to antifungal agents in agar plate growth
tests on yeast extract-peptone-dextrose (YEPD) agar. Where necessary,
the pH of the agar was adjusted with either HCl or NaOH prior to
autoclaving. Serial dilutions from overnight cultures were plated onto
YEPD agar supplemented with antifungal agents or solvent alone using a
steel-pronged replicator. The plates were incubated at 30°C for
two days and growth was
assessed.
Electrolyte leakage experiments.
The yeast
strain INVSc1 was grown to mid log-phase in YEPD and the cells
harvested by centrifugation at 200 x g for 5 min,
washed in sterile distilled water, and divided into aliquots of
approximately 4 x 109 cells. After further
centrifugation, the pellets were resuspended in sterile distilled water
containing
-tomatine, tomatidine, or nystatin. The
conductivity of the cell suspension was monitored with a Jenway 4010
conductivity meter (Jenway, London, United Kingdom) over a period of
6 h. Full lysis was monitored by incubation with chloroform
(0.5% [vol/vol]) or nystatin (100 µM). Control treatments
consisted of solvent alone (0.7%
DMSO).
Gene expression analysis.
RNA was prepared
from yeast cells that had been treated with
-tomatine,
tomatidine, or fenpropimorph at concentrations that gave 10 to 20%
growth inhibition or with DMF solvent alone. An overnight culture of
strain S288C was diluted to an optical density at 600 nm
(OD600) of 0.1. After 1 h of growth at
30°C, the compounds (or solvent alone) were added and the
cultures were incubated for an additional 5 h. Growth
inhibition was monitored by measuring the OD600. Treatments
were carried out in triplicate. Cells were pelleted and frozen in
liquid nitrogen. Acid-washed glass beads (0.5-mm diameter; Sigma) were
added and the cells disrupted using two 20-s cycles at speed setting 6
in the Savant Bio 101 Fast Prep FP120. Total RNA was isolated using the
RNeasy kit (QIAGEN, Inc., Valencia, CA). Each of the 12 RNA samples was
then hybridized to GeneChip yeast genome S98 arrays
(Affymetrix Inc., Santa Clara, CA) following the
manufacturer's instructions. Data were analyzed using the RMA algorithm
(GeneData Inc., Switzerland) and genes annotated according to the
Saccharomyces Genome Database. An analysis of variance of the
RMA expression values (log scale) was conducted for each gene, and gene
expression for each of the three compounds was compared with that of
the untreated control. A Bonferroni multiple-testing correction was
applied to the contrast P value for each gene in order to
minimize false positives.
Northern blot analysis was carried out
by following standard procedures. PCR products for use as probes were
obtained with gene-specific primers based on the coding regions of the
respective genes, purified using a QIAquick nucleotide removal kit
(QIAGEN, Crawley, United Kingdom) and radiolabeled with
[
-32P]dCTP by using a random prime-labeling system
(Rediprime II; Amersham). Hybridization of blots was carried out at
65°C, and filters were washed at 65°C in 0.1x
SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate) plus
0.1% sodium dodecyl sulfate.
Sterol analysis.
Yeast strain
S288C was grown in YEPD medium to saturation. The culture was diluted
15-fold and duplicate aliquots were treated with
-tomatine, tomatidine, or solvent alone. Cells were
harvested when the control culture reached an OD600 of 1.2.
The concentrations of inhibitory compounds used caused 20 to 30% growth
inhibition. Freeze-dried cells were lysed at room temperature by
overnight incubation with 80% ethyl alcohol-6% KOH (wt/vol).
Sterols were extracted into chloroform and analyzed by gas
chromatography/mass spectrometry (GC/MS) as previously described
(27).
Microarray data accession number.
Transcriptome data have been lodged
with NCBI GEO under data deposit number
GSE4669.
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RESULTS
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Inhibition of yeast growth by
-tomatine and its aglycone tomatidine is via distinct modes of action.
Removal of sugars from saponins is
traditionally associated with a loss of biological activity
(16,
24,
30). We tested the
inhibitory activity of
-tomatine (Fig.
