Fungal Genetics and Biology 56 (2013) 1-146

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Fungal Genetics and Biology

ELSEVIER journal homepage: www.elsevier.com/locate/yfgbi

The bacterial secondary metabolite 2,4-diacetylphloroglucinol impairs CrossMark

mitochondrial function and affects calcium homeostasis in Neurospora

crassa

Danielle M. Troppensa, Meiling Chuc, LucyJ. Holcombea, Olive Gleesona, Fergal OGarab, Nick D. Readc 1, John P.
Morrissey^*
Microbiology Department, University College Cork, Cork, Ireland ab Biomerit Research Centre, Biosciences Institute, University College Cork, Cork, Ireland
c
Fungal Cell Biology Group, Institute of Cell Biology, University ofEdinburgh, Rutherford Building, Edinburgh EH9 3JH, UK

A R T I C L E I N F Q

Article history:
ived 6 February 2013 Accepted A B S T R A C T
12 April 2013 Available online T h e bacterial secondary metabolite 2,4-diacetylphloroglucinol (DAPG) is of interest as an active ingredi- ent of
23 April 2013 biological control strains of Pseudomonas fluorescens and as a potential lead pharmaceutical mol- ecule because of its capacity to inhibit
growth of diverse microbial and non-microbial cells. The mechanism by which this occurs is unknown and in this study the filamentous
Keywords: fungus Neurospora crassa was used as a model to investigate the effects of DAPG on a eukaryotic cell. Colony growth, conidial ger- mination
G and cell fusion assays confirmed the inhibitory nature of DAPG towards N. crassa. A number of different fluorescent dyes and fluorescent
ospora crassa protein reporters were used to assess the effects of DAPG treat- ment on mitochondrial and other cellular functions. DAPG treatment led to
Mitochondria changes in mitochondrial morphology, and rapid loss of mitochondrial membrane potential. These effects are likely to be respon- sible for
Calcium the toxicity of DAPG. It was also found that DAPG treatment caused extracellular calcium to be taken up by conidial germlings leading to a
Antimicrobial transient increase in cytosolic free Ca2+ with a distinct con- centration dependent Ca 2+ signature.

2013 Elsevier Inc. All rights reserved

.
1. Introduction

Microbes have the capacity to synthesise a diverse array of secondary

metabolites such as phenolics, polyketides, cationic pep- tides, non-ribosomal
peptides, alkyl quinolones, oxylipins and homoserine lactones. Some of these
metabolites have clearly de- fined and validated functions. Siderophores, for
instance are se- creted to chelate iron for the producing microbe (Cornelis, 2010),
N-acyl homoserine lactones are used to signal within a microbial population (Ng
and Bassler, 2009; Williams and Camara, 2009), and pheromones are used to
induce mating within a species (Jones and Bennett, 2011). Many secondary
metabolites have also been reported to have antimicrobial properties and indeed,
most clinical antibiotics are derived from metabolites naturally produced by
bacteria or fungi (Demain, 2009; Yim et al., 2006). In addition, many more
secondary metabolites are capable of inhibiting micro- bial growth under
laboratory conditions. This has led to the general consensus that the biological
role of most secondary metabolites is to antagonise other microbes in the
environment to gain a compet- itive advantage. More recently, Davies and
colleagues have strongly challenged the prevailing wisdom on the ecological roleof
antibiotics (Yim et al., 2006, 2007). Their thesis, founded on tran- scriptome
analyses, is that many so-called antibiotics act as intra- or inter-microbial signal
molecules at concentrations far below their minimum inhibitory concentrations
(MICs) (Goh et al., 2002). Subsequent work from a number of groups has
provided some support for the notion that certain secondary metabolites with
antibiotic activity at high concentrations have signalling func- tions at lower

