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PY49CH18-Tudzynski ARI 8 July 2011 9:34

Reactive Oxygen Species


in Phytopathogenic Fungi:
Signaling, Development,
and Disease
Jens Heller and Paul Tudzynski
Molecular Biology and Biotechnology of Fungi, Institute of Biology and Biotechnology of
Plants, Westf alische Wilhelms-Universit at M unster, Germany;
email: jens.heller@uni-muenster.de, tudzyns@uni-muenster.de
Annu. Rev. Phytopathol. 2011. 49:36990
The Annual Review of Phytopathology is online at
phyto.annualreviews.org
This articles doi:
10.1146/annurev-phyto-072910-095355
Copyright c 2011 by Annual Reviews.
All rights reserved
0066-4286/11/0908/0369$20.00
Keywords
NADPH oxidases, host-pathogen interaction, oxidative burst,
oxidative stress response, fungal development
Abstract
Reactive oxygen species (ROS) play a major role in pathogen-plant
interactions: recognition of a pathogen by the plant rapidly triggers the
oxidative burst, which is necessary for further defense reactions. The
specic role of ROS in pathogen defense is still unclear. Studies on
the pathogen so far have focused on the importance of the oxidative
stress response (OSR) systems to overcome the oxidative burst or of
its avoidance by effectors. This review focuses on the role of ROS for
fungal virulence and development. In the recent years, it has become
obvious that (a) fungal OSRsystems might not have the predictedcrucial
role in pathogenicity, (b) fungal pathogens, especially necrotrophs, can
actively contribute to the ROS level in planta and even take advantage
of the hosts response, (c) fungi possess superoxide-generating NADPH
oxidases similar to mammalian Nox complexes that are important for
pathogenicity; however, recent data indicate that they are not directly
involved in pathogen-host communication but in fungal differentiation
processes that are necessary for virulence.
369
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PY49CH18-Tudzynski ARI 8 July 2011 9:34
ROS: reactive oxygen
species
NADPH-dependant
oxidase complex
(Nox): superoxide
generating enzyme
system
Oxidative burst:
reactive oxygen species
spike in response to
pathogen attack
INTRODUCTION
Single-celled organisms were the rst to appear
on Earth under an atmosphere very low in
oxygen. The introduction of oxygen into the
atmosphere provided an environment that
allowed the evolution of complex multicellular
organisms with high-energy demands. How-
ever, simultaneously it brought about a new
source of toxins. Although oxygen is by nature a
weak reactant, it has a tendency to readily form
radicals, either by energy transfer reactions or
by electron transfer reactions forming incom-
pletely reduced reactive oxygen species (ROS),
which are highly potent oxidants. Energy
transfer leads to the formation of singlet oxy-
gen (
1
O
2
), whereas electron transfer results in
the sequential reduction to superoxide (O
2

