Available online at www.sciencedirect.
com
Reactive oxygen species generation in fungal development and
pathogenesis
Paul Tudzynski, Jens Heller and Ulrike Siegmund
Reactive oxygen species (ROS) generated by NADPH-
dependent oxidases (Nox) have been shown to function as
signaling molecules and to be essential for many differentiation
processes in mammals and plants. There is growing evidence
that ROS are important for many aspects of fungal life including
vegetative hyphal growth, differentiation of conidial
anastomosis tubes, fruiting body and infection structure
formation, and for induction of apoptosis. Recent results from
studies in fungal saprophytic and pathogenic model systems
have shed new light on the role of Nox in cytoskeleton
organization, the structure of Nox complexes and links to
components of the apical complex, and the localization of Nox
to the endoplasmic reticulum.
Address
Institut für Biologie und Biotechnologie der Pflanzen, Westf. Wilhelms
Universitaet, Schlossplatz 8, D-48143 Muenster, Germany
Corresponding author: Tudzynski, Paul (tudzyns@uni-muenster.de)
Current Opinion in Microbiology 2012, 15:653–659
This review comes from a themed issue on Growth and development:
eukaryotes
Edited by Nicholas J Talbot
For a complete overview see the Issue and the Editorial
Available online 2nd November 2012
1369-5274/$ – see front matter, # 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.mib.2012.10.002
Introduction
Reactive oxygen species (ROS) are normal by-products of
various metabolic pathways localized in different cellular
compartments. Under physiological steady state con-
ditions, ROS are inactivated by a network of antioxidative
defense components, to avoid damage of macromol-
ecules. However, ROS are also involved in intracellular
signaling, in cell–cell interaction and in communication
between organisms. Local bursts of ROS are involved in
various differentiation processes. These complex and
partially conflicting functions require an exact control
of amplitude, duration and localization to ensure speci-
ficity of ROS signaling [1].
The most important enzymatic ROS generating systems
are the NADPH oxidases (Nox), the best-characterized
example being the mammalian gp91phox (also known as
Nox2) responsible for the neutrophil oxidative burst
defense response. Nox2 catalyzes the production of
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superoxide by reduction of oxygen using NADPH as
electron donor and FADH2 and two hemes as cofactors.
Nox activity requires assembly of a complex composed of
several regulatory cytosolic components and the integral
membrane protein flavocytochrome b558, composed of
gp91phox and the adaptor protein p22phox (Figure 1a).
Besides gp91phox, four other Nox with widespread func-
tions occur in human tissues where they are involved in
many cellular differentiation and signaling processes
[2,3]. Nox are also found in a wide range of other organ-
isms [4]. In plants the respiratory burst oxidase homo-
logs (RBOHs) play essential roles in plant development
and response to abiotic stress and biotic interactions [5].
There is increasing evidence that this is also true for
fungi. Filamentous fungi, for example, possess three
distinct subfamilies of Nox [6]. NoxA and NoxB are
homologs of gp91phox, whereas NoxC contains putative
calcium binding EF-hand motifs, a feature of human
Nox5 and RBOHs. The presence of Nox was initially
proposed to correlate with multicellular status. However,
recently a functional Nox-like enzyme was identified in
yeast, questioning this concept [7].
Here we review recent work on occurrence and functions
of Nox complexes in fungi and focus on the various
unsolved questions, especially localization, complex com-
position and molecular mechanisms underlying the
observed functions.
Function of fungal Nox complexes
In fungi Nox are associated with a wide range of functions
(Table 1). The link between Nox activity and differen-
tiation in fungi was first established in Aspergillus nidulans
where NoxA was shown to be necessary for cleistothecia
development [8]. Later studies showed that the corre-
sponding enzyme in Podospora anserina and Neurospora
crassa is necessary for perithecia development. NoxB
(Nox2) in both fungi is important for ascospore germina-
tion [9,10]. In A. nidulans apical dominance is regulated by
ROS, probably from NoxA [11]. In Botrytis cinerea, Scler-
otinia sclerotiorum and Claviceps purpurea, both NoxA and
NoxB are necessary for formation of sclerotia, which are
the basis for the development of fruiting bodies ([12,13];
D. Buttermann, unpublished data), whereas formation of
conidial anastomosis tubes (CATs) in N. crassa and B.
cinerea mainly depends on NoxA (Nox1) [14,15]. Nox in
P. anserina are involved in differentiation of specific
structures, similar to appressoria in plant pathogens, that
are essential for colonization of cellulose [16]. These
results provide a link to the virulence defects of Nox
Current Opinion in Microbiology 2012, 15:653–659
654 Growth and development: eukaryotes

