Ni ic oxide syn hesis by ni a e educ ase is egula ed
du ing de elopmen in
Aspe gillus
Ana T. Ma cos,1Ma ía S. Ramos,1Jose F. Ma cos,2
Lou des Ca mona,2† Joseph S auss3,4 and
Da id Cáno as1,3*
1Depa amen o de Gené ica,Facul ad de Biología,
Uni e sidad de Se illa,Se illa, Spain.
2Depa men o Food Science,Ins i u e o Ag ochemis y
and Food Technology (IATA),Valencia, Spain.
3Fungal Gene ics and Genomics Uni ,Depa men o
Applied Gene ics and Cell Biology,Uni e si y o Na u al
Resou ces and Li e Sciences (BOKU) Vienna,Vienna,
Aus ia.
4Depa men o Heal h and En i onmen ,Bio esou ces,
Aus ian Ins i u e o Technology (AIT),Vienna, Aus ia.
Summa y
Ni ic oxide (NO) is a signalling molecule in ol ed in
many biological p ocesses in bac e ia, plan s and
mammals. Howe e , li le is known abou he ole and
biosyn hesis o NO in ungi. He e we show ha NO
p oduc ion is inc eased a he ea ly s ages o he
ansi ion om ege a i e g ow h o de elopmen in
Aspe gillus nidulans. Full NO p oduc ion equi es a
unc ional ni a e educ ase (NR) gene (niaD) ha is
up egula ed upon induc ion o conidia ion, e en unde
N- ep essing condi ions in he p esence o ammo-
nium. A his s age, NO homeos asis is achie ed by
balancing biosyn hesis (NR) and ca abolism ( la o-
haemoglobins). niaD and la ohaemoglobin hbA a e
ansien ly up egula ed upon induc ion o conidia ion,
and bo h egula o s A eA and Ni A a e necessa y o
his ansc ip ional esponse. The second la ohae-
moglobin gene hbB shows a di e en exp ession
p o ile being mode a ely exp essed du ing he ea ly
s ages o he ansi ion phase om ege a i e g ow h
o conidia ion, bu i is s ongly induced 24 h la e . NO
le els in luence he balance be ween conidia ion and
sexual ep oduc ion because a i icial s ong ele a-
ion o NO le els educed conidia ion and induced he
o ma ion o cleis o hecia. The ni a e-independen
and ni ogen me aboli e ep ession-insensi i e an-
sc ip ional up egula ion o niaD du ing conidia ion
sugges s a no el ole o NR in linking me abolism and
de elopmen .
In oduc ion
Rep oduc ion is an essen ial pa o he biological cycle o
ungi and a mode o dispe sion in he en i onmen .
Among ungi, Aspe gillus nidulans has been used as a
model o ganism o s udy ep oduc ion o decades. A.
nidulans displays wo modes o ep oduc ion: sexual o
asexual. The asexual de elopmen p og am (conidia ion)
is ini ia ed when supe icial hyphae a e exposed o an ai
in e phase. The asexual ep oduc i e s uc u e o A. nidu-
lans is he conidiopho e, which o ms g een-pigmen ed
conidiospo es app oxima ely 24 h a e induc ion o de el-
opmen (Adams e al., 1998; E xebes e e al., 2010; Yu,
2010). A cen al egula o y pa hway encompassing B lA,
AbaA and We A con ols conidia ion (see e iews by
Adams e al., 1998; E xebes e e al., 2010; Yu, 2010, and
e e ences he ein). The i s componen in he egula o y
cascade, B lA, is essen ial o d i e conidia ion (Adams
e al., 1988). b lA is no exp essed du ing ege a i e
g ow h, bu i s induc ion du ing de elopmen is con olled
by a numbe o genes, e.g. he lu y genes (Adams e al.,
1998; Yu e al., 2006). These lu y genes a e exp essed
in ege a i e mycelium and a e able o espond o s imuli
o induce he co-o dina ed ac i a ion o he mas e egu-
la o b lA (E xebes e e al., 2010).
The sexual p og am is p omo ed by signals ha can be
g ouped in ou ca ego ies: absence o ligh , nu ien a ail-
abili y, empe a u e and a mosphe ic gases. Unde labo a-
o y condi ions, pla e sealing limi s he exchange o gases
and esul s in lowe ing he oxygen le els and inc easing
he CO2le els, which p omo es sexual ep oduc ion (Dye
and O’Go man, 2012). A. nidulans is homo hallic, i.e. each
s ain ha bou s bo h ma ing ype genes, MAT1/ma B and
MAT2/ma A (Paole i e al., 2007). Du ing his p og am, A.
nidulans de elops sphe ical ui ing bodies called cleis o-
hecia, which con ain meiospo es called ascospo es (Dye
and O’Go man, 2012). Genes in ol ed in he egula ion o
cleis o hecia o ma ion we e mainly iden i ied h ough A.
nidulans mu an s de ec i e a dis inc s ages o sexual
de elopmen : acleis o hecial s ains such as ΔnsdD and
Accep ed 3 Sep embe , 2015. *Fo co espondence. E-mail da idc@
us.es; Tel. +34954554405; Fax +34954557104. †P esen add ess:
Depa men o Plan Bio echnology, Fundo de De esa da Ci icul u a,
Vila Melhado – A a aqua a, São Paulo, B azil.
Molecula Mic obiology (2016) 99(1), 15–33 ■doi:10.1111/mmi.13211
Fi s published online 14 Oc obe 2015
© 2015 The Au ho s. Molecula Mic obiology published by John Wiley & Sons L d.
This is an open access a icle unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s use, dis ibu ion and
ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed.
Δs uA (Wu and Mille , 1997; Han e al., 2001), mu a ions
ha s op he p opaga ion p og am a e p oduc ion o Hülle
cells such as Δs eA and ΔmedA (Clu e buck, 1969; Vallim
e al., 2000) and s ains ha a e blocked in he ma u a ion
o cleis o hecia (Ecke e al., 2000; Ho mann e al., 2000;
Busch e al., 2001).