1 Corresponding authors.
E-mail address: m.j.morrissey@ucc.ie (J.P. Morrissey).

1087-1845/$ - see front matter 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.fgb.2013.04.006
2 D.M. Troppens et al./Fungal Genetics and Biology 56 (2013) 135-146
concentrations, an effect termed hormesis (Cummins et al., 2009;
Duan et al., 2009; Kuroda et al., 2007; Shen et al., 2008; Yim et
al., 2006). Research in yeast indicates that hor- mesis may be a
common phenomenon. For example, a study of >2000 potential
anti-cancer drugs established that many displayed a hormetic
effect (Calabrese et al., 2008) and recently it was pro- posed that
hormesis effects of hydrogen peroxide could influence cellular
aging in yeast (Mesquita et al., 2010).
Many soil microbes are prolific producers of secondary metab-
l isolates, no clues to intracellular targets were revealed by those
agents to control phytopathogenic fungi (Bloemberg and
Lugten- berg, 2001; Haas and Defago, 2005; Morrissey et al.,
2004; Raaij- makers et al., 2002). Pseudomonas species have
attracted a lot of research as particular strains produce
metabolites, such as phena- zines, cyclic lipopeptides and
polyketides that have antifungal activity in the laboratory and
potential applications in biotechnol- ogy. One secondary
metabolite of particular interest is 2,4-diac- etylphloroglucinol
(DAPG), which is produced mainly by the rhizosphere-associated
bacterium Pseudomonas fluorescens (Ban- gera and Thomashow,
1996; Shanahan et al., 1993). This phenolic metabolite is
synthesised by the activities of the PhlD polyketide synthase and
PhlACB acetlytransferase enzymes (Achkar et al., 2005) and
possesses broad spectrum growth inhibitory activity to- wards
phytopathogenic fungi (Keel et al., 1996; Kwak et al., 2009; Laville
et al., 1992; Mazzola et al., 1995; Vincent et al., 1991), bacteria
(Cronin et al., 1997b; Keel et al., 1992), oomycetes (de Souza et
al., 2003; Fenton et al., 1992), and nematodes (Cronin et al.,
1997a). Because of this broad spectrum activity against plant
patho- gens, many studies have sought to exploit the natural
plant growth/ health promoting activity of P. fluorescens to
develop biocontrol inoculants (reviewed in: (Haas and Defago,
2005; Morrissey et al., 2002; Weller et al., 2007)). Interestingly, a
number of studies have reported that DAPG can also trigger a
global defence response in plants known as induced systemic
resistance (ISR) through a mech- anism yet to be determined
(Iavicoli et al., 2003; Siddiqui and Shau- kat, 2004; Verhagen et
al., 2010; Weller et al., 2007).
The diverse effects of DAPG on microbes and plants raise
inter- esting questions as to its targets, mode-of-action and
ecological function. At one level, it can be considered a classical
antimicrobial, whereas the finding that most phlD+ strains
produce only low lev- els of DAPG, and the intriguing effects on
plants, may be more con- sistent with a signalling role. Some
studies have addressed the question of molecular effects of DAPG
on susceptible organisms. Exposure of the oomycete Pythium
ultimum to DAPG led to inhibi- tion of hyphal growth and
zoospore motility as well as structural changes to the cell
including alteration of the plasma membrane, vacuolization and
cell content disintegration (de Souza et al., 2003). A study of a
library of Saccharomyces cerevisiae (yeast) mu- tants identified
multiple mutants in different cellular processes that were more
sensitive to DAPG than the wild-type strain (Kwak et al., 2010).
These data support the idea that DAPG may affect fundamental
cellular processes, a suggestion also made in other stud- ies
(Jousset, 2006). Direct studies of physiological functions in yeast
demonstrated that DAPG treatment led to loss of mitochon- drial
function, indicating that DAPG may act to depolarise mito-
chondrial membranes (Gleeson et al., 2010). Studying defence
mechanisms can sometimes yield insights into the targets of an
antimicrobial compound but although studies of the fungi
Botrytis cinerea and Fusarium oxysporum implicated degradative
enzymes and ABC transporters in tolerance in some natural
olites, with species within the genera Streptomyces, Bacillus and
Pseudomonas best known in this regard (Challis and Hopwood,
2003; Gross and Loper, 2009; Raaijmakers et al., 2010; Stein,
2005). Whereas the anthropocentric focus with Streptomycetes
has been on the production of antibiotics with clinical potential,
many studies have sought to exploit the intrinsic activities of
specific Bacillus and Pseudomonas strains to develop bacterial
inoculants that can be used in agriculture as natural biocontro
studies (Schouten et al., 2008, 2004).
In this study, Neurospora crassa was selected as a model to
as- sess the effects of DAPG on a filamentous fungus. N. crassa
was the original fungal genetic model and in recent years has
been extensively used as a model organism for fungi and
eukaryotes (Davis and Perkins, 2002). Many cell biology,
microscopy and ge- netic tools are available with N. crassa and
this fungus has proved useful to study the effects of small
molecules such as antifungal peptides and proteins (Binder et al.,
2010; Munoz et al., 2013, 2012; Spelbrink et al., 2004; Thevissen
et al., 1999), azoles (Selker, 1998), chitosan (Palma-Guerrero et
al., 2009, 2010), and other antifungal drugs or metabolites
(Ermolayeva and Sanders, 1995; Pere- ira and Said, 2009; Selker,
1998). In this study, we investigated the effects of DAPG on N.
crassa to determine what processes and func- tions were
impaired or affected by this bacterial metabolite.
2. Materials and methods
2.1. Strains, plasmids and culture conditions
All N. crassa strains used in this study are listed in Table 1.
To measure cytosolic free Ca2+ ([Ca2+]c) levels, either wild type (wt)
containing the pAZ6 aeqS expression vector or mutant strains
con- taining the pAB19 aeqS expression vector were used (Binder
et al., 2010). All strains were cultured and maintained on Vogels
med- ium containing 2% sucrose (Davis, 2000). To select for
mutant strains, medium was supplemented with hygromycin. To
select for aeqS expressing strains, medium was supplemented
with hygromycin for the wild type or ignite (glufosinate) for
mutant strains, respectively. Agar slants and plates were
incubated at either 25 C or 35 C with or without light (as
indicated). To promote conidiation, plates and slants were
incubated at room tem- perature with continuous light.
2.2. Chemicals and dyes
DAPG was synthesised in-house as previously described and
stored as a powder (Shanahan et al., 1993). A stock of 10 mg
DAPG ml-1 in methanol was stored at -20 C. 1,2-Bis(o-amino-
phenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) tetrapotassium
salt (Sigma) was dissolved in distilled water, antimycin A (Cam-
bridge Biosciences) stock solution was dissolved in 96% ethanol
and carbonylcyanide-3-chlorophenylhydrazone (CCCP) (Sigma)
stock solution was dissolved in DMSO. Methylsalicylate and coel-
enterazine stock solutions were dissolved in methanol.
Fluorescent dyes used in this study are listed in Table 2.
2.3. Colony extension assay
The measurement of the radial extension of a colony was
done as previously described (Berepiki et al., 2010), with slight
modifica- tion. A conidial suspension was prepared in sterile
distilled water and filtered, and a 10 pl conidial suspension was
placed onto the centre of an agar plate containing no
 D.M. Troppens et al./Fungal Genetics and Biology 56 (2013) 135-146
supplementation, DAPG at concentrations ranging from 2.5 to 15
pgml-1, or 2% methanol (the DAPG solvent). Four radii were
randomly marked on the underside of each plate. The plates were
then incubated at 35 C in the dark and colony extension was
measured after 28 h of growth. Colony extension was expressed
as percentage of average radius on treatment plates (methanol or
DAPG) compared to control plates (no supplement). The
experiment was done at least in duplicate per concentration and
strain.
2.4. Germination and CAT fusion assay
A conidial suspension was prepared in Vogels medium from 4 to
5-day old cultures incubated at 35 C with light. Filtered conidia
e
3
were counted using a haemocytometer and diluted to 5 x 105-
conidia/ml in Vogels medium which was transferred to an 8-well
culture chamber (Nalg Nunc, Rochester, NY) with 200 pl per well.
DAPG (dissolved in methanol) at the appropriate final concentra-
tion, or equal volumes of methanol or Vogels medium, was added
to the wells. Chambers were transferred to 25 C in the dark and
incubated for 6 h. At least 10 random areas containing conidia
and/or germlings were viewed per treatment using a 60 x
objective on an inverted Eclipse TE2000E microscope with a
DXM1200F camera and ACT-1 image capture software (Nikon,
Kingston- Upon-Thames, Surrey, UK). Germination was expressed
as the per- centage of germinated conidia possessing germ tubes
and/or conidial anastomosis tubes (CATs) compared to the total
amount of conidia/germlings (n > 100). CAT fusion was expressed
as th
4 D.M. Troppens et al./Fungal Genetics and Biology 56 (2013) 135-146

Table 1
Strains used in this study.