Figure 1

(a) flavocytochrome b558 (b)

O2 O2- O2 O2-

p22phox

Pls1
gp91phox NoxA/B ?

p67phox Rac NoxR Rac

p40phox Cdc24
p47phox Bem1 Cla4

Cdc42
Ste20

Current Opinion in Microbiology

Composition of the NADPH oxidase (Nox) complex in fungi and human phagocytic cells. (a) The catalytic center of the phagocyte NADPH oxidase is
the membrane-integrated protein gp91phox complexed with p22phox, which is crucial for the function of gp91phox (the complex is termed cytochrome
b558). In resting cells, gp91phox does not produce superoxide. Activation of the enzyme requires stimulus-induced membrane translocation of the
cytosolic components p40phox, p47phox, p67phox, and Rac. While p47phox, p67phox, and Rac are essential for the activation of the phagocyte NADPH
oxidase, p40phox is a positive regulator. (b) Fungal NoxA and NoxB are homologs of gp91phox. Although the tetraspanin Pls1 shares some structural
similarities with p22phox and might be connected to NoxB, it is no homolog of the adaptor protein. A fungal homolog of p47phox has not been identified,
either. Instead, homologs of the cytosolic p67phox (NoxR) and Rac (Rac) have been identified in fungi. Additionally, the scaffold protein Bem1 was
suggested to be a fungal analog of the regulator p40phox. In several fungi the interaction of single cytosolic components of the Nox complex has been
shown. Bem1 interacts with the Nox regulator NoxR (E. festucae, C. purpurea, B. cinerea), the guanyl-exchange factor Cdc24 (E. festucae, C.
purpurea), the Rho-GTPase Cdc42 (B. cinerea), and the p21-activated protein kinases Cla4 (C. purpurea, B. cinerea) and Ste20 (C. purpurea).
Additionally, an interaction could be shown for NoxR with both, Rac (E. festucae, B. cinerea, C. purpurea) and Cdc24 (C. purpurea), and for Cla4 with
Ste20 (C. purpurea). So far, no direct interaction of the catalytic subunits and the cytosolic components could be shown, and it is not known, whether
or not a synchronous interaction of all the components is needed to activate the fungal Nox complex. (Homologous proteins are shown in the same
color.) [2,4,6,21,29,44].