1A) and its deglycosylated
forms and found that intermediates lacking either of the terminal
sugars (xylose or glucose) have little or no detectable antifungal
activity, as expected (data not shown). Surprisingly, however, we found
that the aglycone tomatidine is a far more potent antifungal agent than
-tomatine (Fig.
1B). Previous studies with
filamentous fungi indicate that
-tomatine is more toxic at
higher pHs when it is in its unprotonated form
(16,
30,
41). Our experiments
confirm this and in addition show that the inhibitory activity of
tomatidine is also pH dependent (Fig.
1B). Yeast growth was
completely inhibited by 0.1 to 1.0 µM tomatidine at pH 7, while
the concentration of
-tomatine required for complete
inhibition at this pH was substantially higher (20 to 40 µM)
(Fig. 1B).
The
inhibitory effects of saponins towards filamentous fungi are generally
ascribed to the ability of these molecules to permeabilize membranes
(24,
30). Our electrolyte
leakage experiments show that
-tomatine causes
dosage-dependent permeabilization of yeast cells (Fig.
1C). In contrast,
tomatidine does not induce electrolyte leakage even at 200 µM,
a concentration well in excess of that required for full inhibition of
growth (Fig. 1B). These
data indicate that
-tomatine and tomatidine have distinct
modes of action.
Genome-wide expression profiling of the response of yeast to tomatidine treatment.
Analysis of changes in gene expression
in response to treatment with antimicrobial agents can yield critical
information about likely modes of action and cellular targets
(1,
4) and is emerging as a
powerful tool in modern chemical genetics. We used genome-wide gene
expression profiling to investigate the effects of tomatidine on yeast.
Preliminary microarray experiments suggested that tomatidine might
affect sterol biosynthesis (data not shown); we therefore included the
sterol biosynthesis inhibitor fenpropimorph as a control in further
experiments. In three independently replicated experiments, yeast
cultures were treated with
-tomatine, tomatidine, or
fenpropropimorph at concentrations selected to give 10 to 20% growth
inhibition. RNA was recovered and transcriptional changes were assessed
using Affymetrix whole genome microarrays. Genes with
1.5-fold mean changes in transcription and a
Bonferroni-corrected P value of <0.05 were identified
as having significant differences in expression. This analysis
identified 271 genes showing differential expression between at least
one of the inhibitor treatments and the control (see Table S1 in the
supplemental material). Of these, 29 showed an increase in expression
in response to tomatidine and none showed reduced expression (Table
2). A survey of these genes revealed clear links with effects on
sterol-related responses. Thirteen of these genes had previously been
shown to be positively regulated by Upc2p, a transcription factor
involved in the regulation of sterol biosynthesis and uptake
(46), while 8 were
involved in sterol biosynthesis and 20 were also upregulated by
fenpropimorph. Sixteen of the 20 genes that were upregulated by both
tomatidine and fenpropimorph were either regulated by Upc2p and/or
involved in sterol biosynthesis. Similar trends in the magnitude of the
changes were evident for both tomatidine and fenpropimorph treatment;
for example, DAN1, DAN4, HES1, and
YPL272C showed the largest changes in both treatments (Table
2).
The high
frequency of Upc2p-regulated genes responding to tomatidine and
fenpropimorph led us to specifically compare the data for the full set
of 87 genes that had been identified by others as being Upc2p regulated
(46). Using a 95%
confidence interval as before but lowering the threshold by omitting
the high-stringency multiple-sampling correction step, we found that
expression of 79% and 56% of this subset of genes were increased in
response to fenpropimorph and tomatidine treatments, respectively
(Table
3). Interestingly,UPC2 showed an increase in expression of 4.6-fold in
fenpropimorph-treated cells and 1.73-fold in tomatidine-treated cells.
A similar analysis was carried out for 22 sterol biosynthetic genes. In
this case, 21 genes and 16 genes showed changes in expression in
response to fenpropimorph and tomatidine, respectively (Fig.
2). Taken together, these data provide strong evidence that
tomatidine inhibits sterol biosynthesis, so causing increased
expression of UPC2 and Upc2p-regulated genes. Upregulation of
expression of the related sterol-responsive transcription factor Ecm22p
(45) was not
observed.
