Download Sulphur and nitrogen regulation of the protease

Microbiology (2008), 154, 1464–1473
DOI 10.1099/mic.0.2007/012005-0
Sulphur and nitrogen regulation of the proteaseencoding ACP1 gene in the fungus Botrytis
cinerea: correlation with a phospholipase D activity
Stéphane G. Rolland3 and Christophe A. Bruel
Correspondence
Christophe A Bruel
[email protected]
Received 25 July 2007
Revised 12 January 2008
Accepted 16 January 2008
Génomique fonctionnelle des champignons pathogènes des plantes, UMR5240 Microbiologie,
Adaptation et Pathogénie, Université Lyon 1, CNRS, Bayer CropScience,
Université de Lyon, 14 Rue Pierre Baizet, 69263 Lyon Cedex 9, France
Sulphur and nitrogen catabolic repressions are regulations that have long been recognized in
fungi, but whose molecular bases remain largely elusive. This paper shows that catabolic
repression of a protease-encoding gene correlates with the modulation of a
phosphatidylethanolamine (PE)-specific phospholipase D (PLD) activity in the pathogenic fungus
Botrytis cinerea. Our results first demonstrate that the ACP1 gene is subject to sulphur catabolic
repression, with sulphate and cysteine inhibiting its expression. Sulphate and cysteine also
cause a decrease of the total cellular PLD activity and, reciprocally, the two PLD inhibitors AEBSF
[4-(2-aminoethyl)benzenesulphonyl fluoride] and curcumin negatively affect ACP1 expression in
vivo. Cysteine moreover inhibits the PE-specific PLD activity in cell extracts. ACP1 is
regulated by nitrogen, but here we show that this regulation does not rely on the proximal AREA
binding site in its promoter, and that glutamine does not play a particular role in the process. A
decrease in the total cellular PLD activity is also observed when the cells are fed ammonia,
but this effect is smaller than that produced by sulphur. RNA-interference experiments finally
suggest that the enzyme responsible for the PE-specific PLD activity is encoded by a gene that
does not belong to the known HKD gene family of PLDs.
INTRODUCTION
Fungi sense the nutritional status of their environment via
multiple mechanisms that first relay the information about
particular nutrients and then trigger the appropriate
cellular response (Gagiano et al., 2002; Eckert-Boulet
et al., 2004; Smith et al., 2003). Three essential nutrients,
namely carbon, nitrogen and sulphur, are present in the
environment as readily usable sources or as complex
molecules from which they can be enzymically extracted by
fungi. In the case of carbon, glucose represents the
favourite source and its perception triggers an important
regulation of the cell physiology. Both high and low
concentrations of glucose can be perceived, and many
components of the signal transduction pathways involved
have been unravelled (Rolland et al., 2002). In the case of
sulphur and nitrogen, sulphate and cysteine on the one
hand, and ammonia and glutamine on the other, are
3Present address: Dartmouth Medical School, Genetics Department,
Remsem 7400 Building, Hanover, NH 03755, USA.
Abbreviations: AEBSF, 4-(2-aminoethyl)benzenesulphonyl fluoride; EST,
expressed sequence tag; GUS, b-glucuronidase; PA, phosphatidic acid;
PC, phosphatidylcholine; PE, phosphatidylethanolamine; PLD, phospholipase D.
1464
considered the favourite sources but the way in which they
are perceived by the fungal cell is essentially unknown.
Fungi can assimilate most of the organic and inorganic
sources of sulphur that are present in their environment,
and this implies different metabolic pathways, each
composed of specific transporters and enzymes. However,
when a fungal cell is exposed to multiple sulphur sources, it
favours the one most readily metabolizable, and the
expression of genes encoding proteins involved in the
assimilation of the others is usually decreased (Jacobson &
Metzenberg, 1977); this phenomenon has been termed
sulphur catabolic repression. In the filamentous fungus
Neurospora crassa, one transcriptional regulator has been
found whose control over the expression of genes coding
for sulphate transporters and some of the enzymes involved
in sulphate assimilation has been demonstrated (Marzluf &
Metzenberg, 1968; McDermott et al., 2004). This regulator,
CYS3 (Fu et al., 1989; Fu & Marzluf, 1990a; Paietta, 1992),
has also been shown to control production of proteases
(Hanson & Marzluf, 1975) and its homologue in
Aspergillus nidulans has been identified (Natorff et al.,
2003). Its consensus target DNA sequence has been
determined and it is found in various gene promoters
(Kanaan et al., 1992; Ellenberger et al., 1992; Li & Marzluf,
1996). In the absence of sulphate or cysteine, CYS3 binds to
Downloaded from www.microbiologyresearch.org by
2007/012005 G 2008 SGM
IP: 88.99.165.207
On: Sun, 11 Jun 2017 23:02:05
Printed in Great Britain
PLD and catabolic repression in fungi
these promoters and activates transcription. In the
presence of sulphate or cysteine, sulphur catabolic
repression seems to operate via ubiquitination of CYS3
and its degradation by the proteasome; this is supported by
results obtained in the yeast Saccharomyces cerevisiae
(Rouillon et al., 2000) and by protein similarities as well
as complementation studies performed with homologous
genes in filamentous fungi (Natorff et al., 1998). The
signalling pathway that connects the presence of favoured
sulphur sources to the changes affecting CYS3 is unknown
but intracellular cysteine, or one of its derived metabolites,
seems to be a necessary intermediate (Jacobson &
Metzenberg, 1977; Natorff et al., 1993).
Filamentous fungi also exhibit nitrogen catabolic repression.
The expression of selected genes involved in nitrogen
metabolism is reduced in cells exposed to readily metabolizable nitrogen sources such as ammonia (Facklam & Marzluf,
1978; Sikora & Marzluf, 1982a, b). One transcriptional
regulator, named NIT2 in N. crassa and AREA in A.
nidulans, has been identified in several fungi (Fu & Marzluf,
1990b, c; Haas et al., 1995; Froeliger & Carpenter, 1996,
Screen et al., 1998), and its binding to DNA is necessary for
the expression of the genes involved in utilization of
nitrogen sources (Scazzocchio, 2000). In the absence of
ammonia, AREA binds to two closely spaced 59-GATA
sequences in various gene promoters and activates their
transcription (Chiang & Marzluf, 1994; Chiang et al., 1994).
Conversely, in the presence of ammonia, AREA activity is
strongly reduced due to low expression of the areA gene,
lower RNA stability (Morozov et al., 2001), interaction with
a negative-acting regulator [NMRA in A. nidulans
(Andrianopoulos et al., 1998) and NMR1 in N. crassa (Fu
et al., 1988)], and/or decreased accumulation in the nucleus
(Todd et al., 2005). Catabolic repression hence occurs.
Again, the signalling pathway that responds to the presence
of ammonia is unknown, but intracellular glutamine or
glutamate have been proposed as possible intermediates
(Marzluf, 1997a; Scazzocchio, 2000; Margelis et al., 2001).