mutants in several plant pathogens. The primary role of
Nox in virulence is probably the generation of spatiotem-
poral ROS spikes necessary for differentiation of struc-
tures such as appressoria and penetration hyphae. The
exact mechanism by which Nox-derived ROS controls
differentiation is still not known. However, recent data
from N. Talbot’s lab provide key new insights into the
potential molecular mechanisms involved in Nox-based
differentiation. Before the differentiation of penetration
hyphae at the base of appressoria of Magnaporthe oryzae, an
actin ring is formed following assembly of septins. This
differentiation step is dependent on the Rho-GTPase
Cdc42 and on activity of both Nox1 and Nox2, the former
for maintenance of the actin network and the latter for
formation of the septin ring ([17], N. Talbot, pers.
commun.). The hypothesis that Nox participate in differ-
entiation processes in fungi by changing the cytoskeleton
organization, as occurs in mammalian systems [2], is
further supported by the recent studies in yeast where
the Nox homolog Yno1 is required for controlling the cell
cycle and actin assembly [7].
In B. cinerea, NoxB appears to be crucial for appressoria
formation. NoxB mutants form non-functional appres-
soria leading to repeated cycles of appressoria formation
and hyphal outgrowth out of appressoria after unsuccess-
ful penetration attempts [1,18].
The situation seems to be different in non-appressoria
forming fungi. In C. purpurea Nox are not necessary for
penetration of host tissue. Here the role of the two com-
plexes is quite different with Nox1 being required for full
and rapid colonization of host tissue and Nox2 for con-
trolled infection. NoxB mutants show vigorous infection of
rye plants that results in prolonged and increased formation
of conidia-containing honeydew (D. Buttermann, pers.
commun.). This is reminiscent of the situation in the
endophyte Epichloe festucae where Nox1 is important for
endophytic, symptomless growth [19]. Perhaps in the
closely related C. purpurea a controlled virulence has devel-
oped, allowing a limited infection of the plant, giving the
host protection against animal herbivory without causing
too much damage. The different functions of the Nox

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 Reactive oxygen species generation in fungal development and pathogenesis Tudzynski, Heller and Siegmund 655

Table 1

Functional analyses of fungal Nox complexes.

Organism Nox components Function Reference

Aspergillus nidulans (saprophyte) NoxA Cleistothecia formation [11]
ROS formation at hyphal tips
Podospora anserina (saprophyte) Nox1 Perithecia formation, cellulose degradation [9,16]
Nox2 Ascospore germination, cellulose degradation
Nox3 No obvious function
NoxR Regulation of Nox1/2
Neurospora crassa (saprophyte) Nox1 Perithecia formation, asexual development, [10]
normal hyphal growth
Nox2 Ascospore germination
Nor1 Regulation of Nox1/2
Saccharomyces cerevisiae (saprophyte) Yno1 Apoptosis; organization of cytoskeleton [7]
Epichloe festucae (endophyte) NoxA Ensures mutalistic interaction/ [19,44,21]
endophytic growth
NoxB No obvious function
NoxR Regulation of NoxA
Magnaporthe oryzae (hemi-biotroph) Nox1 Functional appressoria formation/penetration [36] N. Talbot,
Nox2 Functional appressoria formation/penetration pers. commun.
Nox3 No obvious function
Claviceps purpurea (biotroph) Nox1 Normal infection, sclerotia formation [45] D. Buttermann,
Nox2 Balanced infection, mutant is hypervirulent! pers. commun.
Botrytis cinerea (necrotroph) NoxA Lesion development; sclerotia formation, [12,14,18]
CAT formation
NoxB Penetration, sclerotia formation
NoxR Regulation of NoxA/B
Sclerotinia sclerotiorum (necrotroph) Nox1 Virulence, sclerotia development, [13]
oxalate secretion
Nox2 Sclerotia development
Alternaria alternata (necrotroph) NoxA Virulence, sporulation, resistance [46]
to oxidative stress
Trichoderma harzianum (mycoparasite) Nox1 Biocontrol of kursiv [47]
(secretion of hydrolytic enzymes)