Ea ly wo k in H. Ninnemann’s labo a o y connec ed
ni a e educ ase ac i i y o blue ligh -p omo ed conidia-
ion in Neu ospo a c assa (Klemm and Ninnemann, 1979;
Ninnemann, 1991). The pa hway o he assimila ion o
ni a e has been ex ensi ely s udied in A. nidulans o
se e al decades. Ni a e assimila ion equi es he ac ion
o wo enzymes: ni a e educ ase (NR, encoded by niaD),
media ing he educ ion o ni a e o ni i e; and he ni i e
educ ase (NiR, encoded by niiA) in ol ed in he con e -
sion o ni i e o ammonium. Ni a e assimila ion is gene -
ally ep essed in ungi by he p esence o he p e e ed
ni ogen sou ces ammonium o glu amine and, in A. nidu-
lans, is ac i a ed by ni a e o ni i e h ough he syne gis-
ic ac ion o he pa hway speci ic egula o Ni A and he
gene al egula o o ni ogen me abolism A eA (A s and
Co e, 1973; Caddick e al., 1986; Bu ge e al., 1991;
Ma zlu , 1997; S auss e al., 1998; Mu o-Pas o e al.,
1999; Todd e al., 2005; Be ge e al., 2006; 2008;
Be n ei e e al., 2007; Schinko e al., 2010).
Recen ly, he egula ion o ni a e assimila ion was con-
nec ed o ni ic oxide (NO) me abolism in A. nidulans
(Schinko e al., 2010). NO is widely ecognised o i s ole
as signalling compound om bac e ia o mammals
(Rosselli e al., 1998; He e al., 2004; Kwon e al., 2012;
Se h e al., 2012). This dia omic molecule is a sho -li ed
adical wi h a hal -li e o a ew seconds. I plays essen ial
egula o y oles in a a ie y o biological p ocesses bu
can also cause ni osa i e s ess o cells. Fla ohaemo-
globins a e known o unc ion in NO de oxi ica ion, con-
e ing he eac i e NO adical in o ni a e and
consequen ly p o ec ing agains ni osa i e s ess de i ed
om ei he exogenous o endogenous sou ces (Ga dne
e al., 1998a; Fos e e al., 2009). Di e en ou es o cel-
lula NO biosyn hesis ha e been desc ibed in na u e,
including bo h oxida i e and educ i e pa hways. The oxi-
da i e syn hesis has been epo ed in bac e ia, plan s and
mammals in ol ing he con e sion o L-a ginine and O2
in o ci ulline and NO by he ni ic oxide syn hase
(Alde on e al., 2001; Lamo e e al., 2005; Go en and
Maye , 2007). The educ i e NO syn hesis was epo ed
as i s in legumes (Dean and Ha pe , 1988; Yamasaki,
2000), bu la e was shown o be widesp ead in plan s,
and i in ol es he ac ion o he ni a e educ ase a sa u-
a ing ni i e concen a ions unde educ i e condi ions
(excess o NAPDH) (Yamasaki, 2000; Yamasaki and
Sakihama, 2000; Rockel e al., 2002; Lamo e e al.,
2005). In mammals al e na i e educ i e pa hways o he
con e sion o ni i e o NO ha e been iden i ied in ol ing
di e en enzymes such as cy och ome c, P450 o deoxy-
haemoglobin in he hea and wall essels o by non-
enzyma ic mechanisms (Zweie e al., 2010). No ably,
ungal genomes a e de oid o NO syn hase homologues
(Go en and Maye , 2007; Samalo a e al., 2013) and he
physiological and gene ic mechanisms ha de e mine
changes in he le el o NO in ungi a e poo ly unde s ood
in hese o ganisms. Addi ion o NO chemical dono s
induces asexual spo e de elopmen in he asco-
myce e N. c assa (Ninnemann and Maie , 1996), spo an-
giopho e de elopmen in he zygomyce e Phycomyces
blakesleeanus (Maie e al., 2001), conidia ion in Conio-
hy ium mini ans (Gong e al., 2007) and sexual ui ing
bodies in he ascomyce e A. nidulans (Baidya e al., 2011)
and in he basidiomyce e Flammulina elu ipes (Song
e al., 2000). Magnapo he o yzae syn hesised NO du ing
ge mina ion and ea ly de elopmen ; howe e , he dele ion
o candida e genes in bo h oxida i e and educ i e ou es
did no impai NO biosyn hesis in his ungus (Samalo a
e al., 2013). In A. nidulans he wo la ohaemoglobins
FhbA and FhbB we e shown o be in ol ed in he me abo-
lism o NO o ni a e (Ga dne e al., 1998b; Poole and
Hughes, 2000). In addi ion, hey a e essen ial o p o ec
ni a e educ ase and ni i e educ ase enzymes agains
ni osa i e damage elici ed by g ow h on ni i e a low
en i onmen al pH condi ions (Schinko e al., 2010; 2013).
In his wo k, we p o ide da a indica ing he exis ence o
a ni a e educ ase-dependen pa hway o he syn hesis
o NO in A. nidulans and ound ha NO le els inc ease
immedia ely a e swi ching om ege a i e g ow h o
conidia ion. Bo h la ohaemoglobins a e in ol ed in he
balance o NO du ing he de elopmen al swi ch.
Resul s
Aspe gillus p oduces NO
Se e al echniques ha e been employed o de ec and
quan i y NO p oduc ion (Yamasaki and Sakihama, 2000;
Maie e al., 2001; Planche and Kaise , 2006; Nagano,
2009; Samalo a e al., 2013). We selec ed he
NO-sensi i e luo escen p obes de eloped by Nagano
and collabo a o s (Kojima e al., 1998a,b; 1999). This
app oach was p e iously epo ed o be use ul in ungi
(Ca mona e al., 2012; Samalo a e al., 2013). DAF-FM is
e y sensi i e o NO, wi h a de ec ion limi o 3 nM,
whe eas DAF-FM diace a e (DAF-FM DA) is cell-
pe meable and passi ely di uses ac oss cellula mem-
b anes. Once inside he cells i is deace yla ed o DAF-FM
by es e ases (Kojima e al., 1999). To es whe he hey
could also be employed in Aspe gillus, ungal cells we e
g own in liquid ammonium minimal medium. Ei he
DAF-FM o DAF-FM DA was added o he cul u es. A e
cells we e loaded wi h he dyes o 20 min, he excess o
16
A. T. Ma cos
e al. ■
© 2015 The Au ho s. Molecula Mic obiology published by John Wiley & Sons L d., Molecula Mic obiology,99, 15–33
bo h dyes was washed ou in hal o he samples, and all
o hem we e ans e ed o a 96-well pla e o moni o ing
luo escence. As shown in Fig. 1A–B, he e was an
inc ease in he luo escence o e ime when he excess o
dye we e no washed o he cells. Howe e , when he
cells we e washed, he luo escence eco ded was d as-
ically lowe han in non-washed cells o in con ol
medium. To analyse whe he he moni o ed luo escence
was he esul o he eac ion wi h he NO-sensi i e p obe
inside o ou side he cells, luo escence was imaged
unde a luo escen mic oscope (Fig. 1C). When he cells
we e no washed, luo escence was obse ed bo h
ou side and inside he cells ega dless o he dye
(DAF-FM o DAF-FM DA). A e he excess o dye was
washed ou , luo escence was obse ed p e e en ially
inside he cells. To con i m he biological o igin o he
signal, Aspe gillus cells g own o 16 h in liquid ammo-
nium minimal medium we e au ocla ed. DAF-FM DA was
added o li e cells, dead cells and he minimal medium
and luo escence was eco ded (Fig. 1D). While luo es-
cence in li e cell samples inc eased con inuously wi h
ime, he luo escence gene a ed in he samples con ain-
3
3.5
4
4.5
5
. x 1,000)
Non-washed cells
Washed cells
DAF-FM Diace a e
A
0.5
1
1.5
2
2.5
3
o escence (a.u
Washed
Con ol (medium)
0
0
10
20
30
40
50
60
70
80
90
100
110
120
Flu
Time (min)
18
0)
DAF-FM
B
8
10
12
14
16
ce (a.u. x 1,000
Non-washed cells
Washed cells
0
2
4
6
0
10
20
30
40
50
60
70
80
90
100
110
120
Fluo escen
Washed
Con ol (medium)
shed
DAF-FM Diace a e DAF-FM
Time (min)
C
Non-was
Washed
D
E
7000
8000
Li e ungus
4000
5000
6000
c
ence (a.u.)