Phospholipases D (PLD) are enzymes that respond to
environmental signals to produce the secondary messenger
phosphatidic acid (PA). In eukaryotes, several PLDencoding genes have been isolated in recent years and they
all seem to belong to the HKD gene family (McDermott
et al., 2004). Members of this family preferentially catalyse
the conversion of phosphatidylcholine (PC) into PA and
are also able to perform a transphosphatidylation reaction
with short-chain alcohols. In the yeast S. cerevisiae,
however, a different PLD activity has also been characterized whose calcium dependence, preference for phosphatidylethanolamine (PE) over PC, and incapacity to catalyse
transphosphatidylation make the associated enzyme different from the known HKD family members. The gene that
codes for this non-HKD PLD is still unknown despite the
availability of the whole yeast genome sequence, and it is
proposed to be the first member of a novel PLD gene
family and to present no homology to HKD genes (Tang
et al., 2002).
http://mic.sgmjournals.org
The secretion of lytic enzymes is a hallmark in the biology
of fungi, for these organisms rely on the degradation of
polymers outside the cell for nutrition. The production of
these enzymes is under the control of a sophisticated
regulation system that integrates signals triggered in the cell
by various chemical environmental cues. In saprophytic
species, large panels of enzymes enable them to thrive in
very different ecological niches, and many of these enzymes
are of economic interest. In pathogenic species, these
enzymes participate in the penetration, progression and
survival of the fungus inside its host, and understanding
the regulation of their production is part of the global
quest to understand fungal pathogenicity. In this study, we
explored the regulation of the protease-encoding ACP1
gene of the plant-pathogenic fungus Botrytis cinerea by
sulphur and nitrogen. We report on the discovery of a nonHKD PLD activity that correlates with the activity of the
ACP1 promoter.
METHODS
General techniques. Cultures, Western-blot analysis and b-
glucuronidase (GUS) assays were performed as previously described
(Rolland et al., 2003) except that all sulphate salts in the culture
medium were replaced by chloride salts. Briefly, the fungus was grown
on rich medium on cellophane plates and transferred for 15 h onto
the surface of 2 ml minimal medium containing 2 % glucose, buffered
at pH 4.0 and supplemented as indicated in the text. The medium was
recovered to collect the secreted proteins by precipitation. The
mycelium was frozen in liquid nitrogen and ground to prepare wholecell crude extracts. Antibodies raised against endopolygalacturonases
were used at 1/5000 dilution (Martel et al., 1996).
PLD activity assays. PLD activity was measured according to Hong
et al. (2003) with the following modifications. Briefly, 50 mg crude cell
extract was incubated for 2 h at 28 uC in 100 ml 50 mM HEPES
pH 7.5, 150 mM NaCl, 1 mM CaCl2, 0.25 mM Triton X-100
and 25 mM 1-palmitoyl-2-[1-14C]linoleoylphosphatidylethanolamine
(Amersham). Following separation by TLC using chloroform/
methanol/acetic acid (50 : 25 : 8) as solvent system, radioactive
products were detected using a cyclone phosphoimager (Packard).
Promoter mutagenesis. Versions of the ACP1 promoter 0.4 and
0.6 kb long were generated by using PCR and appropriate primers;
the corresponding DNA fragments were cloned upstream of the GUS
reporter gene. Site-directed mutagenesis was also used to modify the
DNA sequences corresponding to the proximal AREA and CYS3
putative binding sites in the ACP1 promoter. The consensus ‘agataa’
sequence for AREA and ‘atgtcggcat’ sequence for CYS3 were changed
to ‘agggaa’ and ‘atgtggcat’, respectively. These mutations had been
shown to disrupt the nitrogen and sulphur regulation of other genes
(Li & Marzluf, 1996; Ravagnani et al., 1997).
RNA interference (RNAi) cloning. The oliC promoter was amplified
from pLOB-MPD1 (kindly provided by P. Tudzinsky, Westfälische
Wilhelms-Universität, Germany) and cloned upstream of the nos
terminator into pBSSK (Stratagene); the primers used were OliC59
(59-ATATCTAGATGTGGAGCCGCATTCC-39) and OliC39 (59-ATACTGCAGGGATCGATTGTGATGTG-39). A 500 bp DNA fragment
was amplified from B. cinerea genomic DNA using the primers PLD59
(59-AAGCTCTGCAGGATGCCTTCGTCAGCG-39) and PLD39 (59CGCGGATCCTCCCAATTCCTCACATTATCGCCGG-39) designed
on the basis of the EST W0AA034ZA12C1 (Soanes et al., 2002)
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 11 Jun 2017 23:02:05
1465
S. G. Rolland and C. A. Bruel
(predicted to be part of a PLD-encoding gene in B. cinerea). This
fragment was cloned into pTOPO4 (Invitrogen), extracted by
restriction with PstI and cloned between the oliC promoter and the
nos terminator to yield p66. A 300 bp DNA fragment was amplified
from the gfp gene in p18 (Rolland et al., 2003) using GFP59
(59-CTCGGATCCCTTCAAGGACGACGGCAACTACAAG-39) and
GFP39 (59-CTCGGATCCCTGGTAGTGGTCGGCGAGCTGCACG39), digested with BamHI and cloned into p66 upstream of the pld
sequence to yield p67. The same pld sequence was finally cloned into
p67, upstream of the gfp sequence and in the opposite orientation
from that of the other pld sequence, to yield p72. The RNAi cassette
was digested with XbaI and EcoRI and cloned into pBHt2 (Mullins
et al., 2001) to yield p73. Agrobacterium tumefaciens strain LBA1126
containing p73 was used to transform B. cinerea strain BO5-10 as
described previously (Rolland et al., 2003).
sulphate and decreased in a concentration-dependent
manner when sulphate was present (Fig. 1a). Cysteine also
inhibited GUS production whereas alanine had no effect.
Immunodetection of ACP1 secreted by cells exposed to
various amino-acid-supplemented culture media confirmed
these results, and also revealed an inhibitory effect for
methionine, albeit less than that of cysteine (Fig. 1b). These
results demonstrate that availability of sulphur to B. cinerea
influences the activity of the ACP1 promoter, and that
sulphur-containing amino acids act similarly to sulphate.