complexes have therefore evolved to provide adaptation to
specific fungal life styles.
Structure of the Nox complexes
Apart from homologs of gp91phox, the regulatory subunit
p67phox (called NoxR) and the Rho-GTPase Rac, no clear
orthologs of the other mammalian Nox components are
present in fungal genomes. The scaffold protein Bem1
was suggested to be a fungal analog of the Nox complex
organizer p40phox, based on a PB1 protein–protein inter-
action domain [20]. Indeed, in E. festucae not only Rac but
also Bem1 and the guanine nucleotide exchange factor,
Cdc24, interact with NoxR [21,29]. Yeast-two-hybrid
analyses in B. cinerea and C. purpurea indicate that Cdc42
and the p21-activated protein kinases (Pak) Cla4 and
Ste20 are also partners of Nox (D. Buttermann and S.
Giesbert, pers. commun.) (Figure 1b). A key question is
what is the specific role of the different Nox complexes?
Apart from A. nidulans all filamentous fungi possess two or
three Nox homologs that have different functions, though
both are activated by the same regulator, NoxR. Does the
composition of the complexes differ, and how are they
recruited to their specific site of action? In M. oryzae, P.
anserina and B. cinerea a connection between NoxB and a
trans-membrane protein — the tetraspanin Pls1 — was
indicated by the similar phenotype of nox2/B and pls1
mutants [18,22]; a phylogenetic analysis showed a close
correlation between the presence of NoxB and Pls1
encoding genes [23]. Thus, Pls1 could be a good candi-
date for a NoxB-interacting protein involved in its recruit-
ment to the site of action. In B. cinerea the different
impact of the two Nox on formation of CATs was used
to group the potential interaction partners. Bem1 has a
strong effect on CAT formation and hence probably
belongs to the NoxA complex, whereas Cdc42 and Pls1
have minor effects on CAT formation suggesting they
work with NoxB ([18], U. Siegmund, pers. commun.).
While it is clear that ROS production is important for
signaling, the pathways involved are still unclear other
than a general connection with mitogen-activated protein
kinases (MAPK) cascades. Expression of noxA/B in B.
cinerea is controlled by Bmp3, an Slt2 homolog [12]. The
impact of NoxA on CAT formation supports this link,
since in N. crassa CAT formation is controlled by a MAPK
[24]. In P. anserina nuclear localization of PaMpk1, a
homolog of SLT2, a key component of the cell-integrity
pathway, is dependent on PaNox1 with both mutants
showing a similar phenotype [25]. Correspondingly, a
transcriptomic approach showed that in panox and pampk
mutants more than 1000 genes were equally affected.
This is mainly due to a comparable growth pattern,

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656 Growth and development: eukaryotes

Figure 2

(a)

(b)

cytosol
NoxA NoxB
ER lumen

e- e-

Ero1αox
PDIox H2O2
?

O2

GSH
e- Ero1αred
PDIred

GSSG

oxidative protein
folding

timed export of
correctly folded
proteins
Current Opinion in Microbiology

Association of Nox with the endoplasmic reticulum (ER). (a) Cellular localization of BcNoxA: protein localization was determined by epifluorescence
microscopy in germlings of the B. cinerea strain DbcnoxA:GFP-NoxA expressing a gfp-bcnoxA gene fusion. BcNoxA localizes to intracellular
membrane structures including the nuclear envelope, that is, the ER, and partly also to the plasma membrane. (b) Hypothetical scheme of the putative
function of Nox in the ER: Nox are localized in the ER membrane, where they possibly contribute to the ER redox status. Besides GSH (glutathione),
Nox might be a second supplier of electrons to PDI (protein disulphide isomerase). PDI is involved in oxidative protein folding, which is crucial for the
function and the exactly timed and localized export of many proteins. PDI in turn is oxidized, for example, by Ero1 (ER oxidoreduction, an ER-resident
flavoprotein) by transfer of electrons to O2, yielding H2O2 (based on Refs. [28,32,33,34]).

making an attribution of specific pathways controlled by
PaNox1 difficult [26].
Localization
In mammalian cells the different Nox are associated with
the plasmalemma and with quite different cell compart-
ments including the nucleus, nuclear envelope, endoplas-
mic reticulum (ER), specialized endosomes and
mitochondria [3,27,28]. Information on the localization of
Nox in fungi is limited. Localization at the plasmalemma, as
suggested by indirect evidence in E. festucae from the apical
localization of NoxR and complex partners [18,29],
would result in extracellular ROS generation, which could
influence the structure of the cell wall or matrix [4].
Alternatively, ROS could constitute a signal taken up by
sensors and transformed to intracellular signals (Figure 3
and [1]). Still localization experiments are missing using
functional Nox:GFP fusion proteins that are able to comp-
lement the corresponding nox mutants. To our knowledge
the only example where this approach was successful is B.