Strain FGSC number Mating type Genotype Source

Table 2

a PI Sigma H O 1.5 mM 7.5 nM 550 625

Final Solutions diluted in Vogel's liquid mdium. b2Values in brackets are for confocal microscopy.

percentage of conidia or germlings involved in fusion events com-

pared to the total number of conidia/germlings (n > 100) (Roca et
al., 2005). This experiment was done in duplicate.

2.5. Measurement of intracellular Ca2+

A conidial suspension was prepared in Vogels medium from a

7-day old culture of wild-type or mutants strains (Dcch-1, Dfig-1,
Dyvc-1) expressing aequorin were incubated at 35 C with contin-
uous light. A conidial suspension was diluted to 10 6 conidia/ml
in Vogels liquid medium and coelenterazine was added to a final
concentration of 5 pM. 100 pl of conidial suspension was then
transferred to 6 wells of a 96-well microtitre plate per treatment.
The 96-well plates were incubated at 25 C in the dark for 6 h.
Each plate was then gently loaded into a LB96P Microlumat
lumino- meter or a Microlumat Plus LB96V plate luminometer
(Berthold Technology, Bad Wilbad, Germany). To measure
changes in cyto- solic free Ca2+ ([Ca2+]c), 100 pl DAPG at 2x the
final concentration ranging from 15 to 120 pgmr1 was
automatically injected into the wells (6 wells per treatment) and
luminescence was measured for a maximum of 11 min. To
measure the total luminescence of aequorin expressed by cells in
each well, 100 pl of 20% (vol/vol) ethanol containing 3 M CaCl 2
was injected into separate wells to allow total discharge of
aequorin by permeabilisation of the cells. To chelate extracellular
Ca2+, 100 pl BAPTA was added to one control row of wells 10 min
prior to DAPG injection at a final concentration of 7.5 mM. In that
case the injected DAPG was 3x final concentration. To monitor
luminescence and control injections of the plate reader, the PC
running Microsoft Windows based Bert- hold WinGlow software
was used. To convert luminescence values to [Ca2+]c
concentrations, the TermBert software developed by Zelter et al.
(2004) was used. Amplitudes of highest concentrations of DAPG
were tested for statistical difference by performing a t-test (n = 4)
using SigmaStat 3.5.

2.6. Fluorescence microscopy

 D.M. Troppens et al./Fungal Genetics and Biology 56 (2013) 135-146 5

A conidial suspension was prepared in Vogels medium from a 3
to 4-day old culture incubated at 35 C in the light. The conidial
suspension was diluted to 5 x 105 conidia/ml in Vogels medium
and transferred to an 8-well slide culture chamber (Nalg Nunc)
with 180 pl per well and grown for 5 h at 25 C. The slide culture
chamber was then transferred to a Nikon Eclipse TE 2000E
inverted microscope and a fluorescent dye (DASPMI, rhodamine
123, or rho- damine B; Table 2) added to enable imaging of
germlings. Where required, chemical treatments were added at an
appropriate con- centration to bring the final volume in the wells
to 200 pl and the sample was immediately viewed by fluorescence
microscopy. Images of the same region were captured every
minute for a max- imum of 10 min (following DAPG or CCCP
treatment) and 20 min (following antimycin A treatment),
respectively, using a 100x objective. The final concentrations of
DAPG used ranged from 15 to 60 pgmr1, CCCP was added at a
final concentration of 2 pM and antimycin A was added at a final
concentration of 0.5 pg ml-1. Equivalent volumes of methanol or
Vogels medium were added as controls. For pre-treatment with
BAPTA, the conidial suspension was diluted to 106 conidia/ml
with 80 pl added to each well and grown for 5 h. 100 pl of BAPTA
s
(final concentration of 7.5 mM) was then added and cells
incubated for 10 min before addition of dye and DAPG. To assess
the effects of a longer exposure to DAPG, conidia were incubated
for 3 h at 25 C, DAPG was added at a final concentration of 60
pg ml-1 and the conidia were incubated for a further 2 h before
the addition of dye and imaging as described above. An Eclipse
TE 2000E inverted microscope (Nikon) was used for widefield light
microscopy. Fluorescence images were captured with an EM-CCD
Orca camera (Hamamatsu) and Image Pro and MetaMorph
software (Molecular Devices, Sunnydale, CA), respec- tively, and
differential interference contrast (DIC) images were captured with
a DXM1200F camera and ACT-1 image capture software (Nikon).
For confocal microscopy, an inverted TE 2000U Eclipse
microscope (Nikon) coupled to a Radiance 2100 confocal system
with Lasersharp 2000 software v. 5.1 (Bio-Rad Micro- science,
Hemel Hempsted, UK) were used. An H1-GFP expressing strain
(Table 1), in which nuclei were labelled with GFP, was trea- ted
with DAPG for 2 h as described above and stained with rhoda-
mine B. Final fluorescent dye concentrations and the microscope
settings used for fluorescent dyes and GFP are listed in Table 2.
To quantify permeabilisation by DAPG, a conidial suspension wa

stained with propidium iodide (PI) and fluorescein diacetate (FDA), treated with 15 or 60 pg DAPG mi -1 and imaged immediately. As
a control 70% ethanol was added. The percentage of permeabilised conidia was calculated (n > 100).