RT-PCR. The mycelium was frozen in liquid nitrogen, ground and
suspended in 1 ml Trizol reagent (Gibco-BRL). After 5 min vigorous
agitation, 200 ml chloroform was added to the samples and the mixing
was repeated for 5 min. The samples were centrifuged (10 min,
13 000 g, 4 uC) and the supernate was recovered. Total RNAs were
collected by further centrifugation after addition of 500 ml 2propanol. The pellets were washed in 70 % ethanol and suspended
in RNase-free water. cDNA synthesis (1 h at 42 uC) was performed
using AMV-RT reverse transcriptase and random primers (Promega);
RNAs (5 mg) were treated with RQ1 DNase (Promega) for 2 h at
37 uC, mixed with 2-propanol and collected by centrifugation prior to
the reaction. Then 1 ml cDNA was used in a quantitative amplification
reaction using the ‘qPCR and Go’ kit (Qbiogen), the protocol
described by the manufacturer and the following primers: for the pld
gene, 59-TCTCAGGAACACGACACAGTAGCAG-39 and 59-CGGGGGAAAACACATCTTGGACAGC39; for the ACP1 gene, 59-TGATGGTAGCACCGGTAACA-39 and 59-GTGGTGTTGACG-GAGACCTT39; for the tubulin gene, 59-AACTGCAGGTTCCAAACTAACTTGGTTCCTTAC-39 and 59-CGGGATCCCTTAATACTCAGCCTCACCCTC-39.
GUS production was also evident in the fungal cells
(Fig. 2a) when the growth medium contained sulphur but
lacked nitrogen, but this production was lower than that
observed in the absence of sulphur. Glutamine and
glutamate, the first and second established preferred
nitrogen sources for fungi (Marzluf, 1997a), repressed
GUS production as ammonia did. Surprisingly, however,
two non-preferred nitrogen sources (proline and alanine)
also caused total repression, and only small differences
between these various amino acids could be seen when the
Nitrogen regulation of ACP1 appears
unconventional
A405 min–1
(mg protein)–1
(a)
RESULTS
10
5
The protease-encoding ACP1 gene is regulated by
sulphur
Previous studies have shown that the acid-proteaseencoding ACP1 gene is induced by plant extracts and
repressed by nitrogen and neutral or alkaline pH in the two
closely related necrotrophic fungi, B. cinerea and Sclerotinia
sclerotiorum (Girard et al., 2004; Poussereau et al., 2001;
Rolland et al., 2003). To investigate whether this gene also
responds to the availability of sulphur to the fungal cell, we
used a modified B05.10 B. cinerea strain (strain T3)
carrying a transcriptional fusion (pACP1-GUS) able to
drive an intracellular production of b-glucuronidase (GUS)
that parallels that of the endogenous ACP1 (Rolland et al.,
2003). The fungus was grown in rich medium and then
exposed to the absence or presence of sulphate. Acidic
medium was used to avoid repression of the promoter
activity by alkaline pH, and 50 mM ammonia was added to
the medium to prevent transcriptional activation as a result
of nitrogen starvation (Poussereau et al., 2001). The
viability of the mycelium and the amount of proteins
extracted from it were similar under all conditions. The
expression of pACP1-GUS was strong in the absence of
1466
MgSO4 (mM)
0
0.2
1
0
0
Cysteine (mM)
Alanine (mM)
0
0
0
0
0
0
5
0
0
5
(b)
ACP1
MgSO4 (5 mM)
–
+
–
–
Cysteine (5 mM)
Methionine (5 mM)
–
–
–
–
+
–
–
+
Fig. 1. Sulphur catabolic repression of the ACP1 gene. Following
growth in rich medium, the modified B. cinerea strain T3 carrying
the gus gene under the control of the ACP1 gene promoter was
transferred for 15 h to minimal medium (pH 4) supplemented as
indicated. The culture broths and the mycelia were collected to
analyse secreted proteins or to prepare cell extracts. (a) ACP1
expression measured as GUS activity in cell extracts (means±SD
of three independent experiments). (b) Western blot analysis of
secreted proteins using anti-ACP1 polyclonal antibodies.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 11 Jun 2017 23:02:05
Microbiology 154
PLD and catabolic repression in fungi
4
A405 min–1
(mg protein)–1
(a)
15 h
9h
2
1
2
NH4Cl (50 mM)
–
–
–
Glutamine (50 mM)
–
+
– + –
–
– + –
–
–
Glutamate (50 mM)
–
–
– + –
–
Proline (50 mM)
–
–
–
– + –
Alanine (50 mM)
–
–
–
–
– +
(b)
A405 min–1
(mg protein)–1
(c)
0.75
0.50
0.25
NH4Cl (50 mM)
+
+
–
+
+
–
+ + –
+ + –
MgSO4 (1 mM)
+
–
+
+
–
+
+ – +
+ – +
Truncation 1
Truncation 2
cys3*
areA*
Fig. 2. Nitrogen regulation of ACP1. (a) Following growth in rich
medium, B. cinerea strain T3 was transferred for 15 h or 9 h to
nitrogen-free minimal medium buffered to pH 4, containing 1 mM
MgSO4 and further supplemented as indicated. The mycelium was
collected, cell extracts were prepared, and GUS activity was
recorded (means±SD of three independent experiments). (b)
Molecular exploration of the ACP1 promoter regulation. Four
genetic constructs made of various versions of the ACP1 promoter
driving the GUS gene were introduced into the B. cinerea parental
strain B05.10 via A. tumefaciens-mediated transformation. Two
consisted of truncated versions containing the single CYS3
proximal consensus binding site (dot), or both this site and its
AREA counterpart (arrows representing the two closely spaced 59GATA sequences necessary for AREA binding). The two other
constructs consisted of the 0.6 kb promoter into which point
mutations (asterisks) were introduced to modify either the CYS3 or
AREA binding sites. Transformants carrying each construct –
‘truncation 1’ (two isolates), ‘truncation 2’ (two isolates), ‘cys3*’
(three isolates) and ‘areA*’ (three isolates) – were grown on rich
medium and then transferred for 15 h to minimal medium buffered
to pH 4 and supplemented as indicated. Cell extracts were
prepared and GUS activity was measured (means±SD: truncations 1 and 2, two independent experiments; cys3* and areA*,
three independent experiments).
fungus was exposed to them for 9 h instead of 15 h
(Fig. 2a). These results showed that the ACP1 promoter
responds to amino acids as it does to ammonia, but they
indicated that glutamine would not act much differently
http://mic.sgmjournals.org
from other amino acids. As this did not strongly support
glutamine being the real effector in nitrogen repression
(Scazzocchio, 2000), whether ACP1 was subject or not to
conventional nitrogen regulation via AREA became an
issue. We generated two genetic constructs in which
truncated versions of the ACP1 promoter drove the GUS
gene. The first truncation resulted in a 0.6 kb promoter
that kept the single proximal consensus binding sites for
both the nitrogen (AREA) and sulphur (CYS3) regulators.