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 Reactive oxygen species generation in fungal development and pathogenesis Tudzynski, Heller and Siegmund 657

Figure 3

ROS

other ?
ER signals ROS

Respiration other ROS

producer
Mitochondria
MAPK
Nucleus
Actin
?
TF ?
?
Development
? NoxR

Rac NoxA/ Signal

Redox Status Vesicle
NoxB
ROS
ROS
ER

Current Opinion in Microbiology

Summary of the present knowledge on function and localization of fungal NADPH oxidases (Nox). In fungi Nox are located mainly in the endoplasmic
reticulum (ER), in the nuclear envelope and in the plasma membrane. Within the ER Nox might regulate the redox status to ensure proper protein
folding and to regulate development. Additionally, Nox can be transported via vesicles from the ER to the plasma membrane, where they seem to
produce reactive oxygen species (ROS) extracellularly. As the intracellular and extracellular ROS concentration in Nox knockout mutants is not
reduced, ROS produced by Nox probably serve exclusively signaling functions. However, also an influence on the cell wall structure during
development is possible. As shown for M. oryzae, fungal Nox clearly affect the actin cytoskeleton (N. Talbot, pers. commun.), and thus regulate fungal
development. The underlying molecular mechanisms are not yet understood. Besides Nox other intracellular ROS producer must exist that influence
the intracellular and extracellular ROS level. At the transcriptional level Nox seem to be connected to mitogen-activated protein kinases (MAPK) that in
turn can take up signals (ROS) emerging from Nox.

cinerea [18]. Here both NoxA and NoxB localize mainly to
the nuclear envelope and ER in general (Figure 2a).
Whether this represents just a transient localization at
the ER membrane, with the ultimate function being at
the plasma membrane is unknown. Several cellular
differentiation processes such as bud and septum for-
mation require exactly timed and localized secretion of
enzymes or signaling components from the ER [30,31].
However, a direct role in the ER is also possible as
mammalian Nox4 contributes to the ER redox status
[32]. This is crucial for many cellular processes via its
impact on the correct modification and folding of
proteins, especially disulphide bond formation by
protein disulphide isomerases (PDI) which require high
oxidative potential which, apart from ER-specific oxido-
reductases like ERO1, could be supported by Nox
(Figure 2b) [33,34]. A possible ER function of fungal
Nox is substantiated by the recent observation in P.
anserina that mutation of pro41, encoding a homolog of
an ER protein involved in perithecia development in
Sordaria macrospora [35], leads to the same phenotype as
a nox1 deletion (P. Silar, pers. commun.). Also the yeast
homolog Yno1 is mainly localized in ER membranes [7].
In summary, data to date suggest that fungal Nox have
two major cellular locations: the plasmalemma and the
ER (Figure 3).
Alternative ROS sources
Investigations in several fungi indicate that Nox are not
the only ROS generating systems. Deletion of both nox
genes in B. cinerea had no impact on ROS secretion [12]
and in M. oryzae the noxA/B double mutants showed
reduced ROS level in appressoria, but significantly
increased ROS content in hyphae [36]. An increased
ROS level in nox mutants was also observed in P. anserina
[9], demonstrating that there are alternative ROS sources,
and that they are influenced by Nox. Apart from other
enzyme systems (e.g. xanthine oxidases) mitochondria
might be the major source for ROS as shown for mam-
malian systems [37,38], but fungal data are so far limited.
In A. fumigatus the role of mitochondria in oxidative stress
homeostasis and pathogenesis was confirmed [39]. In
mammalian cells glutathione and thioredoxin modulate
mitochondrial ROS production [40], fitting the obser-
vation that in B. cinerea a thioredoxin reductase mutant
secretes significantly more ROS (A. Viefhues, P. Tud-
zynski unpublished data). Another interesting aspect is
the link in mammalian cells between ‘ER-stress’ and