Northern blot analysis confirmed that UPC2,
the sterol biosynthesis genes ERG3 and ERG26, and the
DAN/PAU/TIR genes DAN1, PAU1, and
TIR3 are all upregulated in response to tomatidine treatment
(Fig.
3). In contrast, there was little difference between the
-tomatine
treatments and the DMSO-treated control, although
-tomatine
may cause modest increases in expression of ERG3 and
ERG26. The Northern blot experiments also confirmed previous
studies showing that interference with ergosterol biosynthesis in yeast
(by treatment with sterol biosynthesis inhibitors or by mutation of
sterol biosynthesis genes) results in coordinate upregulation of genes
in the sterol biosynthetic pathway
(1,
4,
46). Treatment with
ergosterol biosynthesis inhibitors that block different steps in
ergosterol biosynthesis (the azole flutriafol and the morpholine
fenpropimorph) resulted in upregulation of all genes tested (Fig.
3). A similar pattern was
seen with the upc2-1 mutant of yeast, which has a
gain-of-function mutation
(28). Five of the six
genes tested were also upregulated in the sterol biosynthesis
erg6, erg3, and erg2 mutants (PAU1
is not responsive in these mutants). These experiments confirm that the
effects of tomatidine on gene expression in yeast closely resemble
those associated with sterol biosynthesis inhibitor treatment and with
the upc2-1
mutation.

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FIG. 3. Northern
blot analysis of effects of chemical treatments and genetic mutations
on gene expression. S. cerevisiae strain S288C was grown in
the presence of -tomatine, tomatidine, or the fungicides
flutriafol and fenpropimorph. Controls included untreated yeast cells
and cells grown in the presence of the solvent used to solubilize the
antifungal compounds (1% DMSO). The erg mutants LPY11
(erg6), LPY25 (erg3), LPY27 (erg2) and the
parent strains KT1357 and KT1358 were grown without drug treatment. The
X2180-1A upc2-1 mutant (UPC20) and corresponding
wild-type strain X2180-1A were also included in these experiments.
Strains were grown aerobically to mid-log phase in YEPD medium. Total
RNA was extracted and analyzed by hybridization with probes specific
for ERG3, ERG26, DAN1, TIR3,
PAU1, and UPC2. The bottom panel indicates rRNA
abundance as assessed by ethidium bromide
staining.
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Effects of tomatidine treatment on sterol content.
The data
presented above are consistent with the hypothesis that
tomatidine inhibits ergosterol biosynthesis. Ergosterol
biosynthesis in yeast proceeds through a pathway from lanosterol to
zymosterol and then to ergosterol via a number of well-defined
enzymatic steps (Fig. 2).
Deletion of genes encoding enzymes in the pathway between zymosterol
and ergosterol does not block sterol synthesis but leads to an altered
sterol profile in the mutant strain
(2,
3,
17,
26). We used GC/MS
analysis to investigate the effect of tomatidine treatment on sterol
composition. The major sterol present in the control yeast cells was
ergosterol, as expected (Fig.
4). The sterol profiles of
-tomatine-treated yeast cells were
similar to that of the control. There was, however, a striking effect
of tomatidine treatment on sterol composition. Tomatidine-treated yeast
cells contained very little ergosterol and instead accumulated
zymosterol (cholesta-8,24-dienol), providing strong
confirmatory evidence that tomatidine blocks
ergosterol biosynthesis. Accumulation of zymosterol
is consistent with inhibition of the C24 methyltransferase
encoded by ERG6 (Fig.
2). The effects of
tomatidine on yeast are likely to be more complicated than this,
however, since tomatidine completely inhibits the growth of yeast,
whereas deletion of ERG6 is not lethal
(17).

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FIG. 4. Tomatidine
inhibits sterol biosynythesis. Analysis of sterol content following
treatment of yeast strain S288C with -tomatine or tomatidine.
Peaks: A, cholesterol (added as an internal standard); B, zymosterol;
C, ergosterol; D,
ergosta-5,7-dienol.
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Sensitivity of sterol biosynthetic mutants.
From the data presented above, we may
expect yeast ergosterol biosynthesis mutants to show altered
sensitivity to tomatidine. To test this, erg2, erg3,
and erg6 mutants
(37) were assessed for
sensitivity to
-tomatine and tomatidine (Fig.