3. Results

3.1. DAPG inhibits colony growth and germination and CAT fusion

To determine the effects of the bacterial metabolite DAPG on the fungus N. crassa, assays were carried out to assess colony
extension rates, conidial germination and CAT fusion in the pres- ence of increasing concentrations of DAPG (Fig. 1). Colony extension
on agar plates containing Vogels medium was inhibited by levels as low as 2.5 pg DAPG ml -1 (65% reduction in colony diam- eter), with
a progressively greater inhibition as the DAPG concentraron increased (Fig. 1A). Treatment of germinating conidia with DAPG also
resulted in dose-dependent inhibition of conidial germi- nation and CAT fusion, whereas the solvent control (2% methanol) had no
effect (Fig. 1B and C). For example, at a concentration of7.5 pg DAPG ml -1, 68% of conidia germinated and 42% of these were involved
in one or more CAT fusion events, compared to 95% and 56%, respectively for the untreated control. From these data it was possible to
calculate an IC50 of ~9 pg DAPG ml -1 for both conidial germination and CAT fusion, which is comparable to the concentrations of DAPG
that inhibit other fungi (Kwak et al., 2009; Schouten et al., 2008, 2004). Given that some strains of P. fluorescens can generate
concentrations as high as 50100 pg DAPG ml-1 in culture (Delany et al., 2000), it is also a bio- logically relevant concentration.

3.2. DAPG treatment disrupts mitochondrial morphology

It has recently been shown that mitochondria are a target of DAPG toxicity in S. cerevisiae (Gleeson et al., 2010) and therefore the
effects of DAPG on mitochondria in N. crassa were investigated. To visualise changes in mitochondrial morphology, the fluorescent
styryl dye DASPMI, which preferentially stains mitochondria (Hickey et al., 2005), was added to N. crassa germlings, which were then
visualised using epifluorescence microscopy. In germlings
AB

.
G
e
r
<f m
in
100
40 at
o io
30
90 n
a) C
20
80 germination and CAT fusion by DAPG in the wild type. (A) Colony extension on agar plates
Fig. 1. Inhibition of colony extension rates, conidial A is normal on Vogels (-DAPG) and
T
70
reduced in the presence ofDAPG. No effect on extension
10 rate was observed in the presence of the solvent control (2% methanol). (B) Conidia
fu germinate and undergo cell fusion in
60
Vogels (-DAPG) but fail to do so when these processes are inhibited when 15 pg ml -1 DAPG is added (+DAPG). Scale bar: 5 pm. (C) Both germination
si and CAT fusion are inhibited
by DAPG in a dose-dependent manner. 2% CO 050 was 0used
O) Methanol in the7.5
MetOH treatment/DAPG
solvent control. [pg/ml]
9 11.4 13.2 15 on
6 D.M. Troppens et al./Fungal Genetics and Biology 56 (2013) 135-146

treated with this dye, mitochondria were either visible as tubular or

J
near spherical structures, with the tubular mitochondrial net-

X'/ _
works more evident at growing hyphal tips (Fig. 2A). When treated
with DAPG, a rapid and dramatic change was seen, with the fluo-
rescence signal becoming dispersed within minutes and some
apparent staining of membranes around large, spherical
structures (Fig. 2A). To ensure that the effects on mitochondrial

M
%
structure and the formation of these spherical structures were
not specific to the DASPMI dye, and to establish whether these
spherical bodies were nuclei, a second dye that stains

y
mitochondria, rhodamine B (Reu- ngpatthanaphong et al., 2003), + DAPG (rho B)
was used in a strain expressing his- tone H1-GFP to label its
nuclei (Freitag et al., 2004). Interestingly, mitochondria stained
with rhodamine B were all near spherical rather than tubular '"'tV -V
indicating that the dye exhibits cytotoxic ef- fects on
mitochondrial morphology. It was seen again, however, that DAPG + DAPG (DASPMI)
treatment led to a profound change in mitochondrial appearance,
with most fluorescence becoming associated with bright dot-like
structures, possibly representing fragmented mitochondria (Fig.
2B). The large spherical structures visible following DASPMI
+ DAPG (GFP)
treatment were also seen after rhodamine B treatment, indicating
that they are not an artefact associated with one specific dye.
Labelling with H1-GFP demonstrated that the large spherical
bodies were nuclei (compare Fig. 2A with Fig. 2B, lower panel).
The visibility of nuclei following treatment with DAPG was a result
- t

%
of some staining of nuclear membranes by DASPMI and
rhodamine B (Fig. 2A and B). When H1-GFP labelled cells were
stained with

- DAPG (DASPMI) - DAPG (rho B)

 D.M. Troppens et al./Fungal Genetics and Biology 56 (2013) 135-146 7

Fig. 2. Effects of DAPG on mitochondrial morphology in conidial germlings of the
wild type imaged by widefield fluorescence microscopy. (A) Germlings with
spherical and tubular mitochondria stained with DASPMI in Vogel's (-DAPG) and
9 min after the addition of DAPG (+DAPG). (B) Germlings of H1-GFP stained with
rhodamine B in Vogel's show mainly spherical mitochondria (-DAPG). In the
presence of DAPG (+DAPG) (2 h exposure) spherical structures are visible, which
match the GFP-labelled nuclei (arrows). The arrow in the lower pane of panel A
indicates an example of the large, spherical structures seen after DAPG
treatment. rho B = rhodamine B Scale bar: 5 im.
rhodamine B in the absence of DAPG, the appearance of nuclei was
similar to that after DAPG treatment indicating that there is no
evidence for an effect of DAPG on nuclear morphology (Fig. S1).
causes a large increase in [Ca2+]c immediately following injection,
but [Ca2+]c quickly re- turns to its resting level, This is manifest
as a highly reproducible Ca2+ signature and has been previously
described (Bencina et al., 2005; Binder et al., 2010; Nelson et al.,
2004). When DAPG was injected, an additional and longer
transient increase in [Ca2+]c was observed following the first
[Ca2+]c transient in response to mechanical perturbation (Fig. 4A).
The amplitude of this secondary [Ca2+]c transient was dose-
dependent and peaked approximately 2 min after addition of
DAPG before slowly returning the original [Ca 2+]c resting level
after ~10 min. This effect appears to be quite specific to DAPG
and a structurally similar molecule, 6-methylsa-