Killed ungus
1000
2000
3000
Fluo es
c
0
0 20 40 60 80 100 120
Time (min)
-1000
4000
9000
14000
19000
24000
29000
0 25 50 75 100 125
Fluo escence (a.u.)
Time (min)
NH4
NH4 + dNO
NH4 + PTIO 100
NH4 + PTIO 500
NH4 + PTIO 1000
Fig. 1. Quan i ica ion o NO p oduced by Aspe gillus.A. nidulans was g own in liquid ammonium minimal medium. DAF-FM DA (A) o
DAF-FM (B) was added o he cul u es and incuba ed o 20 min in he da k. A e cell loading o he dyes, one sample con aining each dye
was washed h ee imes, whe eas con ol samples we e no washed. F esh medium was used as an addi ional con ol o de ec he
backg ound signal. Fluo escence was moni o ed by luo ome y (A and B) o cells we e imaged by luo escen mic oscopy (C). (D) A. nidulans
was g own in liquid ammonium minimal medium o 16 h. One sample was kep as a li ing con ol, whe eas he es o he cells we e killed by
au ocla ing. DAF-FM DA was added o he samples and he luo escence was eco ded by luo ome y. Backg ound signal ob ained wi h esh
medium was sub ac ed om signals ob ained wi h bo h cell samples. (E) Aspe gillus cells we e g own in he p esence o in he absence o
he NO- eleasing compound dNO (1.5 mM) o di e en concen a ions o he NO-sca enge PTIO (100–1000 μM). DAF-FM DA was added o
he samples and luo escence was eco ded by luo ome y. Unde all hese condi ions, ungal g ow h was simila . In all cases, one
ep esen a i e expe imen is shown.
Ni ic oxide syn hesis in
Aspe gillus 17
© 2015 The Au ho s. Molecula Mic obiology published by John Wiley & Sons L d., Molecula Mic obiology,99, 15–33
ing hea -killed ungi emained cons an o e ime, sug-
ges ing ha NO p oduc ion was media ed by li e cells.
I has been p e iously epo ed ha he DAF amily o
NO-sensi i e dyes can also eac wi h asco bic acid and
dehyd oasco bic acid (Zhang e al., 2002a,b). Al hough
his possibili y was al eady dis ega ded by Samalo a and
collabo a o s in Magnapo he (Samalo a e al., 2013),
we es ed his possibili y in Aspe gillus by employing
he NO-sca enge 4,4,5,5- e ame hylimidazoline-l-oxyl3-
oxide (PTIO). Addi ion o di e en concen a ions o PTIO
esul ed in a dose-dependen dec ease o luo escence
compa ed wi h he con ol sample in he absence o he
NO sca enge (Fig. 1E). Addi ion o he NO- eleasing
compound de aNONOATE (dNO) p oduced a high
inc ease o luo escence, as expec ed. Collec i ely, i
shows ha DAF-FM and DAF-FM DA we e able o de ec
he NO p oduced by Aspe gillus, bo h in acellula and
ex acellula ly. We selec ed DAF-FM DA o he es o he
s udy o ha ing a be e signal a io be ween samples
con aining cells and con ol media in ou 96-well pla e
assays (3.3 wi h DAF-FM DA e sus 1.5- old wi h
DAF-FM).
NO syn hesis du ing g ow h in liquid media pa ially
depends on niaD
I has been known o a long ime ha ungi can p oduce
NO (Ninnemann and Maie , 1996); howe e , he mecha-
nisms o NO syn hesis ha e emained obscu e so a . In
o de o s udy a possible ole o he ni a e assimila ion
pa hway in he biosyn hesis o NO, ou expe imen al se
up was as ollows: he A. nidulans wild ype s ain was
g own in liquid media con aining ni a e, ni i e o ammo-
nium as sole ni ogen sou ce o 16 h; hen NO le els
we e ollowed o up o 200 min by addi ion o he
NO-sensi i e luo escen dye DAF-FM DA di ec ly o he
g ow h medium and by subsequen luo ome ic quan i i-
ca ion (Fig. 2A). The le el o luo escence in ensi y
inc eased in he samples con aining ungal cells o e
ime, o a highe ex en han in con ol samples (which
con ain only he cul u e media). Sub ac ion o he back-
g ound signal de i ed om he g ow h media con i med
ha , in e es ingly, NO le els we e highe in ammonium
han in ni i e o ni a e-con aining media (Fig. 2B, wild
ype s ain).
We hen in es iga ed whe he a ni a e educ ase-
dependen ou e o he syn hesis o NO is ope a ional in
A. nidulans, simila o he si ua ion desc ibed in plan s
(Dean and Ha pe , 1988; Yamasaki and Sakihama, 2000;
Rockel e al., 2002). We employed a ΔniaD mu an lacking
ni a e educ ase. Ob iously his mu an canno g ow on
ni a e as sole ni ogen sou ce, bu i can g ow on ni i e,
which is, in plan s, he subs a e o he ni a e educ ase
o p oduce NO. No majo di e ences we e ound in he
NO le els be ween he wild ype and he mu an s ain on
ammonium (Fig. 2B). Howe e , when bo h s ains we e
g own in ni i e, he ΔniaD mu an p oduced 30% less NO
han he wild ype, indica ing ha his enzyme pa icipa es
in he p oduc ion o NO and is esponsible o a ound 30%
o his me aboli e syn hesis when ni i e se ed as
N-sou ce. On ni a e medium, he wild ype s ain showed
he smalles amoun o NO compa ed wi h he o he wo
ypes o ni ogen (Fig. 2B). In o de o es also NO p o-
duc ion o ΔniaD on ni a e, he s ains we e p e-g own on
ammonium o 12 h and hen ans e ed o ni a e-
con aining ammonium liquid medium o back o sole
ammonium medium as con ol. A e 6 h o g ow h unde
hese condi ions, DAF-FM DA was added o he cul u es,
and luo escence was quan i ied (Fig. 2C). The wild- ype
s ain showed highe NO le els han he mu an when he
medium was supplemen ed wi h ni a e indica ing again
ha NR is in ol ed in he p oduc ion o NO h ough he
ni a e assimila ion ou e.