5). Clear differences were observed. The erg3 and erg6
mutants had enhanced resistance to tomatidine, while the erg2
mutant showed wild-type sensitivity. All three mutants were resistance
to nystatin as shown previously
(37) but differed in
sensitivity/resistance to
-tomatine, depending on the
particular genetic lesion. Similar results were obtained with a second
set of erg mutants derived from a different parent strain
(23,
32), indicating that
these effects are not specific to the particular genetic background of
the parental strain (data not shown). These data suggest a complex
interaction between the effects of erg mutations and
tomatidine resistance/sensitivity that may be attributable in part to
interactions of these compounds with different membrane
sterols.
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DISCUSSION
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Biological activity
of saponins is normally defined in terms of ability to complex with
sterols, permeabilize membranes, or inhibit the growth of fungi
(24). Removal of one or
more sugars from the C3 sugar chain is generally associated
with loss of biological activity and/or detoxification
(30). However,tomatidine and other
-tomatine hydrolysis
products can interfere with induced defense responses in
plants, indicating that these compounds also have biological activity
of some kind (7,
25). Here we have shown
that tomatidine (the aglycone of
-tomatine) has potent
antifungal activity towards yeast and that this activity does not
involve membrane permeabilization. Using a combination of gene
expression analysis and GC/MS, we have demonstrated that tomatidine
(but not
-tomatine) mimics the effects of sterol biosynthesis
inhibitors. Tomatidine-treated cells accumulate zymosterol rather than
ergosterol, which is consistent with inhibition of the sterol
C24 methyltransferase Erg6p
(5,
17,
32). There may be
additional targets, however, since tomatidine completely inhibits the
growth of yeast, whereas the deletion of ERG6 is not lethal,
and tests of the sensitivity of erg mutants to tomatidine
indicate that the differential effects of this compound on different
erg mutants are complex. In summary, our experiments indicate
that tomatidine has a distinct mode of action to
that of
-tomatine and that it targets the
sterol biosynthetic pathway. This property appears to be a feature of
steroidal alkaloid aglycones since other steroidal alkaloid aglycones
such as solanidine were also inhibitory while the triterpene aglycones
that we tested (oleanic acid, ß-amyrin, and hederagenin) were
not (data not shown). Future experiments to determination the precise
mode of action of tomatidine will include isolation of mutants with
altered tomatidine sensitivity.
The ergosterol biosynthetic
pathway is a key target for chemical control of fungal pathogens of
plants and animals, and there is constant pressure to identify new
compounds with novel modes of action to combat the development of
resistance to existing fungicides
(14,
34,
48). The major fungicides
that target sterol biosynthesis are azoles (Erg11p), morpholines
(Erg24p, Erg2p, and Erg5p), and allylamines (Erg1p).
Fungicides that target Erg6p (sterol C24 methyltransferase)
are not in agricultural or clinical use although this enzyme can be
inhibited by azasterols and other substrate analogues
(33,
35,
39,
40). Interestingly, the
deletion of ERG6 increases the rate of passive drug diffusion
in yeast, making the cells more susceptible to a broad range of
chemicals (12). Thus,
compounds that target Erg6p are likely to have a dual effect on fungi
by inhibiting sterol biosynthesis and by facilitating uptake of other
antimicrobial compounds. This has clear relevance for natural
situations, since synergism between antifungal plant compounds is a
well-known phenomenon. It also has direct relevance for strategies for
control of pathogens in agriculture and in the clinic that involve use
of combinations of chemicals. erg6 mutants are hypersensitive
to the triterpene saponin avenacin A-1, which is consistent with this
prediction (our unpublished data). However, they have enhanced
resistance to tomatidine and
-tomatine (Fig.
5).
Steroidal
alkaloids are found in a variety of Solanaceous plants, both as
precursors in the synthesis of steroidal
glycoalkaloid saponins and as secondary metabolites
per se. They can also be generated as a consequence of
hydrolysis of steroidal glycoalkaloid saponins by fungal pathogens, and
this may result in interference with induced defense mechanisms
(7). From this study, the
strategies employed by certain fungal pathogens of tomato, such as
Fusarium oxysporum f. sp. lycopersici of
"detoxifying"
-tomatine to tomatidine
(30), would seem to be
counterintuitive, since tomatidine is a highly toxic metabolite.