3.3. DAPG treatment causes the loss of mitochondrial

membrane potential

To assess whether the DAPG-induced alterations in mitochon-

drial structure seen with DASPMI and rhodamine B were associ-
ated with a loss in mitochondrial membrane potential, a time
course experiment was performed using confocal microscopy. Be-
cause the potentiometric dye rhodamine B appeared to have some
cytotoxic effects on mitochondria, an alternative potentiometric
dye, rhodamine 123 (Hickey et al., 2005), was used. This dye,
which was rapidly taken up by cells and fluoresces only in
normally charged mitochondrial membranes, is commonly used
to measure mitochondrial membrane potential. When
mitochondria in un- treated conidial germlings were visualised
with rhodamine 123, they appeared spherical or tubular,
consistent with them being functional mitochondria (Fig. 3A).
When treated with 60 ig DAPG ml-1 the intensity of mitochondrial
fluorescence decreased and disappeared completely within 10
min (Fig. 3B), whereas no effect on mitochondrial fluorescence
intensity was observed in the control which was only treated with
Vogels liquid medium. Similar effects were observed using lower
concentrations of 15 ig DAPG ml-1 (data not shown). To further
investigate these findings, two known inhibitors of mitochondrial
function, antimy- cin A and CCCP, were used to treat conidial
germlings in the same manner. Antimycin A binds specifically to
the Complex III of the respiratory chain and inhibits electron
transfer, whereas CCCP is an uncoupler of oxidative
phosphorylation and ATP synthesis. When antimycin A was
added, a loss of mitochondrial fluorescence intensity was also
observed but this was less pronounced and slower than DAPG
treatment (data not shown). In contrast, the effects of CCCP were
very similar to those of DAPG with a rapid decrease of
mitochondrial fluorescence (Fig. 3C). These data are consistent
with DAPG acting as a chemiosmotic uncoupler and dis- rupting
mitochondrial membrane potential in a manner analogous to
CCCP. It is also possible that the loss of mitochondrial membrane
potential is a consequence of the effects of DAPG on
mitochondrial structure.

3.4. DAPG affects intracellular Ca2+ homeostasis

Several previous studies have suggested that DAPG may affect

multiple cellular processes and given the central importance of
Ca2+ homeostasis in filamentous fungi (Binder et al., 2010;
Nelson et al., 2004), it was decided to determine whether DAPG
treatment had any effects on Ca 2+ levels in conidial germlings.
Cytosolic free Ca2+ ([Ca2+]c) concentrations can be routinely
measured in conidial germlings of N. crassa germlings using the
heterologously-ex- pressed, bioluminescent Ca2+-reporter
aequorin (Binder et al., 2010). In this assay, test compounds in
Vogels liquid medium are injected into the wells of a 96-well plate
containing conidial germlings. This mechanical perturbation
8 D.M. Troppens et al./Fungal Genetics and Biology 56 (2013) 135-146

before

1 min

10 min

V
*V
'K

Control DAPG
CCCP
Fig. 3. DAPG-induced loss of mitochondrial membrane potential in conidial germlings. Time course experiment using confocal microscopy to show brightfield images (top) and
fluorescence images (below) before, and 1 min and 10 min after treatment. (A) Treatment with liquid Vogel's medium has no effect on mitochondrial fluorescence intensity. (B)
Treatment with DAPG leads to rapid loss of mitochondrial fluorescence within 10 min similar to (C) treatment with CCCP, an uncoupler of ATP synthesis, scale bar: 5 pm.