The second truncation removed an extra 0.2 kb, eliminating the AREA binding site. As shown in Fig. 2(b), and
despite the weaker strength of the shorter promoters, both
versions of the promoter led to minimal GUS production
in the presence of both nitrogen and sulphur, and
increased production in the absence of sulphur. By
contrast, this production was not increased in the absence
of nitrogen in cells carrying the 0.4 kb version of the
promoter. To strengthen this result, we next targeted the
0.6 kb promoter through site-directed mutagenesis, and
specifically modified the CYS3 or AREA consensus binding
sites according to reports demonstrating the negative
impact of the chosen mutations on gene regulation by
these factors (see Methods). The first construct (mutant
cys3*) drove a GUS production that responded to nitrogen
limitation, but no longer to sulphur limitation (Fig. 2b),
indicating that sulphur regulation occurs through the
targeted CYS3 binding site. In contrast, the second
construct (mutant areA*) still drove a GUS production
that responded to both nitrogen and sulphur limitation.
This suggests that nitrogen regulation of ACP1 relies on a
200 bp region of its promoter that contains the single
proximal AREA binding site, but that the regulation would
not proceed through this site. GUS production measured
in the transformants carrying the areA* mutation was
higher than in the other transformants, probably indicating
different insertions of the four genetic constructs in the
genome of each transformant, and, as a consequence,
different levels of expression.
Two PLD inhibitors affect the expression of the
ACP1 gene
While exploring the mechanism of ACP1 regulation by
nitrogen, we discovered that AEBSF [4-(2-aminoethyl)benzenesulphonyl fluoride] interfered with the expression of
that gene. AEBSF had no effect on the activity of the GUS
enzyme in vitro (data not shown), but it abolished GUS
production when added to cell cultures (Fig. 3a). Identical
results were obtained when the cells were continuously
exposed to AEBSF (15 h), or treated for only 1 h and then
transferred to control medium (14 h) (Fig. 3a). AEBSF also
caused a drop in the amount of ACP1 protein secreted by
the fungus, while not affecting other secreted lytic enzymes
such as polygalacturonases (Fig. 3b). Known targets of
AEBSF are serine proteases and PLDs (Andrews et al.,
2000), and serine proteases are secreted by both
S. sclerotiorum (Billon-Grand et al., 2002) and B. cinerea
(G. Billon-Grand, personal communication). However
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 11 Jun 2017 23:02:05
1467
S. G. Rolland and C. A. Bruel
29
ACP1
10
92
53.9
35.4
29
5
AEBSF (2.5 mM)
AEBSF-1 h (2.5 mM)
– + –
– – +
AEBSF (2.5 mM)
PGs
–
+
A405 min–1
(mg protein)–1
(c)
0.50
2
0.25
1
pH of the culture
5 5 5
4 4 4
AEBSF (2.5 mM)
– + –
– + –
AEBSF-Sepharose (2.5 mM)
– – +
Curcumin (1 mM)
– – +
Fig. 3. Inhibition of ACP1 expression by PLD inhibitors. (a)
Following growth in rich medium, B. cinerea strain T3 was
transferred to nitrogen-free minimal medium buffered to pH 4 and
supplemented with AEBSF; the drug was either kept in the
medium for 15h or removed after 1 h (AEBSF-1 h) by replacing
the medium with minimal medium buffered to pH 4. The mycelium
was collected, cell extracts were prepared, and GUS activity was
recorded (means±SD of three independent experiments). (b)
Western blot analysis of secreted proteins using anti-ACP1 (top)
or anti-polygalacturonase (bottom) polyclonal antibodies. (c) Effect
of curcumin and immobilized AEBSF. The fungus was treated as in
(a) and GUS activity was monitored. The chemical bond between
AEBSF and the Sepharose beads is unstable below pH 5;
therefore the experiment had to be performed at this pH rather
than pH 4. The difference in GUS production observed in the
experiments arises from the lower activity of the ACP1 promoter at
pH 5 than at pH 4.
unlikely was the involvement of these secreted enzymes
made by the irreversible effect of 1 h treatment with
AEBSF, this was tested. As AEBSF retains its inhibitory
action on proteases when coupled to beads (Ohkubo et al.,
1994), we used AEBSF coupled to Sepharose beads as a
complex whose size would be incompatible with crossing
the fungal cell wall. We observed no inhibition of GUS
production (Fig. 3c). We also used polyclonal anti-serine
protease antibodies with inhibitory effect on the secreted
protease of B. cinerea in vitro (G. Billon-Grand, personal
communication), and again no inhibition of GUS production was observed (data not shown). AEBSF therefore
seemed not to target a secreted serine protease. To
discriminate between AEBSF targeting a serine protease
or a PLD inside the fungus cell, we used curcumin, another
known PLD inhibitor that does not act on serine proteases
(Yamamoto et al., 1997). The low solubility of curcumin
prevented its use at high concentration, but 1 mM caused
1468
partial inhibition of GUS production (Fig. 3c). Altogether,
these results suggested that AEBSF could mimic the
presence of ammonium in the growth medium and that
it would affect the production of ACP1 by acting upon an
intracellular PLD.
A PE-specific PLD activity is detected under
conditions of ACP1 expression
If a PLD were involved in the regulation of ACP1
expression, we reasoned that its activity would be
modulated by the same environmental conditions as those
which affect that expression. B. cinerea was grown in rich
medium and then exposed to both nitrogen and sulphur
starvation conditions. Fungal cell extracts were prepared
and fed radioactive PC, and the production of PA was
measured. No such production was observed (data not
shown). PE was then used instead of PC and a PAproducing activity was measured when calcium was present
in the reaction (Fig. 4a). In comparison, cells supplied with
nitrogen and sulphur exhibited half of this activity, and
cells exposed to starvation conditions in the presence of
AEBSF exhibited the same half-reduced level of activity
(Fig. 4a). In parallel, AEBSF caused 44 % inhibition of PA
production when added in vitro to a PLD reaction
performed with extracts originating from control cells
(exposed to starvation conditions; Fig. 4b). We tested the
capacity of the enzyme to catalyse a transphosphatidylation
(a)
(b)
Relative activity (%)
(b)
A405 min–1
(mg protein)–1
(a)
PE
PA
Origin
MgSO4 (1 mM)
+ NH4Cl (50 mM)
–
+
–
AEBSF (2.5 mM)
–
–
+
26.5
(±4.5)
15
(±0.8)
12
(±0.3)
mmol PA
(mg prot.)–1
(±SD)
100
50
AEBSF (2.5 mM)
–
+
Fig. 4. AEBSF-sensitive PA production correlates with the
nutrient availability to the cell. Following growth on rich medium,
the B. cinerea parental strain B05.10 was transferred for 15 h to
minimal medium buffered to pH 4 and lacking or containing
sulphur (1 mM) and nitrogen (50 mM). AEBSF was added as
indicated. The mycelium was collected and cell extracts were
prepared to perform phospholipase activity assays. (a)
Radioanalysis of PA production from radioactive PE after TLC;
the quantitative results of two independent reactions are shown in
the table below. (b) Inhibition of PA production by AEBSF in
extracts prepared from cells grown in the absence of sulphur and
nitrogen; AEBSF was directly added to the in vitro assay;
means±SD of three independent reactions are presented.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 11 Jun 2017 23:02:05
Microbiology 154
PLD and catabolic repression in fungi
reaction by adding butan-1-ol to the assay, and we found
that this reaction did not occur (data not shown). These
results indicate that cells deprived of nitrogen and sulphur
respond by doubling their production of PA via an AEBSFsensitive PE-specific PLD.