Ou da a sugges ed a pu a i e ole o niaD in medi-
a ing he syn hesis o NO. P e ious da a easoned ha
he la ohaemoglobin genes could be in ol ed in he
ca abolism and hus de oxi ica ion o NO (Schinko e al.,
2010). The dele ion o bo h la ohaemoglobin genes,
hbA and hbB, did no a ec NO le els in ammonium
medium (Fig. 2C–D, compa e NO le els wi h he wild
ype s ain), bu he e was a d as ic inc ease in NO le els
much highe han in he wild ype (up o 3.5- old
inc ease) when ni a e was p esen in he medium
(Fig. 2D). Impo an ly, e en in he p esence o he
ep essing ni ogen sou ce ammonium, ni a e was an
ob ious sou ce o NO p oduc ion. This is in ag eemen
wi h indings by Schinko e al. (2010), who showed ha
he ansc ip ion o hbA is independen om A eA unc-
ion and ha ull induc ion o his gene also occu s unde
induced- ep essed condi ions (i.e. ammonium plus
ni a e). When he ni a e educ ase niaD was dele ed in
he la ohaemoglobin dele ion mu an (ΔniaD Δ hbA
Δ hbB), he inc ease o NO in he p esence o ni a e was
no obse ed, con i ming ha a ni a e pa hway ope a es
o syn hesise NO in A. nidulans and ha his pa hway
equi es a unc ional niaD gene.
Dele ion o niaD esul s in d as ic educ ion o NO
p oduc ion du ing g ow h on solid media
P e ious ansc ip omic da a ob ained in ou labo a o y
sugges ed ha he ni a e u ilisa ion pa hway was
exp essed du ing he induc ion o conidia ion (Cano as
e al., 2014), which allowed us o hypo hesise ha , in
addi ion o ege a i e g ow h, NO could also be p oduced
h ough he ni a e pa hway du ing de elopmen . Fo his
eason, we we e in e es ed in in es iga ing whe he NR
was also ele an o NO p oduc ion on solid medium. We
18
A. T. Ma cos
e al. ■
© 2015 The Au ho s. Molecula Mic obiology published by John Wiley & Sons L d., Molecula Mic obiology,99, 15–33
compa ed he NO le els in he wild ype and he ΔniaD
s ain. Bo h s ains we e g own on solid medium con ain-
ing di e en ni ogen sou ces (ammonium, p oline o
p oline supplemen ed wi h ni a e). P oline allows he
g ow h o he wild ype and also he ΔniaD s ain, and i is
a neu al (non- ep essing, non-inducing) ni ogen sou ce
o he ni a e assimila ion sys em. Su p isingly, we ound
ha NO le els o he ΔniaD mu an on ammonium solid
medium was only 41% o he wild ype (Fig. 3A), whe eas
in liquid ammonium medium bo h exhibi ed simila le els
(Fig. 2). On p oline solid medium, he ΔniaD mu an p o-
duced less NO compa ed wi h he wild ype. When ni a e
was added o he p oline solid media, he wild ype
inc eased NO p oduc ion (P<0.05; S uden ’s - es ),
whe eas in he ΔniaD s ain educed le els we e ound:
30% less ela i e o p oline media (P<0.05; S uden ’s
- es ), and 55% less NO han he wild ype in p oline plus
ni a e (P<0.01; S uden ’s - es ). In his case, we
obse ed ha he NO le els in he ni i e educ ase de ec-
i e mu an (niiA4) we e simila o he wild- ype le els
(Fig. 3A). The e o e, all hese da a sugges ha he e is a
niaD-dependen pa hway o he syn hesis o NO bo h in
liquid and on solid media and ha addi ional pa hway(s)
o he syn hesis o NO mus ope a e in A. nidulans.
0
20
40
60
80
100
120
NH4 NO2 NO3
Pe cen age ( % luo escence / OD595)
A
B
0
2000
4000
6000
8000
10000
12000
14000
0 50 100 150 200
Fluo escence (a.u.)
Time (min)
Con ol_NH4
A. nidulans_NH4
Con ol_NO2
A. nidulans_NO2
Con ol_NO3
A. nidulans_NO3
Wild ype
ΔniaD
n.d.
NH4+NO3-
NO2-
Con ol NH4+
Con ol NO3-
Con ol NO2-
A. nidulans
NH4+
A. nidulans
NO3-
A. nidulans
NO2-
0
10
20
30
40
50
60
70
80
90
100
Fluo escence (a.u.) / mg d y weigh
C
0
50
100
150
200
250
300
350
Fluo escence (a.u.) / mg d y weigh
D
WT
ΔniaD
Δ hbAB ΔniaD
Δ hbAB
NH4+NH4+
+ 10 mM NO3-
NH4+NH4+
+ 10 mM NO3-
NH4+NH4++
10 mM NO3-
NH4++
100 mM NO3-
NH4+NH4++
10 mM NO3-
NH4++
100 mM NO3-
Fig. 2. NO quan i ica ion in mycelium in liquid medium.
A. A. nidulans wild ype was inocula ed in media con aining he indica ed compounds as sole ni ogen sou ces. DAF-FM DA was added o he
cul u es and non-inocula ed media (con ol samples). Media, which was no inocula ed, was employed as con ol o de e mine he biological
o igin o he NO p oduced. Fluo escence was eco ded o e ime. One ep esen a i e expe imen is shown.
B. A. nidulans wild ype (ligh g ey ba s) and ΔniaD (da k g ey ba s) s ains we e g own in liquid media wi h he indica ed ni ogen sou ces.
The ΔniaD s ain could no g ow in ni a e, and consequen ly, NO p oduc ion was no es ed in his sample (n.d.). Da a shown co espond o
one ime poin a e addi ion o DAF-FM DA (100 min) and a e e e ed o he wild- ype s ain g own in ammonium media (100%). Da a shown
a e he mean and he s anda d e o o he mean o a leas h ee independen biological eplica es. N.d.: no de e mined.