However, a previous study involving assessment of the relative toxicity
of
-tomatine, ß2-tomatine
(
-tomatine lacking the terminal glucose), and
tomatidine to a range of pathogenic and nonpathogenic fungi has shown
that fungal pathogens of Solanaceous plants are
in general more resistant to tomatidine than nonpathogens
(41). The mechanism of
this resistance is not known. Nevertheless, the ability of F.
oxysporum f. sp. lycopersici to withstand the toxic
effects of tomatidine may confer a competitive advantage over other
fungi that attempt to inhabit the same Solanaceous host plant,
particularly since tomatidine may predispose sensitive microbes to the
antimicrobial effects of other low-molecular-weight compounds.
Importantly, tomatidine has recently been shown to suppress induced
defense responses in suspension-cultured tomato cells
(25). Future work that
addresses the physiological effects of
-tomatine and its
hydrolysis products on plant cells is expected to shed light on
signaling processes associated with the establishment of plant-fungus
interactions and on the relationship of these to sterol
homeostasis.
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ACKNOWLEDGMENTS
|
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We thank D. Ristorcelli for
sterol analysis, K. Lecoq and H. Clake for preparation of RNA samples
for microarray analysis, K. Tatchell, L. Parks, A. Keesler, and H.
Riezman for kindly supplying yeast strains, and A. Heese-Peck for
valuable discussion.
The John Innes Centre is supported by the
Biotechnology and Biological Sciences Research Council and the
Sainsbury Laboratory by the Gatsby Charitable Foundation. Research in
J.M.'s laboratory is funded from grants from the Irish HEA PRTLI
program and from Enterprise Ireland (SC/02/517;
IP/2005/0268).
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FOOTNOTES
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* Corresponding
author. Present address: Department of Metabolic Biology, John Innes
Centre, Norwich NR4 7UH, United Kingdom. Phone: 44 1604 450407. Fax: 44
1603 450011. E-mail:
anne.osbourn{at}bbsrc.ac.uk. 
Supplemental material for this article may be found at http://aac.asm.org/. 
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REFERENCES
|
|---|
- Agarwal,
A. K., P. D. Rogers, S. R. Baerson,
M. R. Jacob, K. S. Barker, J. D. Cleary,
L. A. Walker, D. G. Nagle, and A. M.
Clark. 2003. Genome-wide expression profiling of the
response to polyene, pyrimidine, azole, and echinocandin antifungal
agents in Saccharomyces cerevisiae. J. Biol.
Chem.
278:34998-35015.[Abstract/Free Full Text]
- Arthington,
B. A., L. G. Bennett, P. L. Skatrud,
C. J. Guynn, R. J. Barbuch, C. E.
Ulbright, and M. Bard. 1991. Cloning, disruption and
sequence of the gene encoding yeast C-5 sterol desaturase.Gene
102:39-44.[CrossRef][Medline]
- Ashman,
W. H., R. J. Barbuch, C. E. Ulbright,
H. W. Jarrett, and M. Bard. 1991. Cloning
and disruption of the yeast C-8 sterol isomerase gene.Lipids
26:628-632.[Medline]
- Bammert,
G. F., and J. M. Fostel. 2000.
Genome-wide expression patterns in Saccharomyces cerevisiae:
comparison of drug treatments and genetic alterations affecting
biosynthesis of ergosterol. Antimicrob. Agents
Chemother.
44:1255-1265.[Abstract/Free Full Text]
- Bard,
M., R. A. Woods, D. H. Barton, J. E.
Corrie, and D. A. Widdowson. 1977. Sterol
mutants of Saccharomyces cerevisiae: chromatographic analyses.Lipids
12:645-654.[Medline]
- Bloecher,
A., and K. Tatchell. 2000. Dynamic localization of
protein phosphatase type 1 in the mitotic cell cycle of
Saccharomyces cerevisiae. J. Cell
Biol.