The PE-specific PLD activity is modulated by both
sulphur and nitrogen
PLD [mmol PA
(mg protein)–1]
(b)
30
20
10
MgSO4 (1 mM)
Cysteine (5 mM)
– + – –
– – + –
AEBSF (2.5 mM)
– – – +
(d)
PLD [mmol PA
(mg protein)–1]
(c)
15
10
5
NH4Cl (mM)
AEBSF (2.5 mM)
0 50 0
– – +
30
20
10
Cysteine (5 mM)
Alanine (5 mM)
PLD [mmol PA
(mg protein)–1]
(a)
PLD [mmol PA
(mg protein)–1]
Having shown that sulphur and nitrogen together act upon
the ACP1 promoter and modulate a PLD activity, we next
investigated the effect of these nutrients individually. The
fungus was grown in rich medium and then deprived of
sulphate. Almost twofold higher PE-specific PLD activity
was measured in these cells when compared to cells
supplied with sulphate, cysteine or AEBSF (Fig. 5a).
Furthermore, cysteine caused a 40 % inhibition of PA
production in vitro whereas alanine had no effect (Fig. 5b).
Since this inhibition could have been attributable to the
reducing power of cysteine, DTT was tested at the same
concentration; it showed no effect. Fungal cells grown in
rich medium and then deprived of nitrogen exhibited a
slightly (20 %) higher PLD activity when compared to cells
supplied with ammonia or AEBSF (Fig. 5c). Neither
glutamine nor alanine caused a significant reduction in
PLD activity (data not shown), and these amino acids had
no effect when added to the enzyme assay in vitro (Fig. 5d).
Altogether, these results demonstrate that a correlation
– + –
– – +
20
15
The single putative PLD-encoding gene in B.
cinerea seems not to be involved in ACP1 or PA
production
In order to validate our results genetically, we searched the
B. cinerea EST database COGEM for a DNA fragment
showing homology to a PLD gene and we found one
annotated EST (number W0AA034ZA12C1). In conjunction, we also searched the now available genome sequence
of this fungus (Broad Institute) and found only one gene
showing homology to any known PLD genes in other
organisms; this gene corresponds to the EST cited above.
Oligonucleotides were designed to target the EST DNA
sequence and to amplify the related DNA fragment from
B. cinerea. This fragment was then used to create a PLD-RNAi
construct (Fig. 6a) that was introduced into B. cinerea strain
BO5.10 by A. tumefaciens-mediated transformation (Rolland
et al., 2003). Several transformants carrying the RNAi
construct were obtained. They all grew identically to the
parental strain and they all achieved normal asexual
reproduction (data not shown). As expected, the ectopic
integration of the construct into the fungus genome (Rolland
et al., 2003) resulted in different levels of interference (90 % in
transformant R1) that were detected by quantitative real-time
PCR in different transformants (Fig. 6b). However, these
interferences had little or no effect on ACP1 transcription
(Fig. 6c) and did not cause a reduction in PE-specific PLD
activity (Fig. 6d). Unless the very small level of expression of
the PLD gene in transformant R1 was sufficient to ensure
wild-type level of PA production, this result rather suggests
that the targeted gene does not encode the PLD responsible
for the nutrient-modulated PA production observed in this
study. Interestingly, this result also suggests that the AEBSFinsensitive production of PA observed in this study probably
does not originate from the activity of a standard PLD, as this
activity too would have been reduced in the transformants.
10
5
Glutamine (5 mM)
Alanine (5 mM)
DISCUSSION
– + –
– – +
Fig. 5. Modulation of the PLD activity by sulphur and nitrogen.
Following growth in rich medium, B. cinerea strain T3 was
transferred for 15 h to a nitrogen-containing (50 mM; a, b) or
sulphur-containing (1 mM; c, d) minimal medium buffered to pH 4,
and supplemented as indicated. The mycelium was collected and
cell extracts were prepared to perform phospholipase activity
assays (a, c; three independent experiments). The effect of
cysteine, glutamine and alanine (5 mM each) on PA production
in vitro was also monitored (two independent experiments); the
control reaction was performed on extracts from cells grown in the
absence of sulphur (b) or nitrogen (d). Means±SD are presented.
http://mic.sgmjournals.org
exists between the availability of sulphur or nitrogen to the
cell and a decrease in the conversion of PE into PA.
However, the two nutrients trigger a response of different
intensity.
ACP1 is a protease secreted by the necrotrophic fungi S.
sclerotiorum and B. cinerea during plant infection (BillonGrand et al., 2002) or upon exposure to plant tissues
(Poussereau et al., 2001; Rolland et al., 2003). Following
the demonstration that the transcription of the ACP1 gene
is under the control of pH and nitrogen (Poussereau et al.,
2001; Rolland et al., 2003), we establish here that it also
responds to changes in sulphur availability to the cell, and
that it does so through a single CYS3 binding site present
in the proximal 400 bp of its promoter. As already reported
for other fungal proteases (Cohen et al., 1975; Farley &
Ikasari, 1992; Katz et al., 1994), ACP1 is produced in
response to sulphur deprivation and this probably
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 11 Jun 2017 23:02:05
1469
S. G. Rolland and C. A. Bruel
(a)
(b)
PLD/TUB
expression
(c)
0.8
0.8
0.4
0.4
ACP1/TUB
expression
(d)
mmol PA
(mg prot.)–1
30
20
P R1 R2 R6
10
P R1 R2 R6
P R1 R2 R6
Fig. 6. Analysis of PLD RNAi transformants of B. cinerea BO5.10
(a) Plasmid used in the A. tumefaciens-mediated transformation of
the fungus. The PLD gene fragments are separated by a linker
made of a fragment of the gfp gene and the construct is driven by
the OliC promoter. The hygromycin (hph) resistance gene is driven
by the TrpC promoter and both genes lie between the left and right
borders of the bacterial ‘transfer DNA’. (b, c) Quantitative
measurements of the targeted PLD gene expression (b) and of
the ACP1 gene (c) in the parental strain (P) and three different
transformants (R1, R2 and R6). The data are from two independent
experiments in which the tubulin-encoding gene (tub) was used as
an internal control for gene expression. (d) PA production in the
parental and transformed strains (means±SD). The strains were
grown on rich medium and then transferred for 15 h to nitrogenfree, sulphur-free minimal medium buffered to pH 4.