licylate did not induce this secondary increase in [Ca 2+]c at equiv-
alent concentrations (Fig. 4B). To establish whether the increase
in [Ca2+]c was a consequence of Ca 2+ uptake from outside the cell
or Ca2+ release from intracellular stores, the Ca 2+ chelator BAPTA
was added prior to treatment with DAPG (Fig. 4C). In this case,
both the [Ca2+]c increase caused by mechanical perturbation and
that caused by DAPG were abolished, indicating that the DAPG-
induced [Ca2+]c transient requires an import of Ca2+ from the
external medium. To further explore this, the DAPG-induced
[Ca2+]c transient was assessed in deletion mutants of three genes
that encode the following Ca 2+ channel proteins: CCH-1, which is
a high affinity cytoplasmic membrane channel; FIG-1, which is a
low affinity cytoplasmic membrane channel, and YVC-1, which is
a vacuolar Ca2+ channel (Bencina et al., 2009; Zelter et al., 2004).
The mutants lacking CCH-1 or FIG-1 still exhibited the DAPG-
induced [Ca2+]c transient, although some alteration in the Ca 2+
signature was seen in both mutants (Fig. 4D and F). Deletion of
the yvc-1 gene had no discernible effect on the [Ca 2+]c transient
confirming that this channel plays no role in releasing Ca 2+ from
internal Ca2+ stores (Fig. 4E). Thus, although CCH-1 and FIG-1
may play some role in the import of Ca2+, neither of these
proteins is solely involved in the secondary [Ca2+]c transient seen
following DAPG treatment.
 D.M. Troppens et al./Fungal Genetics and Biology 56 (2013) 135-146
To assess whether these Ca2+ channel proteins play a role in
DAPG toxicity, the sensitivity of the Ca 2+ channel protein mutants
to DAPG was compared with the wild-type. In addition, a mutant
lacking another putative Ca2+ channel protein, namely MID-1,
was tested for its DAPG sensitivity. When mutants were grown on
Vogels agar medium for 28 h in the presence of different con-
centrations of DAPG, no significant increase in sensitivity or toler-
ance to this compound was observed (Fig. 5). However, when
themutants were grown for longer (48 h) in the presence of the
high- est concentration (15 pgmr1) of DAPG used in this
experiment, increased sensitivity of all of the mutants was noted
with Dmid- 1 being the most sensitive (Fig. 6). Nevertheless, a link
between the DAPG-induced [Ca2+]c transient and DAPG toxicity
was not found: when cells were pre-treated with BAPTA to abolish
the [Ca2+]c transient (Fig. 4C), this did not prevent the cells from
losing their rhodamine 123 fluorescence after DAPG was added
(Fig. S2).
Given the effects that DAPG has on mitochondrial
membranes, whether the DAPG-induced [Ca2+]c transient could
9
be a conse- quence of plasma membrane permeabilisation by
DAPG, was determined. Conidia were treated with DAPG or
ethanol in the presence of the live/dead cell dyes propidium
iodide (PI) and fluo- rescein diacetate (FDA) (Hickey et al., 2005).
Whereas ethanol caused 100% of cells to be permeabilised, less
than 3% of cells were permeabilised by DAPG treatment (Fig. S3),
indicating that the increase in [Ca2+]c is unlikely to be the result
of membrane perme- abilisation. The ~2 min lag between
injection of DAPG (when the secondary transient [Ca 2+]c increase
was observed) and the subse- quent recovery of the [Ca 2+]c to its
resting level is further evidence that the plasma membrane is not
compromised by DAPG treatment.
4. Discussion
N. crassa displays a comparable level of sensitivity to DAPG as
various phytopathogenic fungi and budding yeast (Gleeson et al.,
2010; Kwak et al., 2009; Schouten et al., 2008, 2004), confirming
that it is an appropriate model to assess DAPG mode-of-action
.

4. The effects of DAPG on [Ca2+]c in conidial germlings. (A) A secondary [Ca2+]c transient induced by DAPG is dose-dependent in the wild type. (B) The secondary [Ca 2+]c transient is specific to DAPG because it
is not induced by the structurally similar molecule, 6-methylsalicylate. (C) Treatment with the Ca 2+ chelator, BAPTA, demonstrates that the DAPG-induced increase in [Ca 2+]c originates from the external
medium. Lacle of Ca2+ channels. (D and F) The DAPG-induced, transient increases are dose-dependent in the cch-1, yvc-1 and fig-1 mutants, respectively, but their Ca2+ signatures differ slightly from those in
the wild type (compare with Fig. 4A).

e capacity, in a genetically tractable fungus with a large suite of cell biology and determinethe specific effects of DAPG on conidial germlings of N. crassa. We show
genetic tools, to measure impacts on fungal cell growth, development and that DAPG inhibits fungal growth by impairing mito- chondrial function. DAPG
physiology makes N. crassa an excellent experimental system for this purpose. In was also found to transiently elevate [Ca2+]c but no evidence linking this
this study, various phys- iological probes and mutant strains were employed to increased [Ca2+]c with DAPG toxicity was obtained.
 response to DAPG indicating that this is likely to be a primary sub- cellular
target of the metabolite. The reduction in fluorescence de- tected with rhodamine
123 is consistent with a loss of mitochondrial membrane potential, similar to
that seen with agents that uncouple oxidative phosphorylation from ATP genera-
tion by dissipating the proton gradient (e.g. CCCP and 2,4 dinitro- phenol).
Indeed, it is notable, that DAPG shares structural and chemical properties, for
130 example, a phenolic moiety and lipophilic nature,
wt with known uncouplers (Draber et al., 1972;
120
A fig-1 Heytler, 1979; Tollenaere, 1973). Previously, a loss
110 A yvc-1 of mitochondrial membrane potential was
100 A cch-1
observed with the carbocyanine dye DiOCl6(3) in
the yeast S. cerevisiae (Gleeson et al., 2010) and
90 A mid-1
effects on other fungi and oomycetes would also
80 be consistent with the uncoupling of oxidative
70 phosphorylation and ATP synthesis (de Souza et
al., 2003; Schouten et al., 2008, 2004). Recent
60
biochemical studies in yeast found that the effects
50 of CCCP and DAPG on respiratory function were
40 similar, providing further support for the
30 suggestion that DAPG has proton ionophoric
activity (Troppens et al., 2013). The capacity to act
20 as an uncoupling agent also offers an explanation
10 0 for why DAPG inhibits the growth of diverse

5 10 15
DAPG [pg/mi]

Fig. 5. No inhibition of colony extensin in the wild type and Ca 2+ channel protein mutant strains over 28 h. The lack of the Ca 2+ channel proteins FIG-1, YVC-1, CCH-1 and MID-
1 in each the deletion mutants does not change the sensitivity to DAPG when grown in the presence of different concentrations of DAPG and incubated for 28 h.

wt control 15 |jg/ml DAPG

organisms that indude bacteria, fungi, oomycetes and helminths, as the
requirement to maintain electrochemical gradients to generate energy is uni-
versal. The precise means by which uncoupling occurs has not been established,
4.1. D though the structural similarity to dinitrophenol may suggest a mechanism of H+
APG inhibits
growth by transport across the inner mitochon- drial membrane. In addition to affecting
membrane potential, the structure and possibly integrity of the mitochondria
were altered by DAPG. Several studies in N. crassa have found a link between
mitochondrial morphology, cytoskeletal elements and growth (Prokisch et al.,
2000). For example ropy mutants which are defective in the dynein-dynactin
A fig-1
motor protein complex, exhibited altered microtubule organisation and have dot-
impairing like mitochondria in N. crassa (Minke et al., 1999), not dissimilar to those
observed in this study. In both S. cerevisiae and Schizosaccharomyces pombe the
requirement of specific outer membrane proteins for normal mitochondrial
morphology has been demonstrated (Burgess et al., 1994; Fritz et al., 2001). A
significant mitochondrial membrane potential is required for protein transfer into
A yvc-1 mitochondria (Schleyer et al., 1982) and it is possible that disrupting this mem-
mitochondrial brane potential triggers the morphological changes that are ob- served following
function DAPG treatment