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658 Growth and development: eukaryotes
induction of mitochondrial ROS production [33]. An
understanding of this process could help to understand
the deregulation of ROS production in fungal Nox
mutants.
Conclusions/perspectives
Since the emerging importance of the redox status for
many essential aspects of fungal biology and the apparent
central impact of Nox complexes research in this field has
been stimulated. We anticipate that many of these out-
standing questions will soon be answered. From our point
of view the following aspects deserve special attention:
1. Localization and assembly of the Nox complexes and a
focus on growth/developmental stages in which the Nox
complexes are essential. This will require detailed
reporter gene studies, with functional fusion constructs
(in the corresponding mutant background), BIFC
analyses, and the use of mutants of the possible
complex partners.
2. The possible function of Nox in the ER by using, for
example, the redox sensor technology (roGFP) recently
adopted in our laboratory for B. cinerea [41]; the impact
of Nox on the ER redox status and the orientation of
Nox in the ER membrane could be analyzed.
3. Complex composition: using adequate techniques
membrane proteins partners of NoxA/B must be
identified; direct interaction between NoxR and the
catalytic subunits still has to be shown.
4. Positioning of the Nox complexes in the cellular
regulatory network: the impact of the cell-integrity
MAPK pathway [12] and Rho GTPases [42] on nox
expression should be analyzed in detail; in addition,
regulatory circuits identified in mammalian cells like
lipid signaling are likely to be also relevant in fungi [43].
Acknowledgements
We thank P. Silar, N. Talbot, D. Buttermann, S. Giesbert, A. Herrmann, A.
Viefhues for providing unpublished data, and B. Scott for critical reading of
the manuscript. Work in our lab is supported by the Deutsche
Forschungsgemeinschaft (DFG; Tu50-17).
References and recommended reading
Papers of particular interest, published within the period of review,
have been highlighted as:
 of special interest
 of outstanding interest
1. Heller J, Tudzynski P: Reactive oxygen species in
phytopathogenic fungi: signaling, development, and disease.
Annu Rev Phytopathol 2011, 49:369-390.
2. Brown DI, Griendling KK: Nox proteins in signal transduction.
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An excellent review on the wide distribution and potential functions of
Nox.
5. Marino D, Dunand C, Puppo A, Pauly N: A burst of plant NADPH
 oxidases. Trends Plant Sci. 2012, 17:9-15.
Very concise overview of the state of art in plant Nox research.
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oxygen species and development in microbial eukaryotes.
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 Weinberger M, Stolze K, Grousl T, Hasek J et al.: Yno1p/Aim14p,
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reactive oxygen species generation, apoptosis, and actin
cable formation in yeast. Proc. Natl. Acad. Sci. U. S. A. 2012,
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A breakthrough paper providing evidence for an NADPH oxidase-like
enzyme in yeast, breaking the link between Nox activity and multicellularity.
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NADPH oxidases NOX-1 and NOX-2 require the regulatory
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First paper linking Nox activity with a newly described form of fungal
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New aspects of Nox function in fungal differentiation, linking development
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 Steinberg G, Talbot NJ: Septin-mediated plant cell invasion by
the rice blast fungus, Magnaporthe oryzae. Science 2012,
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A breakthrough paper, especially if the impact of Nox on this process is
considered (is not yet mentioned in this paper).
18. Siegmund U, Heller J, van Kan J, Tudzynski P: The NADPH oxidase
 complexes in Botrytis cinerea: evidence for a close association
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The first localization of a catalytic Nox subunit using a functional reporter
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 Reactive oxygen species generation in fungal development and pathogenesis Tudzynski, Heller and Siegmund 659
21. Takemoto D, Tanaka A, Scott B: A p67phox-like regulator is
recruited to control hyphal branching in a fungal-grass
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Very nice perspective paper.
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Current Opinion in Microbiology 2012, 15:653–659