C. A. nidulans wild ype and ΔniaD s ains we e g own in ammonium liquid medium o 12 h. The mycelia we e il e ed, washed and
ans e ed o ammonium (con ol) and ammonium +10 mM ni a e. The cul u es we e g own o 6 addi ional hou s. DAF-FM DA was added o
quan i y NO p oduc ion, and luo escence was eco ded o 60 min. Da a shown a e he mean and he s anda d e o o he mean o wo
independen biological eplica es.
D. A. nidulans Δ hbA Δ hbB and Δ hbA Δ hbB ΔniaD s ains we e g own in ammonium liquid medium o 12 h. The mycelia we e il e ed,
washed and ans e ed o ammonium (con ol), ammonium +10 mM ni a e, and ammonium +100 mM ni a e. The cul u es we e g own o 6
addi ional hou s. DAF-FM DA was added o quan i y NO p oduc ion, and luo escence was eco ded o 60 min. Da a shown a e he mean
and he s anda d e o o he mean o wo independen biological eplica es.
Ni ic oxide syn hesis in
Aspe gillus 19
© 2015 The Au ho s. Molecula Mic obiology published by John Wiley & Sons L d., Molecula Mic obiology,99, 15–33

Wi h he aim o con i m he sou ce o NO, he la ohae-
moglobin mu an s we e used again o educe NO ca abo-
lism and hus o inc ease he o e all le els o his
me aboli e. The h ee s ains we e g own on solid media
con aining p oline as a sole ni ogen sou ce; he luo es-
cence eco ding was s a ed by addi ion o DAF-FM, and
hen di e en ni ogen sou ces we e indi idually added o
he assay as he eco ding con inued. As shown in
Fig. 3B, addi ion o ni a e did no ha e majo e ec s on
Δ hbA Δ hbB. Howe e , addi ion o ni i e elici ed an
inc ease in he luo escence. The ni i e educ ase mu an
(Δ hbA Δ hbB niiA4), on he o he hand, exhibi ed an
inc ease in he NO le els a e he addi ion o ei he ni a e
o ni i e, bu no ammonium, demons a ing ha he ni i e
educ ase (NiR) was no esponsible o he syn hesis o
NO. Addi ion o ei he ni a e o ni i e o he Δ hbA Δ hbB
ΔniaD mu an did no inc ease he NO le els o e he
con ol sample; u he mo e, his s ain showed educed
NO le els in he con ol condi ions (p oline) compa ed
wi h he wild ype (Δ hbA Δ hbB) and he Δ hbA Δ hbB
niiA4 s ains (Fig. 3B). Addi ion o ammonium o any o he
s ains did no change he NO le els signi ican ly o e he
con ol condi ion (p oline), as expec ed. The e o e, aken
oge he , ou da a demons a e ha he ni a e educ ase
is pa ially esponsible o he syn hesis o NO employing
ni i e as subs a e.
NO le els a e highe on solid medium han in
liquid medium
Du ing he p e ious expe imen s, we obse ed a highe
p oduc ion o NO du ing g ow h on solid media han in
liquid media. In o de o compa e he NO le els in bo h
ype o media, he A. nidulans wild- ype s ain was g own
in liquid o solid media con aining ammonium (con ol) o
ammonium supplemen ed wi h 10 o 100 mM ni a e o
18 h. DAF-FM was added o he samples, and luo es-
cence was eco ded o 200 min. In e es ingly, p oduc ion
o NO was much highe when A. nidulans was g own on
solid media han in subme ged cul u e unde hese
expe imen al condi ions (Fig. 4A). Addi ionally, he inc e-
men in he p oduc ion o NO in he p esence o ni a e
was also signi ican ly highe on solid media.
In o de o ind ou whe he NO was p oduced o accu-
mula ed in hyphae o conidiopho es, Aspe gillus was
g own on solid medium. DAF-FM DA was added and
allowed o pe mea e he cells o 20 min be o e washing
ou he excess o he dye. Visualisa ion unde he luo es-
cen mic oscope e ealed ha bo h hyphae and conidi-
opho es we e s ained; howe e , he signal was s onge in
he conidiopho es han in he hyphae (Fig. 4B). Unde his
low magni ica ion and using a luo escen mic oscope, he
signal appea ed o be g ea e in s e igma a cells (me ulae
and phialides) han in he s alk cells. Con ocal mic oscopy
con i med ha he luo escen signal could be de ec ed in
he s e igma a cells (Fig. 4C–E), bu also in he esicle
and s alk cells, and in hyphae (Fig. 4C).
Fungal g ow h media es ni i e and ni a e syn hesis in
ammonium media
The high le els o NO obse ed on ammonium we e
in iguing, bu he ac ha he ΔniaD s ain p oduced less
NO han he wild ype on ammonium solid medium
(Fig. 3A) posed wo in e es ing ques ions. The i s one is:
can NO be syn hesised om ni a e in a medium con ain-
0
20
40
60
80
100
120
% Fluo escence (a.u.) / OD595
Δ hbAB ΔniaD Δ hbAB Δ hbAB niiA4
P ol
P ol + NO3-
P ol + NO2-
P ol + NH4+
ΔniaD
WT
niiA4
P ol
P ol + NO3-
NH4+
A
B
0
25
50
75
100
125
150
175
% Fluo escence (a.u.) / OD595
Fig. 3. NO quan i ica ion on solid medium.
A. A. nidulans wild- ype, ΔniaD and niiA4 s ains we e g own on
solid media con aining he indica ed ni ogen sou ces a 10 mM
(ammonium o ni a e) o 3 mM (p oline) a 37°C o 18 h. DAF-FM
DA was added o quan i y NO p oduc ion, and luo escence was
eco ded o 100 min. Da a shown a e e e ed o he wild- ype
s ain g own on ammonium media (100%). Da a shown a e he
mean and he s anda d e o o he mean o a leas h ee
independen biological eplica es.
B. A. nidulans Δ hbA Δ hbB, Δ hbA Δ hbB ΔniaD and Δ hbA Δ hbB
niiA4 s ains we e g own on p oline solid media o 18 h. DAF-FM
DA was added o quan i y NO p oduc ion. Then, he indica ed
ni ogen sou ces we e added, and luo escence was eco ded o
60 min. Da a shown a e e e ed o he con ol s ain (Δ hbA Δ hbB)
g own on p oline wi hou he addi ion o any o he ni ogen sou ce.
E o ba s ep esen he s anda d e o o he mean.