Using three different

mitochondrial
Acch-1
probes,
detrimental
effects of DAPG
on mitochondrial
function were
A mid-1 apparent, with
the rapid
. In this study, it was found that, in response to DAPG, there is a transient
increase in the average [Ca2+]c in a population of conidial germlings of N. crassa.
This Ca2+ originates from outside the cell and as no significant membrane
Fig. 6. Inhibition of growth and signs of stress in colonies of the wild type and Ca 2+
channel protein mutant strains when grown for 48 h. Cultures were incubated for 48
h in the absence and in the presence of 15 pgmr 1 DAPG and visualised by
photography.
3contributes to a general stress-response that helps the cell cope with the
effects of DAPG. Although DAPG did not specifically affect the growth of the
4.2. 4.2. DAPG induces a transient [Ca2 3+]c increase Dcch-1, Dmid-1 or Dfig-1 mutants, it is notable that after 48 h of incubation in its
pres- ence, the mutant colonies as well as those of the wild-type were showing
signs of stress (Fig 6). In S. cerevisiae, Ca2+ is a well known second messenger
2The increase in [Ca +] 2
c may act as an intracellular signal that involved in mediating stress responses and it has been shown in fungal cells that
permeabilisation occurs, it must enter the cell via a channel or transporter of
some kind. Although Ca2+ signalling and homeostasis are of fundamental
importance for many processes, Ca2+ uptake in fungi is little understood. cch- 1
Encodes a protein that is clearly recognisably a plasma mem- brane Ca 2+
channel, and many studies have demonstrated that Cch1p in the budding yeast
is a high affinity Ca2+ channel (HACS) that responds to stress, including ER
stress, and other stimuli (Courchesne et al., 2011; Fischer et al., 1997; Hong et
al., 2010; Liu et al., 2006). In addition, mid-1 encodes a protein that appears to
be sometimes associated with Cch1p (Hong et al., 2013) but that may also be
independently regulated - for example, it is some- times described as being part
of a mechanosensitive channel (Lew et al., 2008; Peiter et al., 2005). A third gene,
fig-1, encodes a protein that is involved in Ca 2+ uptake during mating (Muller et
al., 2003; Yang et al., 2011). Neither Mid1p nor Fig1p display any of the typical
features associated with Ca2+ channels and it is not known whether they are
themselves transporters or regulators of true channel proteins. In S. cerevisiae,
there are additional com- ponents of this Cch1p high affinity channel (Martin et
al., 2011), and one recent study suggests that at least one additional Ca 2+-
channel exists in that yeast (Groppi et al., 2011). Homologues of budding yeast
CCH1, MID1 and FIG1 are found in filamentous fungi (Bencina et al., 2009; Zelter
et al., 2004) and mutants defective in these proteins were used in this study to
try to ascertain the signif- icance of changes in [Ca 2+]c levels for DAPG toxicity in
N. crassa, and specifically, whether the increase in [Ca2+]c levels exacerbated the
effects of DAPG. The Dcch-1, Dmid-1 or Dfig-1 mutants all dis- played increased
sensitivity to DAPG in terms of growth inhibition suggesting that, at least
individually, they are not involved in DAPG toxicity. Furthermore, none of the
Ca2+ channel mutants showed a markedly different Ca 2+ signature in response to
DAPG treatment. In addition, preventing Ca 2+ uptake by chelating extracellular
Ca2+ (BAPTA addition) did not have any impact on the kinetics of mitochondrial
impairment. It was interesting that the increase in [Ca 2+]c still occurred in the
Dcch-1 and Dfig-1 mutants, indicating that a different system involved in Ca2+
uptake from the external medium is induced by DAPG. Difficulties in generating
a Dmid-1 strain expressing the aequorin reporter (Chu & Read, unpublished
data) meant that this mutant could not be tested in the present study but given
that budding yeast Mid1p commonly works with Cch1p, the most likely
explanation would seem to be that an as- yet undescribed channel or transporter
mediates the DAPG-in- duced Ca2+ import across the plasma membrane. In
summary, our results provide no clear link between DAPG-induced [Ca 2+]c in-
crease and DAPG toxicity although the growth of the Ca 2+ channel mutants was
more sensitive to DAPG than the wild type.
The DAPG-induced [Ca2+]c transient is not involved in DAPG toxicity and
understanding its significance will require further work. There are three possible
roles that this transient [Ca2+]c increase may serve:
endoplasmic reticulum (ER) stress activates the Cch1p- Mid1p channel
complex that acts to restore ER Ca 2+ levels (Hong et al., 2010, 2013). The
best-known mode-of-action of Ca2+ involves its binding to calmodulin,
which then inter- acts with the protein phosphatase calcineurin leading to
activation of various target proteins, including transcription factors (Cyert,
2003; Kraus and Heitman, 2003).
(2) The [Ca2+]c transient may be a consequence of the effects of DAPG on
mitochondria. Although there are no data from fungi, one study with sea
urchin sperm found that impairing mitochondrial function led to an
uptake of extracellular Ca2+ in a mechanism that commenced with
mitochondrial Ca2+ release (Ardon et al., 2009). This is a possibility that
should be explored but the differences between the Ca 2+ signal-
ling/homeostatic machinery of animal and fungal cells (Bor- kovich et al.,
2004; Galagan et al., 2003), and the rather unique cell-type used in that
study (the sea urchin sperm cell) advise caution in extrapolating between
animal and fungal systems.
(3) The transient [Ca2+]c increase could be a distinct response induced by
DAPG via an unknown mechanism. This option is supported by the
observation that the Cch1p channel is not required for Ca 2+ uptake in the
response of N. crassa to DAPG (Fig. 4).
Whichever of these three possible roles that the DAPG-induced [Ca 2+]c has,
this still leaves the questions of how the DAPG activates the channel(s)
responsible for Ca2+ uptake into germlings, what channel(s) is/are used to take