represents a way for the fungus to harvest cysteine or
methionine, both organic sulphur sources, from proteins
present in the environment. Our results moreover indicate
that ACP1 expression is higher when the fungus lacks
sulphur than when it lacks nitrogen and, conversely, a 50fold higher nitrogen concentration than that of sulphur is
required to repress transcription. The regulation of ACP1
by sulphur seems therefore to be stronger than that exerted
by nitrogen.The transcriptional activators CYS3 and AREA
respectively mediate gene regulation by sulphur and
nitrogen in fungi. The presence of each nutrient activates
an unknown signalling pathway whose outcome is the
removal from the nucleus, and/or the degradation, of the
corresponding activator, and this leads to sulphur or
nitrogen catabolic repression. This study shows that cells
exposed to sulphur and/or nitrogen limitation exhibit a
specific conversion of PE to PA (PC is not reactive) that is
sensitive to the PLD inhibitor AEBSF both in vivo and in
vitro. A direct correlation is shown in this study between
1470
this activity and that of the ACP1 promoter. Moreover, two
PLD inhibitors, AEBSF and curcumin, can mimic the
presence of sulphur or nitrogen in the environment of
B. cinerea, as deduced from the repression of the ACP1
promoter in cells exposed to these molecules. Finally, the
smaller response of the ACP1 promoter to the lack of
nitrogen than to that of sulphur is mirrored by a smaller
modulation of the PLD activity by nitrogen than by
sulphur in vivo. Altogether, these data reveal the parallel
modulation of a PE-specific PLD activity and the ACP1
promoter. The concomitant inhibition of the PLD and the
ACP1 promoter by AEBSF in vivo suggests that the PLD
modulation could be involved in sulphur and nitrogen
repression of the ACP1 gene by turning the information
relating to the presence of these nutrients into a drop in
intracellular PA production. The total PLD activity
measured in this study is modulated up to 50 % in
response to the presence or absence of sulphur and
nitrogen. Such changes in PLD activity in response to
stimuli have been observed in yeast (Waksman et al., 1996).
In fungi, PA is produced either by PLD or by phospholipase C plus diacylglycerol kinase, and PLD alone is
inhibited by AEBSF (Andrews et al., 2000). Under our
experimental conditions, the maximal AEBSF-sensitive
production of PA accounted for 50 % of the total cellular
production of PA, and the AEBSF-insensitive counterpart
was unaffected in a mutant whose unique PLD-encoding
gene has been repressed by RNA interference. Half the
production of PA therefore probably originates from a
phospholipase C plus diacylglycerol kinase activity, and
this activity is not modulated by nutrients.
Sulphur catabolic repression can be triggered by mineral
sources, such as sulphate, or by organic sources, such as
cysteine or methionine (Marzluf, 1997b). According to the
current model describing this phenomenon, the intracellular pool of cysteine could act as an intermediate in the
process. The PLD activity uncovered in this study is
modulated in vivo by exposure of cells to sulphate, cysteine
and methionine, and it is inhibited in vitro by cysteine,
whereas alanine or DTT have no effect. These data
strengthen the idea that the PLD activity discovered in
this study could respond to changes in sulphur availability
to the cell, and they would be consistent with the current
model of sulphur catabolic repression (Marzluf, 1997b).
More work is however required to establish whether
cysteine acts directly or indirectly on the PLD activity.
Amino acids as well as ammonia lead to nitrogen catabolic
repression (Marzluf, 1997a), and an intracellular pool of
glutamine is proposed as an intermediate in the process
(Scazzocchio, 2000; Margelis et al., 2001). Here we have
shown that amino acids and ammonia modulate the
activity of the ACP1 promoter. Curiously, however,
glutamine seems not to play a special role in nitrogen
regulation of ACP1. When compared to other amino acids
tested, it is slightly more potent, but the difference is small
and the PLD activity discovered in this study is not
inhibited by glutamine in vitro. We used directed
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 11 Jun 2017 23:02:05
Microbiology 154
PLD and catabolic repression in fungi
mutagenesis to further investigate nitrogen repression of
ACP1, and we found that disrupting one of the tandem
GATA sequences that constitute the only canonical AREA
binding site present in its promoter did not affect nitrogen
regulation. Although the possibility remains that AREA
could function through the binding of a single GATA site
(Merika & Orkin, 1993), our data indicate that the
nitrogen regulatory system for ACP1 is unconventional.
Our search of the recently released B. cinerea genome
revealed a single gene with significant homology to the
currently known PLD genes. We used this gene to
successfully develop RNA interference in B. cinerea but
the diminution of this gene’s expression (up to 90 %) had
no effect on ACP1 expression and did not affect the PLD
activity discovered in this study. Together with our
demonstration that this PLD does not catalyse transphosphatidylation with short-chain alcohols as acceptors, this
result suggests that this PLD could be a new member of the
non-HKD family of PLDs. This family of PLDs is
exemplified by the yeast PLD2 protein and, possibly, a
prokaryotic PLD (Ogino et al., 2001). PLD2 (Tang et al.,
2002; Waksman et al., 1997) exhibits the same biochemical
characteristics as the PLD discovered in this study and,
despite the accessibility of the yeast genome, its encoding
gene remains elusive because its sequence is unrelated to
the defined HKD family of PLD genes, whose representative in yeast is PLD1 (Hairfield et al., 2001).
In conclusion, the regulation of the B. cinerea ACP1 gene
by sulphur has been demonstrated. By studying the
regulation of this gene by nutrients, we have also shown
a correlation between the response of this gene’s promoter
to sulphur or nitrogen and the activity of a PE-specific
PLD. This PLD could be a new component in the signalling
pathways that underlie sensing of these nutrients and
catabolic repression in fungi, and PA could hence represent
a secondary messenger in this system. One possible target
of PA is the TOR kinase (Fang et al., 2001), and the
acknowledged role of this enzyme in nutrient signalling
(Rohde & Cardenas, 2004) would now merit being
explored in connection with PA, sulphur and nitrogen.
Finally, the PLD uncovered in this study could belong to
the recently discovered non-HKD family of PLDs, and the
identification of its encoding gene is of particular interest.
ACKNOWLEDGEMENTS
This work was supported by the CNRS and the University Lyon 1.
S. R. was supported by a grant from the French MENRT. We are
grateful to C. Rascle for her technical support.
REFERENCES
Andrianopoulos, A., Kourambas, S., Sharp, J. A., Davis, M. A. &
Hynes, M. J. (1998). Characterization of the Aspergillus nidulans nmrA
gene involved in nitrogen metabolite repression. J Bacteriol 180,
1973–1977.
Billon-Grand, G., Poussereau, N. & Fèvre, M. (2002). The
extracellular proteases secreted in vitro and in planta by the
phytopathogenic fungus Sclerotinia sclerotiorum. J Phytopathol 150,
507–511.
Chiang, T. Y. & Marzluf, G. A. (1994). DNA recognition by the NIT2
nitrogen regulatory protein: importance of the number, spacing, and
orientation of GATA core elements and their flanking sequences upon
NIT2 binding. Biochemistry 33, 576–582.