20
A. T. Ma cos
e al. ■
© 2015 The Au ho s. Molecula Mic obiology published by John Wiley & Sons L d., Molecula Mic obiology,99, 15–33
ing ammonium as sole ni ogen sou ce? This sugges s
ha ni a e could be p esen in his medium. To es his
hypo hesis, we employed he A. nidulans ΔniaD s ain,
which does no me abolise ni a e allowing i s accumula-
ion, and quan i ied bo h ni a e and ni i e du ing g ow h in
he media. The ΔniaD s ain was g own on he su ace o
a pe i dish con aining liquid media supplemen ed wi h
ammonium as sole ni ogen sou ce, as p e iously
desc ibed (Ruge -He e os e al., 2011). Unde hese con-
di ions, A. nidulans conidia ion p oceeded as on solid
media. Nei he ni a e no ni i e could be de ec ed in he
cul u e media be o e he inocula ion wi h he ungal
spo es. Bu su p isingly, bo h ni a e and ni i e we e ound
in he cul u e media a e 19, 24 and 48 h o ungal g ow h
(Fig. 5). When he expe imen was pe o med wi h a wild-
ype s ain, ni a e and ni i e we e also ound in he media
bu a lowe le els, due o he me abolism o ni a e by an
ac i e ni a e educ ase (Supplemen a y Fig. S1).
niaD is induced du ing conidia ion
The second ques ion is: i acco ding o he heo y o
ni ogen egula ion, he ni a e educ ase gene niaD mus
be ep essed in a media con aining he ep essing ni o-
gen sou ce ammonium (A s and Co e, 1973), how is he
ni a e educ ase ac i e in hese condi ions? T ansc ip-
omics da a ob ained p e iously sugges ed ha he ni a e
u ilisa ion pa hway was exp essed du ing induc ion o
de elopmen on ammonium ni a e medium (Cano as
e al., 2014). To u he con i m his, we i s ob ained
0
2000
4000
6000
8000
10000
12000
14000
16000
0 50 100 150 200
Fluo escence (a.u.) / OD595
Time (min)
SOL AMM
SOL AMM + 10 mM NIT
SOL AMM + 100 mM NIT
LIQ AMM
LIQ AMM + 10 mM NIT
LIQ AMM + 100 mM NIT
SOL NH4+
SOL NH4+ + 10 mM NO3-
SOL NH4+ + 100 mM NO3-
LIQ NH4+
LIQ NH4+ + 10 mM NO3-
LIQ NH4+ + 100 mM NO3-
D
E
C
DIC DAF-FM MERGE
A
B
Fig. 4. NO p oduc ion is highe on solid
medium han in liquid medium.
A. A. nidulans wild- ype s ain was g own in
liquid o solid media o 18 h. In liquid
medium, A. nidulans was g own in ammonium
liquid medium o 12 h. The mycelia we e
il e ed, washed and ans e ed o ammonium
(LIQ NH4+), ammonium +10 mM ni a e (LIQ
NH4++10 mM NO3−) and ammonium +
100 mM ni a e (LIQ NH4++100 mM NO3−).
The cul u es we e g own o 6 addi ional
hou s ( o al g ow h ime was 18 h). On solid
media spo es we e inocula ed on solid media
con aining ammonium (SOL NH4+), ammonium
+10 mM ni a e (SOL NH4++10 mM NO3−)
and ammonium +100 mM ni a e (SOL NH4+
+100 mM NO3−), and he pla es we e
incuba ed a 37°C o 18 h. DAF-FM DA was
added o all cul u es o quan i y NO
p oduc ion, and luo escence was eco ded
o 200 min. The plo shown is he a e age o
wo eplica e samples o a ep esen a i e
expe imen .
B–E. Aspe gillus wild- ype s ain was g own
on solid ammonium medium o 24 h. Aga
cubes con aining ungal biomass we e
subme ged in a DAF-FM DA solu ion o
20 min and washed h ee imes.
Conidiopho es we e imaged by luo escence
mic oscopy (B) o con ocal mic oscopy
(maximal p ojec ion in C, and single planes in
D–E).
Ni ic oxide syn hesis in
Aspe gillus 21
© 2015 The Au ho s. Molecula Mic obiology published by John Wiley & Sons L d., Molecula Mic obiology,99, 15–33
wild- ype ege a i e mycelia g own in ammonium liquid
medium o 18 h, and hen, conidia ion was induced by
ans e ing he mycelia o solid media con aining ammo-
nium o ni a e as a sole ni ogen sou ce (Fig. 6). Two
hou s a e he induc ion o conidia ion, niaD mRNA le els
we e oughly 70- old highe on ni a e solid media han in
ege a i e mycelia and, s ikingly, niaD was also an-
sc ibed on ammonium solid media. Howe e , he ni a e-
induced le el was 8.6- old highe compa ed wi h
ammonium. Longe induc ion o conidia ion on ni a e
solid media did no esul in much u he inc ease o niaD
exp ession ( om 70- o 92- old induc ion compa ed wi h
ege a i e mycelia). Howe e , longe induc ion o conidi-
a ion on ammonium media inc eased he induc ion o
niaD om 8.5- o 23- old ela i e o ege a i e mycelia.
We also quan i ied he le els o he la ohaemoglobin
mRNAs a e he induc ion o conidia ion. As p e iously
epo ed, hbA was induced by ni a e (Schinko e al.,
2010), and he e was no signi ican a ia ion in he
exp ession le els in ammonium du ing he i s hou s o
conidia ion (Fig. 6). On he o he hand, he hbB gene was
sligh ly ep essed immedia ely a e induc ion o asexual
de elopmen ega dless o he ni ogen sou ce and
d opped h ee old a e 2 h (Fig. 6). Down egula ion o
hbB was simila in bo h ni ogen sou ces ammonium and
ni a e, sugges ing ha hbB is no cons i u i e, bu a he
de elopmen ally egula ed.
NO le els a e inc eased du ing he i s hou s
o conidia ion
The highe le els o NO p oduced du ing g ow h on solid
media compa ed wi h liquid media sugges s ha he e
migh be a connec ion be ween NO and de elopmen .
Indeed he exp ession pa e n o hbB poin s o an ea ly
egula ion o he NO le els du ing he ansi ion om
ege a i e g ow h o conidia ion. In o de o s udy his
possible connec ion be ween NO and asexual de elop-
men , we i s quan i ied he NO le els a he ea ly s ages
o he ansi ion om ege a i e g ow h o conidia ion. The
wild- ype s ain and he mu an lacking bo h la ohaemo-
globins (Δ hbA Δ hbB), which canno de oxi y NO, we e
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
0h 19h 24h 48h
NO3- o NO2- (ppm)
NO3-
NO2-
Fig. 5. Quan i ica ion o ni a e and ni i e in he cul u e media. A.
nidulans ΔniaD s ain was g own on he su ace o a pe i dish
con aining liquid media o he indica ed ime poin s. Ni a e (ligh
g ey ba s) and ni i e (da k g ey ba s) we e quan i ied wi h he
G iess eagen . Nei he ni a e no ni i e was de ec ed in he media
be o e ungal g ow h.