149:125-140.[Abstract/Free Full Text]
- Bouarab,
K., R. Melton, J. Peart, D. Baulcombe, and A. Osbourn.2002
. A saponin-detoxifying enzyme mediates suppression of
plant defences. Nature
418:889-892.[CrossRef][Medline]
- Bowyer,
P., B. R. Clarke, P. Lunness, M. J. Daniels, and
A. E. Osbourn. 1995. Host range of a plant
pathogenic fungus determined by a saponin detoxifying enzyme.Science
267:371-374.[Abstract/Free Full Text]
- Chappell,
J. 2002. The genetics and molecular genetics of
terpene and sterol origami. Curr. Opin. Plant Biol.
5:151-157.[CrossRef][Medline]
- Dewey,
R. E., R. F. Wilson, W. P. Novitzky, and
J. H. Goode. 1994. The AAPT1 gene of soybean
complements a cholinephosphotransferase-deficient mutant of yeast.Plant Cell
6:1495-1507.[Abstract]
- Dixon,
R. A. 2001. Natural products and plant
disease resistance. Nature
411:843-847.[CrossRef][Medline]
- Emter,
R., A. Heese-Peck, and A. Kralli. 2002. ERG6 and PDR5
regulate small lipophilic drug accumulation in yeast cells via distinct
mechanisms. FEBS Lett.
521:57-61.[CrossRef][Medline]
- Francis,
G., Z. Kerem, H. P. S. Makkar, and K. Becker.2002
. The biological action of saponins in animal systems:
a review. Br. J. Nutr.
88:587-605.[CrossRef][Medline]
- Francois,
I. E., A. M. Aerts, B. P. Cammue, and K.
Thevissen. 2005. Currently used antimycotics:
spectrum, mode of action and resistance occurrence. Curr. Drug
Targets
6:895-907.[CrossRef][Medline]
- Frederick,
D. L., and K. Tatchell. 1996. The REG2 gene
of Saccharomyces cerevisiae encodes a type 1 protein
phosphatase-binding protein that functions with Reg1p and the Snf1
protein kinase to regulate growth. Mol. Cell. Biol.
16:2922-2931.[Abstract]
- Friedman,
M. 2002. Tomato glycoalkaloids: role in the plant and
the diet. J. Agric. Food Chem.
50:5751-5780.[CrossRef][Medline]
- Gaber,
R. F., D. M. Copple, B. K. Kennedy, M.
Vidal, and M. Bard. 1989. The yeast gene ERG6 is
required for normal membrane function but is not essential for
biosynthesis of the cell-cycle-sparking sterol. Mol. Cell.
Biol.
9:3447-3456.[Abstract/Free Full Text]
- Giaever,
G., P. Flaherty, J. Kumm, M. Proctor, C. Nislow, D. F.
Jaramillo, A. M. Chu, M. I. Jordan, A. P.
Arkin, and R. W. Davis. 2004. Chemogenomic
profiling: identifying the functional interactions of small molecules
in yeast. Proc. Natl. Acad. Sci. USA
101:793-798.[Abstract/Free Full Text]
- Hanausek,
M., P. Ganesh, Z. Walaszek, C. J. Arntzen, T. J.
Slaga, and J. U. Gutterman. 2001. Avicins, a
family of triterpenoid saponins from Acacia victoriae (Bentham),
suppress H-ras mutations and aneuploidy in a murine skin carcinogenesis
model. Proc. Natl. Acad. Sci. USA
98:11551-11556.[Abstract/Free Full Text]
- Haralampidis,
K., G. Bryan, X. Qi, K. Papadopoulou, S. Bakht, R. Melton, and A.
Osbourn. 2001. A new class of oxidosqualene cyclases
directs synthesis of antimicrobial phytoprotectants in monocots.Proc. Natl. Acad. Sci. USA
98:13431-13436.[Abstract/Free Full Text]
- Haridas,
V., C. J. Arntzen, and J. U. Gutterman.2001
. Avicins, a family of triterpenoid saponins from
Acacia victoriae (Bentham), inhibit activation of nuclear
factor-kappaB by inhibiting both its nuclear localization and ability
to bind DNA. Proc. Natl. Acad. Sci. USA
98:11557-11562.[Abstract/Free Full Text]
- Haridas,
V., M. Higuchi, G. S. Jayatilake, D. Bailey, K. Mujoo,
M. E. Blake, C. J. Arntzen, and J. U.
Gutterman. 2001. Avicins: triterpenoid saponins from
Acacia victoriae (Bentham) induce apoptosis by mitochondrial
perturbation. Proc. Natl. Acad. Sci. USA
98:5821-5826.[Abstract/Free Full Text]
- Heese-Peck,
A., H. Pichler, B. Zanolari, R. Watanabe, G. Daum, and H. Riezman.2002
. Multiple functions of sterols in yeast endocytosis.Mol. Biol. Cell.