Chiang, T. Y., Rai, R., Cooper, T. G. & Marzluf, G. A. (1994). DNA
binding site specificity of the Neurospora global nitrogen regulatory
protein NIT2: analysis with mutated binding sites. Mol Gen Genet
245, 512–516.
Cohen, B. L., Morris, J. E. & Drucker, H. (1975). Regulation of two
extracellular proteases of Neurospora crassa by induction and by
carbon-nitrogen and sulfur-metabolite repression. Arch Biochem
Biophys 169, 324–330.
Eckert-Boulet, N., Stein Nielsen, P., Friis, C., Moreira dos Santos, M.,
Nielsen, J., Kielland-Brandt, M. C. & Regenberg, B. (2004).
Transcriptional profiling of extracellular amino acid sensing in
Saccharomyces cerevisiae and the role of Stp1p and Stp2p. Yeast 21,
635–648.
Ellenberger, T. E., Brandl, C. J., Struhl, K. & Harrison, S. C. (1992).
The GCN4 basic region leucine zipper binds DNA as a dimer of
uninterrupted alpha helices. Cell 71, 1223–1237.
Facklam, T. J. & Marzluf, G. A. (1978). Nitrogen regulation of amino
acid catabolism in Neurospora crassa. Biochem Genet 16, 343–350.
Fang, Y., Vilella-Bach, M., Bachmann, R., Flanigan, A. & Chen, J.
(2001). Phosphatidic acid-mediated mitogenic activation of mTOR
signaling. Science 294, 1942–1945.
Farley, P. C. & Ikasari, L. (1992). Regulation of the secretion of
Rhizopus oligosporus extracellular carboxyl proteinase. J Gen Microbiol
138, 2539–2544.
Froeliger, E. H. & Carpenter, B. E. (1996). NUT1, a major nitrogen
regulatory gene in Magnaporthe grisea, is dispensable for pathogenicity. Mol Gen Genet 251, 647–656.
Fu, Y.-H. & Marzluf, G. A. (1990a). cys-3, the positive-acting sulfur
regulatory gene of Neurospora crassa, encodes a sequence-specific
DNA-binding protein. J Biol Chem 265, 11942–11947.
Fu, Y.-H. & Marzluf, G. A. (1990b). nit-2, the major nitrogen regulatory
gene of Neurospora crassa, encodes a protein with a putative zinc finger
DNA-binding domain. Mol Cell Biol 10, 1056–1065.
Fu, Y.-H. & Marzluf, G. A. (1990c). Site-directed mutagenesis of the
‘zinc finger’ DNA-binding domain of the nitrogen-regulatory protein
NIT2 of Neurospora. Mol Microbiol 11, 1847–1852.
Fu, Y. H., Young, J. L. & Marzluf, G. A. (1988). Molecular cloning and
characterization of a negative-acting nitrogen regulatory gene of
Neurospora crassa. Mol Gen Genet 214, 74–79.
Fu, Y.-H., Paietta, J. V., Mannix, D. G. & Marzluf, G. A. (1989). cys-3,
the positive-acting sulfur regulatory gene of Neurospora crassa,
encodes a protein with a putative leucine zipper DNA-binding
element. Mol Cell Biol 9, 1120–1127.
Gagiano, M., Bauer, F. F. & Pretorius, I. S. (2002). The sensing of
nutritional status and the relationship to filamentous growth in
Saccharomyces cerevisiae. FEMS Yeast Res 2, 433–470.
Andrews, B., Bond, K., Lehman, J. A., Horn, J. M., Dugan, A. &
Gomez-Cambronero, J. (2000). Direct inhibition of in vitro PLD
Girard, V., Fèvre, M. & Bruel, C. (2004). Involvement of cyclic AMP in
activity by 4-(2-aminoethyl)-benzenesulfonyl fluoride. Biochem
Biophys Res Commun 273, 302–311.
the production of the acid protease Acp1 by Sclerotinia sclerotiorum.
FEMS Microbiol Lett 237, 227–233.
http://mic.sgmjournals.org
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 11 Jun 2017 23:02:05
1471
S. G. Rolland and C. A. Bruel
Molecular cloning and analysis of nre, the major nitrogen regulatory
gene of Penicillium chrysogenum. Curr Genet 27, 150–158.
novel membrane-bound phospholipase D from Streptoverticillium
cinnamoneum, possessing only hydrolytic activity. Biochim Biophys
Acta 1530, 23–31.
Hairfield, M. L., Ayers, A. B. & Dolan, J. W. (2001). Phospholipase D1
Ohkubo, I., Huang, K., Ochiai, Y., Takagaki, M. & Kani, K. (1994).
is required for efficient mating projection formation in Saccharomyces
cerevisiae. FEMS Yeast Res 1, 225–232.
Dipeptidyl peptidase IV from porcine seminal plasma: purification,
characterization, and N-terminal amino acid sequence. J Biochem 116,
1182–1186.
Haas, H., Bauer, B., Redl, B., Stoffler, G. & Marzluf, G. A. (1995).
Hanson, M. A. & Marzluf, G. A. (1975). Control of the synthesis of a
single enzyme by multiple regulatory circuits in Neurospora crassa.
Proc Natl Acad Sci U S A 72, 1240–1244.
Paietta, J. V. (1992). Production of the CYS3 regulator, a bZIP DNA-
Hong, S., Horiuchi, H. & Ohta, A. (2003). Molecular cloning of
binding protein, is sufficient to induce sulfur gene expression in
Neurospora crassa. Mol Cell Biol 12, 1568–1577.
phospholipase D gene from Aspergillus nidulans and characterization
of its deletion mutants. FEMS Microbiol Lett 224, 231–237.
Poussereau, N., Creton, S., Billon-Grand, G., Rascle, C. & Fèvre, M.
(2001). Regulation of acp1, encoding a non-aspartyl acid protease
Jacobson, E. S. & Metzenberg, R. L. (1977). Control of arylsulfatase
expressed during pathogenesis
Microbiology 147, 717–726.
in a serine auxotroph of Neurospora. J Bacteriol 130, 1397–1398.
Kanaan, M. N., Fu, Y.-H. & Marzluf, G. A. (1992). The DNA-binding
domain of the Cys-3 regulatory protein of Neurospora crassa is
bipartite. Biochemistry 31, 3197–3203.
of
Sclerotinia
sclerotiorum.
Ravagnani, A., Gorfinkiel, L., Langdon, T., Diallinas, G., Adjadj, E.,
Demais, S., Gorton, D., Arst, H. N. & Scazzocchio, C. (1997). Subtle
characterization of an Aspergillus nidulans gene encoding an alkaline
protease. Gene 150, 287–292.
hydrophobic interactions between the seventh residue of the zinc
finger loop and the first base of an HGATAR sequence determine
promoter-specific recognition by the Aspergillus nidulans GATA
factor AreA. EMBO J 16, 3974–3986.