NO3- NH4+
0.000
0.005
0.010
0.015
0.020
0.025
Rela i e exp ession
hbB
0 h 2 h 4 h 6 h
Time
(
h
)
0
2
4
6
8
10
12
14
16
Rela i e exp ession
hbA
0.0
0.5
1.0
1.5
2.0
Rela i e exp ession
niaD
Fig. 6. Exp ession o he la ohaemoglobins and ni a e educ ase
genes in media con aining ni a e o ammonium as sole ni ogen
sou ce ea ly du ing conidia ion. A. nidulans wild- ype s ain was
g own ege a i ely in ammonium liquid minimal medium o 18 h
and hen ans e ed o ni a e (da k g ey ba s) o ammonium (ligh
g ey ba s) solid medium o induce conidia ion. Samples we e aken
a he indica ed ime poin s o RNA isola ion. hbA, hbB and niaD
exp ession was quan i ied by eal ime RT-PCR. Da a we e
no malised agains he exp ession o he ß- ubuline gene (benA).
The plo s show he a e age and s anda d e o o he mean o he
ela i e exp ession alues in a leas h ee independen
expe imen s.
22
A. T. Ma cos
e al. ■
© 2015 The Au ho s. Molecula Mic obiology published by John Wiley & Sons L d., Molecula Mic obiology,99, 15–33
g own in ammonium liquid medium, and hen ans e ed
o solid media o induce conidia ion as abo e. Samples
we e aken a 1 h in e als, and NO was quan i ied wi h
DAF-FM. As can be seen in Fig. 7A–B, NO inc eased
a e 1 h o induc ion compa ed wi h ege a i e g ow h in
all he condi ions. In ac , we obse ed his inc ease a e
induc ion o conidia ion o 1 h, and i emained ele a ed
a leas o 7 h (da a no shown). The e we e no majo
di e ences in he NO le els be ween ammonium and
ni a e g ow h condi ions du ing he ea ly induc ion o
conidia ion.
We we e in e es ed in c ea ing a highe ange o NO
le els o he ollowing expe imen s. The e o e, we added
he NO- eleasing chemical compound de aNONOa e
(dNO), which was p e iously es ed o inc eased le els o
NO (Fig. 1E), and checked o an addi ional inc ease o
NO le els (Fig. 7C). In his case, we ound a d as ic
inc ease o NO in bo h s ains, as expec ed, and NO
le els we e h ee old highe in he mu an han in he wild
ype.
Conidia ion (asexual de elopmen ) is ep essed by
ele a ed NO le els
In o de o ge insigh in o he e ec s o NO du ing ep o-
duc ion, we i s es ed he e ec s o di e en NO le els on
conidia ion using he wild- ype s ain and he mu an
lacking bo h la ohaemoglobins (Δ hbA Δ hbB). Conidia-
ion was induced as desc ibed abo e by ans e ing eg-
e a i e mycelia o bo h s ains o solid media wi h o
wi hou dNO. Based on he esul s shown abo e, we
expec ed ha hese condi ions and s ains gene a ed a
g adien o NO. As NO le els quickly inc eased a e
induc ion o conidia ion, we quan i ied he le els unde
hese condi ions and obse ed he expec ed g adien o
inc easing NO le els (Fig. 8A). A e 3 days o incuba ion,
he numbe o conidia was coun ed. Conidia ion was
sligh ly educed in he double mu an compa ed wi h he
wild ype in he con ol ni a e media (Fig. 8B). Addi ion o
he NO-dono dNO o he media esul ed in a d as ic
inc ease o NO le els, especially in he mu an s ain, and
a u he educ ion o conidia ion (ca. 60%) bo h in he wild
ype (P<0.1; S uden ’s - es ) and in he mu an s ain
(P<0.05; S uden ’s - es ). Obse a ion o he ungal
g ow h on he solid media wi h a s e eoscopic mic oscope
e ealed a educ ion in he densi y o conidiopho es (Fig.
S2). These esul s sugges ha an inc ease o NO co e-
la es wi h a dec ease in conidia ion.
Sexual de elopmen is induced by NO
Conidia ion and sexual de elopmen a e no mally bal-
anced in A. nidulans, meaning ha when one o hem
inc eases, he o he one dec eases (Adams e al., 1998;
Rod iguez-Rome o e al., 2010; Dye and O’Go man,
2012). To s udy he e ec s o NO in sexual de elopmen
o A. nidulans, we ollowed a simila s a egy as desc ibed
abo e bu g owing s ains unde condi ions o induce
sexual de elopmen (i.e., sealed pla es in he da k).
Again, his s a egy also allowed c ea ing he NO g adien
unde he condi ions o induce sexual de elopmen
(Fig. 8C). Opposi e o he e ec s obse ed in conidia ion,
he e was an inc ease in he numbe o cleis o hecia ( he
sexual ep oduc i e s uc u es o A. nidulans)in he
mu an s ain compa ed wi h he wild ype in he con ol
medium (Fig. 8D). When he NO- eleasing compound
dNO was added o he media, he numbe o cleis o hecia
e en inc eased in he pa en al s ain compa ed wi h he
con ol media (Fig. 8D). Simila esul s we e ob ained by
Baidya e al. (2011). This inc ease in he numbe o cleis-
o hecia co ela ed wi h he inc ease o NO. Howe e ,
0
200
400
600
800
1000
1200
1400
Fluo escence (a.u.)
0
200
400
600
800
1000
1200
1400
Fluo escence (a.u.)
0
2000
4000
6000
8000
10000
VEG 1 h 2 h 3 h 4 h
Fluo escence (a.u.)
Ammonium
Ni a e
Ni a e + dNO
Wild ype
Δ hbA Δ hbB
A
B
C
Time (h)
Fig. 7. NO quan i ica ion a e induc ion o de elopmen . A.
nidulans wild- ype s ain (ligh g ey ba s) and a Δ hbA Δ hbB
mu an (da k g ey ba s) we e g own ege a i ely in ammonium
liquid medium o 18 h (VEG) and hen ans e ed o solid media
con aining di e en ni ogen sou ces o induce de elopmen :
ammonium a a e as con ol ni ogen sou ce (A), sodium ni a e
(B) and sodium ni a e supplemen ed wi h NO- eleasing chemical
compound dNO (C). Samples we e aken a di e en imes, and he
luo escence p oduced by he eac ion o NO and DAF-FM DA was
quan i ied. Please no e he di e en g aph scales in A–C.