),
hydrogen peroxide (H
2
O
2
), and hydroxyl radi-
cal (OH

) (51). Within the cell, these and other


short-lived ROS can react nonspecically and
rapidly with macromolecules, including DNA,
proteins, lipids, and carbohydrates. ROS cause
molecular damage such as DNA mutations,
lipid peroxidation, and protein oxidations,
eventually leading to cell death and progressive
aging of the organism (12). Because ROS are
continuously produced as byproducts of var-
ious metabolic pathways localized in different
cellular compartmentsmainly in mitochon-
dria, peroxisomes, and chloroplaststhey have
historically been observed as a harmful but
unavoidable outcome of an aerobic lifestyle.
Under physiological steady state conditions,
these molecules are scavenged by different
antioxidative defense components. However, if
the equilibrium between production and scav-
enging of ROS is perturbedfor example, by
adverse environmental factorsintracellular
levels of ROS can rise, resulting in changes
in the cell transcriptome. In addition, local
bursts of ROS have been shown to be involved
in various differentiation processes. This
indicates that in spite of their toxic effect, most
organisms have adapted to the existence of
ROS and have evolved processes to use them as
second messengers to transduce extracellular
signals to the nucleus (81). However, ROS are
not only involved in intracellular signaling,
they also play a role in cell-cell interaction
and in the interaction of different organisms.
In plant-microbe and phagocyte-pathogen
interactions, ROS are mainly involved in
recognition or defense reactions.
In this review, we discuss the role of ROS
in the interaction of phytopathogenic fungi and
plants. In the past fewyears, many excellent and
comprehensive reviews have been published,
mainly describing the importance of ROS for
defense reactions of plants against pathogens
(76, 84, 98, 107, 108). Here, we focus on the
effects of ROS in the fungal partner during the
interaction and in this context outline the im-
portance of ROSfor signaling and development
in lamentous fungi.
Reactive Oxygen Species
Generating Systems
Mitochondria are the major source of intracel-
lular ROS. However, there are several other en-
zymatic and nonenzymatic systems involved in
cellular ROS production (36). The most impor-
tant enzymatic ROS generating system is the
NADPH-dependent oxidase complex (Nox).
The best-studied member of this group of en-
zymes is the mammalian gp91
phox
(also known
as Nox2) responsible for the neutrophil oxida-
tive burst defense response (57). Nox2 catalyzes
the production of superoxide by a one-electron
reduction of oxygen using NADPH as electron
donor. Generation of ROS by Nox requires the
assembly of a multi-subunit complex. This is
composed of the regulatory cytosolic compo-
nents p40
phox
, p47
phox
, p67
phox
, and the small
GTPase Rac, which come together as a com-
plex, and the integral membrane protein avo-
cytochrome b
558
, composed of the catalytic sub-
unit gp91
phox
and the adaptor protein p22
phox
,
which are localized in endosomal membranes.
Upon cell stimulation, the p47
phox
subunit is
phosporylated and the entire cytosolic complex
is recruited to the membrane, where it asso-
ciates with the two membrane-bound compo-
nents to assemble the active oxidase complex
and produce O
2

. O
2

can be converted to a
370 Heller

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PY49CH18-Tudzynski ARI 8 July 2011 9:34
MAPK:
mitogen-activated
protein kinase
number of other reactive oxidants that are used
by phagocytes to kill invading microorganisms.
Along with gp91
phox
, six other enzymes of the
Nox family have been described in different hu-
man tissues (57). Therefore, the generation of
ROS is not a unique characteristic of phago-
cytes but probably a general feature of all hu-
man cells. Nox enzymes are also found in a wide
range of other organisms, where presence of
this enzyme has beenproposedtocorrelate with
multicellular status (54).
Reactive Oxygen Species Scavenging
Most likely all organisms have to cope with
oxidative stress conditions during their life-
span. These conditions can be caused either
by ROS produced as byproducts during normal
metabolic processes or by an environment with
unfavorable ROS levels. Therefore many or-
ganisms have evolved oxidative stress response
(OSR) mechanisms to scavenge elevated in-
tracellular ROS levels. These mechanisms can
generally be divided into enzymatic and nonen-
zymatic systems.
Nonenzymatic defense systems typically
consist of small soluble molecules that are
oxidized by ROS and thereby remove oxi-
dants from solution. They include the ma-
jor cellular redox buffer glutathione (GSH, a
tripeptide -L-glutamyl-L-cysteinyl-glycine),
but also other compounds like phytochelatins,
ascorbic acid, polyamines, avonoids, alkaloids,
andcarotenoids (46). GSHis a ubiquitous thiol-
containing reductant that maintains the intra-
cellular redox homeostasis by reducing cellu-
lar disulde bonds and detoxifying damaging
molecules, such as xenobiotics and heavy met-
als. During the reaction, GSH is converted to
its oxidizedform, glutathione disulde (GSSG).
However, the cell maintains a fairly high intra-
cellular concentration of GSH and a high ratio
of GSH to GSSG by the action of glutathione
reductase, using NADPHas the electron donor
(6).
Enzymatic ROS scavenging mechanisms
include superoxide dismutase (SOD) and vari-
ous peroxidases, such as glutathione peroxidase
(GPX), peroxiredoxin, and catalase (CAT).
SODs dismutate O
2