Li, Q. & Marzluf, G. A. (1996). Determination of the Neurospora crassa
Rohde, J. R. & Cardenas, M. E. (2004). Nutrient signaling through
CYS3 sulfur regulatory protein consensus DNA-binding site: aminoacid substitutions in the CYS3 bZIP domain that alter DNA-binding
specificity. Curr Genet 30, 298–304.
Rolland, F., Winderickx, J. & Thevelein, J. M. (2002). Glucose-sensing
Margelis, S., D’Souza, C., Small, A. J., Hynes, M. J., Adams, T. H. &
Davis, M. A. (2001). Role of glutamine synthetase in nitrogen metabolite
Rolland, S., Jobic, C., Fèvre, M. & Bruel, C. (2003). Agrobacterium-
Katz, M. E., Ricea, R. N. & Cheetham, B. F. (1994). Isolation and
repression in Aspergillus nidulans. J Bacteriol 183, 5826–5833.
TOR kinases controls gene expression and cellular differentiation in
fungi. Curr Top Microbiol Immunol 279, 53–72.
and signalling mechanisms in yeast. FEMS Yeast Res 2, 183–201.
endopolygalacturonases from Sclerotinia sclerotiorum: multiplicity of
the complex enzyme system. Curr Microbiol 33, 243–248.
mediated transformation of Botrytis cinerea, simple purification of
monokaryotic transformants and rapid conidia-based identification
of the transfer-DNA host genomic DNA flanking sequences. Curr
Genet 44, 164–171.
Marzluf, G. A. (1997a). Genetic regulation of nitrogen metabolism in
Rouillon, A., Barbey, R., Patton, E., Tyers, M. & Thomas, D. (2000).
the fungi. Microbiol Mol Biol Rev 61, 17–32.
Feedback-regulated degradation of the transcriptional activator Met4
is triggered by the SCFMet30 complex. EMBO J 19, 282–294.
Martel, M.-B., Létoublon, R. & Fèvre, M. (1996). Purification of
Marzluf, G. A. (1997b). Molecular genetics of sulfur assimilation in
filamentous fungi and yeasts. Annu Rev Microbiol 51, 73–96.
Marzluf, G. A. & Metzenberg, R. (1968). Positive control by the cys-3
locus in regulation of sulfur metabolism in Neurospora. J Mol Biol 33,
423–437.
Scazzocchio, C. (2000). The fungal GATA factors. Curr Opin
Microbiol 3, 126–131.
Screen, S., Bailey, A., Charnley, K., Cooper, R. & Clarkson, J. (1998).
McDermott, M., Wakelam, M. & Morris, A. (2004). Phospholipase D.
Isolation of a nitrogen response regulator gene (nrr1) from
Metarhizium anisopliae. Gene 221, 17–24.
Biochem Cell Biol 82, 225–253.
Sikora, L. & Marzluf, G. A. (1982a). Regulation of L-amino acid
Merika, M. & Orkin, S. H. (1993). DNA-binding specificity of GATA
oxidase and D-amino acid oxidase in Neurospora crassa. Mol Gen
Genet 186, 33–39.
family transcription factors. Mol Cell Biol 13, 3999–4010.
Morozov, I. Y., Galbis-Martinez, M., Jones, M. G. & Caddick, M. X.
(2001). Characterization of nitrogen metabolite signalling in
Aspergillus via the regulated degradation of areA mRNA. Mol
Microbiol 42, 269–277.
Mullins, E. D., Chen, X., Romaine, P., Raina, R., Geiser, D. M. & Kang, S.
(2001). Agrobacterium-mediated transformation of Fusarium oxy-
sporum: an efficient tool for insertional mutagenesis and gene transfer.
Phytopathology 91, 173–180.
Natorff, R., Piotrowska, M. & Paszewski, A. (1998). The Aspergillus
Sikora, L. A. & Marzluf, G. A. (1982b). Regulation of L-phenylalanine
ammonia-lyase by L-phenylalanine and nitrogen in Neurospora crassa.
J Bacteriol 150, 1287–1291.
Smith, D. G., Garcia-Pedrajas, M. D., Gold, S. E. & Perlin, M. H.
(2003). Isolation and characterization from pathogenic fungi of genes
encoding ammonium permeases and their roles in dimorphism. Mol
Microbiol 50, 259–275.
Soanes, D. M., Skinner, W., Keon, J., Hargreaves, J. & Talbot, N. J.
(2002). Genomics of phytopathogenic fungi and the development of
nidulans sulfur regulatory gene sconB encodes a protein with WD40
repeats and an F-box. Mol Gen Genet 257, 255–263.
bioinformatic resources. Mol Plant Microbe Interact 15, 421–427.
Natorff, R., Balinska, M. & Paszewski, A. (1993). At least four
and regulation of yeast Ca2+-dependent phosphatidylethanolaminephospholipase D activity. Eur J Biochem 269, 3821–3830.
regulatory genes control sulphur metabolite repression in Aspergillus
nidulans. Mol Gen Genet 238, 185–192.
Natorff, R., Sienko, M., Brzywczy, J. & Paszewski, A. (2003).
Tang, X., Waksman, M., Ely, Y. & Liscovitch, M. (2002). Characterization
Todd, R. B., Fraser, J. A., Wong, K. H., Davis, M. A. & Hynes, M. J.
(2005). Nuclear accumulation of the GATA factor AreA in response
The Aspergillus nidulans metR gene encodes a bZIP protein which
activates transcription of sulphur metabolism genes. Mol Microbiol
49, 1081–1094.
to complete nitrogen starvation by regulation of nuclear export.
Eukaryot Cell 4, 1646–1653.
Ogino, C., Negi, Y., Daiso, H., Kanemasu, M., Kondo, A., Kuroda, S.,
Tanizawa, K., Shimizu, N. & Fukuda, H. (2001). Identification of a
Identification and characterization of a gene encoding phospholipase
D activity in yeast. J Biol Chem 271, 2361–2364.
1472
Waksman, M., Eli, Y., Liscovitch, M. & Gerst, J. E. (1996).
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 11 Jun 2017 23:02:05
Microbiology 154
PLD and catabolic repression in fungi
Waksman, M., Tang, X., Eli, Y., Gerst, J. E. & Liscovitch, M. (1997).
Yamamoto, H., Hanada, K., Kawasaki, K. & Nishijima, M. (1997).
Identification of a novel Ca2+-dependent, phosphatidylethanolamine-hydrolyzing phospholipase D in yeast bearing a disruption in
PLD1. J Biol Chem 272, 36–39.
Inhibitory effect of curcumin on mammalian phosholipase D activity.
FEBS Lett 417, 196–198.
http://mic.sgmjournals.org
Edited by: B. A. Horwitz
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 11 Jun 2017 23:02:05
1473