13:2664-2680.[Abstract/Free Full Text]
- Hostettmann,
K., and A. Marston. 1995. Saponins. Chemistry and
pharmacology of natural products. Cambridge University Press,
Cambridge, United
Kingdom.
- Ito,
S., T. Eto, S. Tanaka, N. Yamauchi, H. Takahara, and T. Ikeda.2004
. Tomatidine and lycotetraose, hydrolysis products of
alpha-tomatine by Fusarium oxysporum tomatinase, suppress
induced defense responses in tomato cells. FEBS Lett.
571:31-34.
- Jensen-Pergakes,
K. L., M. A. Kennedy, N. D. Lees, R.
Barbuch, C. Koegel, and M. Bard. 1998. Sequencing,
disruption, and characterization of the Candida albicans
sterol methyltransferase (ERG6) gene: drug susceptibility studies in
erg6 mutants. Antimicrob. Agents Chemother.
42:1160-1167.[Abstract/Free Full Text]
- Kelly,
S. L., D. C. Lamb, B. C. Baldwin,
A. J. Corran, and D. E. Kelly.1997
. Characterization of Saccharomyces
cerevisiae CYP61, sterol delta22-desaturase, and inhibition by
azole antifungal agents. J. Biol. Chem.
272:9986-9988.[Abstract/Free Full Text]
- Lewis,
T. L., G. A. Keesler, G. P. Fenner, and
L. W. Parks. 1988. Pleiotropic mutations in
Saccharomyces cerevisiae affecting sterol uptake and
metabolism. Yeast
4:93-106.[CrossRef][Medline]
- Lum,
P. Y., C. D. Armour, S. B. Stepaniants,
G. Cavet, M. K. Wolf, J. S. Butler, J. C.
Hinshaw, P. Garnier, G. D. Prestwich, A. Leonardson, P.
Garrett-Engele, C. M. Rush, M. Bard, G. Schimmack,
J. W. Phillips, C. J. Roberts, and D. D.
Shoemaker. 2004. Discovering modes of action for
therapeutic compounds using a genome-wide screen of yeast
heterozygotes. Cell
116:121-137.[CrossRef][Medline]
- Morrissey,
J. P., and A. E. Osbourn. 1999.
Fungal resistance to plant antibiotics as a mechanism of pathogenesis.Microbiol. Mol. Biol. Rev.
63:708-724.[Abstract/Free Full Text]
- Mortimer,
R. K., and J. R. Johnston. 1986.
Genealogy of principal strains of the yeast genetic stock center.Genetics
113:35-43.[Abstract/Free Full Text]
- Munn,
A. L., A. Heese-Peck, B. J. Stevenson, H. Pichler,
and H. Riezman. 1999. Specific sterols required for
the internalization step of endocytosis in yeast. Mol. Biol.
Cell
10:3943-3957.[Abstract/Free Full Text]
- Nes,
W. D., D. Guo, and W. Zhou. 1997.
Substrate-based inhibitors of the
(S)-adenosyl-L-methionine:delta24(25)- to delta24(28)-sterol
methyl transferase from Saccharomyces cerevisiae. Arch.
Biochem. Biophys.
342:68-81.[CrossRef][Medline]
- Odds,
F. C., A. J. Brown, and N. A. Gow.2003
. Antifungal agents: mechanisms of action.Trends Microbiol.