to H
2
O
2
and act as the
rst line of defense. H
2
O
2
can then be con-
verted to H
2
O by the action of GPX and CAT.
ROS scavenging systems are crucial for sup-
pressing toxic ROS levels in a cell, and the OSR
has to be regulated very tightly. In yeast and
ssion yeast, there are several signaling compo-
nents that control the OSR at a transcriptional
level, including a mitogen-activated protein ki-
nase (MAPK) cascade (Hog1/Sty1) and a redox
sensitive transcription factor (Yap1/Pap1) (39,
44). The MAPK pathway is activated not only
in response to oxidative stress, but in response
to multiple stresses (18). After activation, the
MAPK acts as a transcription factor itself or
activates the transcription machinery by phos-
phorylating other transcriptionfactors and pro-
teins, resulting inmodulationof proteinactivity
and altered gene expression (21).
The mode of action of Yap1 is based on
redox-sensitive nuclear export: H
2
O
2
mediates
the oxidation of a peroxiredoxin that oxidizes
the highly conserved Cys-residues of the
transcription factor, causing nuclear retention.
Upon exposure to oxidative stress, Yap1
becomes localized to the nucleus, where it
induces the expression of OSR genes (53). In
ssion yeast, Pap1 and Sty1 seem to have both
overlapping and specialized roles during oxida-
tive stress, with Pap1 dominating the response
to low ROS levels and Sty1 dominating the
response to high ROS levels (113). Moreover,
activation of particular pathways depends on
the nature of the oxidant (18). However, in
a recent study, G onzalez-P arraga et al. (33)
proposed that in Candida albicans there are
Cap1 ( =Yap1)-independent OSRmechanisms
that include the transcription factors Skn7
and Msn2/4 (29). In addition, the thioredoxin
system is one of the major control systems for
cellular redox homoeostasis. It is composed
of two enzymes, the thioredoxin (Trx) and its
thioredoxin reductase (TrxR). The reduced
dithiol [Trx-(SH)
2
] is able to directly reduce
disuldes of target proteins, which is required
for several intracellular processes like sulfur
assimilation, detoxication of ROS, protein
www.annualreviews.org ROS in Phytopathogenic Fungi 371
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PY49CH18-Tudzynski ARI 8 July 2011 9:34
RBOH: respiratory
burst oxidase homolog
repair, and redox regulation of enzymes and
transcription factors (7).
Although the OSR has been studied ex-
tensively in prokaryotes and eukaryotes, the
information available on lamentous fungi is
fragmentary. However, in Botrytis cinerea, As-
pergillus fumigatus, Aspergillus nidulans, and
Cochliobolus heterostrophus Hog1 homologs ap-
pear to be essential for the OSR (23, 43, 49,
94). As in yeast, Hog1 homologs in these fungi
also seem to be involved in controlling cellu-
lar responses to several different stresses rather
than being specic just for oxidative stress. In
contrast, Yap1 homologs seem to be the main
regulators of OSR genes in lamentous fungi
(61, 62, 104). Furthermore, homologs of the re-
sponse regulator Skn7 are involved in the OSR
as they confer H
2
O
2
resistance and regulate
CAT gene expression in Aspergillus species (55,
111). However, the Skn7 homolog of the phy-
topathogenic fungus Magnaporthe oryzae is nei-
ther involved in OSR nor is it important for
pathogenicity (74). Other cellular components
controlling the OSR in lamentous fungi re-
main to be identied.
REACTIVE OXYGEN SPECIES IN
EARLY PLANT MICROBE
INTERACTIONS
In the early events of plant-microbe interac-
tions, the rapid and transient production of
ROS (oxidative or respiratory burst) by plants
is mainly caused by membrane-associated res-
piratory burst oxidase homologs (RBOHs),
whichare homologs of the mammaliangp91
phox
(109). This enzyme complex is involved in both
symbiotic and pathogenic interactions. There-
fore, this typically apoplastic ROS production
could be considered as a specic signal during
the interaction process (63). Nevertheless, the
course of ROS formation differs between sym-
biotic and pathogenic interactions. Avirulent
pathogens induce a biphasic ROS accumulation
in plants with a transitory, low amplitude rst
phase that usually takes place within minutes
after the rst contact and a second one con-
sisting of a sustained phase that occurs hours
afterward and that is usually associated with the
establishment of defense reactions and the hy-
persensitive response (2, 35, 83). Just the rst
burst has been observed in virulent pathogens
and in symbiotic interactions, suggesting there
is suppression of the second wave of ROS (97).
The mechanism that underlies this suppression
is still unknown. Nevertheless, the second re-
sponse obviously plays an important role for the
exclusion of pathogens showing that ROS are
key factors in the establishment of plant defense
responses (63).
Mutualistic Interactions
The best-studied microbial symbiotic interac-
tions of plants are those between plants of the
family Leguminosae and bacteria (rhizobia)
(75). Although rhizobia colonize roots in a
way that resembles colonization by pathogenic
microorganisms, almost no host plant defense
reactions are triggered during successful sym-
bioses. In these symbiotic interactions, there is
an initial response by the host to the bacteria,
but they then appear to suppress the plant
defense responses (9, 79, 112). Accordingly,
the involvement of ROS in this interaction
has been shown: Alfalfa responds to infection
with Sinorhizobium meliloti by production of
superoxide and hydrogen peroxide (89). The
symbionts use protecting mechanisms like
ROS-scavenging enzymes to counteract the
plant defense response and particularly sus-
tained ROS accumulation (45). However, since
both application of extracellular ROS and inhi-
bition of plant NADPH oxidases by dipheny-
lene iodonium (DPI) treatment suppressed
the root hair curling and infection thread
formation, ROS are suggested to regulate,
rather than directly form, part of the defense
system in rhizobia-legume symbioses (82).
Mutualistic fungus-plant interactions have
been analyzed in detail using functional genetic
and genomics approaches. There is increasing
evidence that during the process of plant root
colonization by mycorrhizal fungi, ROS play
an important role; e.g., volatile compounds
secreted by the trufe Tuber melanosporum
372 Heller