Ni ic oxide syn hesis in
Aspe gillus 23
© 2015 The Au ho s. Molecula Mic obiology published by John Wiley & Sons L d., Molecula Mic obiology,99, 15–33
ligh o conidia ion condi ions, o al e na i ely hey we e
sealed and incuba ed in he da k o sexual de elopmen al
condi ions. Plugs we e cu ou om he pla e 72 h a e induc-
ion o de elopmen . Conidia in he plugs we e esuspended
in Tween 0.1% bu e and coun ed. Fo coun ing cleis o he-
cia, pla es incuba ed o 15 days we e i s sp ayed wi h 70%
e hanol o acili a e he isualisa ion o he sexual s uc u es
and hen pho og aphed. Da a shown a e he a e age o a
leas ou independen expe imen s.
NO quan i ica ion
Ni ic oxide was quan i ied in samples by using he
NO-sensi i e luo escen dye DAF-FM DA (In i ogen) ollow-
ing he manu ac u e ’s ins uc ions excep in Fig. 1, in which
DAF-FM (Sigma) was also employed. In Fig. 1, ungal cells
we e g own in liquid ammonium minimal medium o 16 h, and
50 μlo 5μM DAF-FM DA o DAF-FM we e added o 100 μlo
each sample. Cells we e loaded wi h he dye o 20 min and
washed h ee imes wi h a 0.05% ween solu ion (when indi-
ca ed). Samples we e ans e ed o 96-well pla es, and
luo escence was eco ded in a Syne gy HT Mul i-mode
Mic opla e Reade (Bio ek) equipped wi h FITC il e se s (λEm
488 nm, λEx 520 nm). In Fig. 2Aand B, conidia we e inocula ed
in o 100 μl o minimal media a a inal concen a ion o
105conidia ml−1in 96-well pla es, and he pla es we e incu-
ba ed wi h shaking a 37°C o 16 h o allow ae a ion o he
cul u e. A e incuba ion, DAF-FM DA was added o he cul-
u es o a inal concen a ion o 2.5 μM, and luo escence was
eco ded in POLARs a Omega luo ome e (MG Lab ech)
equipped wi h FITC il e se s (λEm 488 nm, λEx 520 nm) o up
o 200 min. Da a we e analysed wi h Omega Con ol ( e sion
1.10) so wa e. Fo he es o he expe imen s, he s ains
we e g own in ammonium liquid medium o 12 h, hen ans-
e ed o liquid media con aining he app op ia e ni ogen
sou ces and incuba ion con inued o 6 addi ional hou s. Cells
we e loaded wi h he dye o 20 min and ans e ed o 96-well
pla es. Al e na i ely, he s ains we e g own in ammonium
liquid medium o 18 h and hen ans e ed o solid media o
he indica ed ime poin s. Pla es we e incuba ed unde he
condi ions desc ibed abo e o conidia ion o sexual de elop-
men . Samples we e aken om he pla es a he indica ed
ime poin s, subme ged in a solu ion o 5 μM DAF-FM DA o
1 h. Samples we e cen i uged, and he supe na an was
ans e ed o a 96-well pla e o luo escence quan i ica ion.
Fo he o he expe imen s pe o med on solid media in Figs 3
and 4, 105conidia we e inocula ed on 96-well pla es con ain-
ing solid media con aining he indica ed ni ogen sou ces and
incuba ed o 18 h be o e quan i ica ion o NO. In Fig. 3B,
ungal cells we e g own in p oline solid media o 18 h, luo-
escence quan i ica ion s a ed a e addi ion o DAF-FM DA
and hen he indica ed ni ogen sou ces we e added and
luo escence quan i ica ion con inued o 2 addi ional hou s. In
all cases 2.5 μM DAF-FM DA was added o he samples.
Fluo escence was de ec ed and quan i ied in a Syne gy HT
Mul i-mode Mic opla e Reade (Bio ek) equipped wi h GFP
il e se s. Da a we e analysed wi h Gen5TM Da a Analysis
So wa e. In all expe imen s, da a we e no malised o d y
weigh o OD595 as indica ed. Expe imen s we e epea ed a
leas wo imes and pe o med in duplica es o iplica es,
depending on he expe imen .
Mic oscopy
Aspe gillus was g own ei he on liquid ammonium medium o
14–16 h o solid ammonium medium o 24 h. A solu ion o
5μM DAF-FM DA(solid and liquid samples) o DAF-FM (liquid
samples) was added o he mycelium. A e cell loading o he
dye o 20 min, he excess o dye was washed ou wi h a 0.05%
Tween 80 solu ion. Images we e eco ded in an Olympus
IX2-UCB in e ed luo escence mic oscope equipped wi h a
FITC il e se (λEm 488 nm; λEx 520 nm). Images in Fig. 1C
we e aken using iden ical se ings o compa a i e pu poses.
Images in Fig. 4B we e p ocessed and me ged using Adobe
ImageReady CS2 (Adobe Sys ems Inco po a ed, CA, USA).
Con ocal images we e ob ained in a ZEISS LSM 7 DUO
con ocal mic oscope wi h an exci a ion a 488 nm using an
OPSS lase beam. Con ocal images we e p ocessed using
ZEN Li e 2012 (Zeiss, Ge many).
Ni a e and ni i e measu emen s in he
cul u e supe na an
Ni a e and ni i e we e quan i ied in he cul u e media using
he G iess eagen as p e iously desc ibed (Schinko e al.,
2010).
Acknowledgemen s
We would like o hank Michael Hynes o he niiA4 s ain,
Tho s en Schinko o help ul discussions, Modes o Ca ballo
o help wi h he luo ome ic assays (Se icio de Biología,
Cen o de In es igación Tecnología e Inno ación, Uni e sidad
de Se illa) and Juan Luis Ribas o help wi h he con ocal
mic oscopy (Cen o de In es igación Tecnología e Inno ación,
Uni e sidad de Se illa). Wo k in Valencia was suppo ed by
g an numbe BIO2012-34381 om he MINECO o JFM, and
in Vienna was suppo ed by g an numbe LS12-009 om he
n( +b) Lowe Aus ia Science Fund and M01693-B22 om
he FWF. We would like o hank he anonymous e e ees o
help ul sugges ions o imp o e his manusc ip .
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Suppo ing in o ma ion
Addi ional suppo ing in o ma ion may be ound in he online
e sion o his a icle a he publishe ’s web-si e.
Ni ic oxide syn hesis in
Aspe gillus 33
© 2015 The Au ho s. Molecula Mic obiology published by John Wiley & Sons L d., Molecula Mic obiology,99, 15–33