up extracellular Ca2+, and what are the downstream consequences of the
increased [Ca2+]c?
In fungi, multiple distinct stimuli employ Ca2+ as a second mes- senger (Aiello
et al., 2002; Araki et al., 2009; Binder et al., 2010; Greene et al., 2002; Kallies et
al., 1998; Moser et al., 1996; Viladev- all et al., 2004) so understanding how
particular responses are reg- ulated by changes in [Ca 2+] is a great challenge.
Some insights may come from plant and animal biology where the concept of
Ca2+ signatures has been developed further (Cheung et al., 2011; McAinsh and
Pittman, 2009; Webb et al., 1996). Depending on the spatial and temporal
dynamics of the intracellular Ca 2+ signal, different phenotypic outcomes are
possible. It seems likely that Ca 2+ signatures encoding information that can be
decoded by the signal-response machinery will also feature in fungi. In this
regard, Ca2+ pulses with species and age specific signatures were recently
observed at the subcellular level in filamentous fungi using the genetically
encoded fluorescent Ca2+ reporter cameleon (Kim et al., 2012). However, the
information (if any) encoded in these signatures was not determined. In relation
to the Ca2+ signature observed by aequorin luminescence measurements of cell
popula- tions in the present study, the kinetics of the transient [Ca 2+]c increase
that is induced by DAPG in N. crassa is interesting as the slow return its resting
level is not typical of what has previously been observed in this fungus (Binder et
al., 2010).
4.3. Implications for understanding the ecological role ofDAPG
This study identified two, possibly unlinked, effects of DAPG on fungi:
antimicrobial activity by inhibition of mitochondrial func- tion and stimulation of
intracellular Ca2+ uptake, thus affecting Ca2+ homeostasis. The mitochondrial
impairment conforms to the traditional view of DAPG as an antibiotic produced
by P. fluorescens as a defence against protozoan predation or to antagonise other
microbes and thereby gain a competitive advantage. In this re- spect, DAPG
would be broadly similar to a number of other micro- bial metabolites that impair
mitochondrial function. For example, the F(1)F(0) ATP synthase (inhibited by
oligomycin), complex III (inhibited by antimycin A, ilicicolin H, funiculosin and
strobilurin) and complex II (inhibited by atpenins) are all specifically impaired by
bacterial and fungal metabolites (Bartlett et al., 2002; Gutierrez- Cirlos et al.,
2004; Miyadera et al., 2003; Slater, 1973; vonJagow and Link, 1986). It is far
from certain, however, that DAPG is pro- duced at sufficient levels in the
rhizosphere to mediate significant antibiotic effects, and, in fact, the pattern of
DAPG production is more reminiscent of a signal than an antibiotic, as DAPG
levels rise sharply in late exponential phase and then rapidly decline as the
molecule is turned over (Bottiglieri and Keel, 2006; Delany et al., 2000). The
possibility that DAPG serves as a signal is intriguing and in that regard, the
finding that DAPG affects Ca 2+ homeostasis in a eukaryotic (fungal) cell is very
interesting because of the known importance of the Ca2+ second messenger in
plant-microbe interactions. Both the rhizobium-legume symbiosis and the sym-
biosis between mycorrhizal fungi and diverse plants involves microbial signal-
induced Ca2+oscillations in the plant cell (Downie, 2010; Navazio et al., 2007).
The effect of the Ca2+ oscillations is to activate patterns of gene expression that
are required for develop- ment of the symbiotic state. It has also been reported
that a [Ca2+]c transient in plant cells is part of the plant-pathogen interaction
that is required for the activation of systemic resistance by plants (Lecourieux et
al., 2006). Although speculative at this stage, a role for DAPG in Ca 2+ signalling
is an intriguing concept that should be further explored.
4.4. Conclusions
By assessing the effects of DAPG on N. crassa, we have now established that
the antibiotic activity of DAPG is a consequence of its capacity to act as an
uncoupler in the mitochondrion. This has important implications for the
development of biological control inoculants as it confirms that the activity of
DAPG-producing strains will be broad spectrum. Furthermore, this is a distinct
mode-of-action from other antifungal agents. It also suggests that resistance will
be a rare event as the mitochondrial membrane is a not easily altered target. It
will be interesting to see if some of the natural Fusarium isolates that show
reduced susceptibility to DAPG (Schouten et al., 2008) are also more tolerant to
 other uncou- pling agents. It is also noteworthy that anti-cancer and pro-apop-
totic effects have been attributed to various uncoupling agents (Han et al., 2008;
Pardo-Andreu et al., 2011). In this study, a second physiological effect of DAPG
on fungi, namely alteration of intracel- lular Ca 2+ homeostasis by inducing
uptake of Ca2+ from the extra- cellular environment via an unknown
channel/transport system, has also been observed. This raises the possibility
that DAPG acts as a bacterial signal that is perceived by eukaryotic cells. If so,
this could have important ramifications for the interaction of DAPG- producing
bacteria with fungi, protozoa and/or plants. Genetically tractable fungi like N.
crassa, or indeed S. cerevisiae, offer a means to further explore all of these
possibilities.
nowledgments
Funding support for this Project was provided by a Biotechnol- ogy and
Biological Sciences Research (BBSRC) Council Grant (15/ P18594) to N.D.R. and
by Grants from the Science Foundation Ire- land (08/RFP/GEN1295), European
Commission (O36314) and the Irish Department of Agriculture and Marine
(BEAU/BIOD/01) to J.P.M. and F.OG. Technical support from Pat Higgins is
gratefully acknowledged.
pendix A. Supplementary material
Supplementary data associated with this article can be found, in the online
version, at http://dx.doi.org/10.1016Zj.fgb.2013.04.006.
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