11:272-279.[CrossRef][Medline]
- Oehlschlager,
A. C., R. H. Angus, A. M. Pierce,
H. D. Pierce, Jr., and R. Srinivasan. 1984.
Azasterol inhibition of delta 24-sterol methyltransferase in
Saccharomyces cerevisiae. Biochemistry
23:3582-3589.[CrossRef][Medline]
- Oleszek,
W., and A. Marston. 2000. Saponins in food, feedstuffs
and medicinal plants. Phytochemical Society of Europe, Amsterdam, The
Netherlands.
- Palermo,
L. M., F. W. Leak, S. Tove, and L. W.
Parks. 1997. Assessment of the essentiality of ERG
genes late in ergosterol biosynthesis in Saccharomyces
cerevisiae. Curr. Genet.
32:93-99.[CrossRef][Medline]
- Papadopoulou,
K., R. E. Melton, M. Leggett, M. J. Daniels, and
A. E. Osbourn. 1999. Compromised disease
resistance in saponin-deficient plants. Proc. Natl. Acad. Sci.
USA
96:12923-12928.[Abstract/Free Full Text]
- Pierce,
A. M., A. M. Unrau, A. C. Oehlschlager,
and R. A. Woods. 1979. Azasterol inhibitors
in yeast. Inhibition of the delta 24-sterol methyltransferase and the
24-methylene sterol delta 24(28)-reductase in sterol mutants of
Saccharomyces cerevisiae. Can. J.
Biochem.
57:201-208.[Medline]
- Pierce,
H. D., Jr., A. M. Pierce, R. Srinivasan,
A. M. Unrau, and A. C. Oehlschlager.1978
. Azasterol inhibitors in yeast. Inhibition of the
24-methylene sterol delta24(28)-reductase and delta24-sterol
methyltransferase of Saccharomyces cerevisiae by
23-azacholesterol. Biochim. Biophys. Acta
529:429-437.
- Sandrock,
R. W., and H. D. VanEtten. 1998.
Fungal sensitivity to and fungal degradation of the phytoanticipan
alpha-tomatine. Phytopathology
88:137-143.
- Simon,
J. A., and A. Bedalov. 2004. Yeast as a
model system for anticancer drug discovery. Nat. Rev.
Cancer
4:481-492.[CrossRef][Medline]
- Thevissen,
K., B. P. Cammue, K. Lemaire, J. Winderickx, R. C.
Dickson, R. L. Lester, K. K. Ferket, F. Van Even,
A. H. Parret, and W. F. Broekaert.2000
. A gene encoding a sphingolipid biosynthesis enzyme
determines the sensitivity of Saccharomyces cerevisiae to an
antifungal plant defensin from dahlia (Dahlia merckii).Proc. Natl. Acad. Sci. USA
97:9531-9536.[Abstract/Free Full Text]
- Thevissen,
K., K. K. Ferket, I. E. Francois, and B.
P. Cammue. 2003. Interactions of antifungal plant
defensins with fungal membrane components. Peptides
24:1705-1712.[CrossRef][Medline]
- Vik,
A., and J. Rine. 2001. Upc2p and Ecm22p, dual
regulators of sterol biosynthesis in Saccharomyces cerevisiae.Mol. Cell. Biol.
21:6395-6405.[Abstract/Free Full Text]
- Wilcox,
L. J., D. A. Balderes, B. Wharton, A. H.
Tinkelenberg, G. Rao, and S. L. Sturley.2002
. Transcriptional profiling identifies two members of
the ATP-binding cassette transporter superfamily required for sterol
uptake in yeast. J. Biol. Chem.
277:32466-32472.[Abstract/Free Full Text]
- Wink,
M. 2003. Evolution of secondary metabolites from an
ecological and molecular phylogenetic perspective.Phytochemistry
64:3-19.[CrossRef][Medline]
- Zhang,
J.-D., Y.-B. Cao, Z. Xu, H.-H. Sun, M.-M. An, L. Yan, H.-S. Chen, P.-H.
Gao, X.-M. Jia, and Y.-Y. Jiang. 2005. In
vitro and in vivo antifungal activities of the eight
steroidal saponins from Tribulis terrestris L. with potent
activity against fluconazole-resistant fungi. Biol. Pharm.
Bull.
28:2211-2215.[CrossRef][Medline]
Antimicrobial Agents and Chemotherapy, August 2006, p. 2732-2740, Vol. 50, No. 8
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