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PY49CH18-Tudzynski ARI 8 July 2011 9:34
Arbuscular
mycorrhiza: most
widespread form of
mycorrhiza that
include formation of
intricately branched
haustoria in cortex
cells of plant roots, the
arbuscules
PAMP: pathogen-
associated molecular
pattern
PTI: PAMP-
triggered immunity
Biotrophic fungi:
pathogens feeding and
depending on living
cells
Necrotrophic fungi:
pathogens able to kill
their host cells and to
feed on dead plant
tissue
Appressoria:
unicellular fungal
penetration organs in
which high osmotic
pressure is generated
induce an oxidative burst in Arabidopsis thaliana
(100). In arbuscular mycorrhizal interactions,
the most widespread terrestrial symbiosis (14)
accumulation of ROS, catalase, peroxidase, and
SODtranscripts has been observed (26, 32, 56).
A proteomics study of the interaction of two
Glomus species and Medicago truncatula iden-
tied several fungal proteins involved in redox
homoeostasis, including the Cu/Zn-SOD,
thioredoxin peroxidase, thioredoxin reductase,
and other enzymes (85). In the ericoid mycor-
rhizal fungus Oidiodendron maius, disruption of
a sod gene caused increased sensitivity against
external ROS and a reduction in mycorrhiza-
tion (1). These data strongly suggest that ROS
and redox homeostasis in general are crucial
for establishment and maintenance of the my-
corrhization process. Similarly, ROS has been
shown to be important in fungal endophytic
partnerships; e.g., for the interaction of Epichloe
festucae and perennial ryegrass growth of the
hyphal tip, and branching is proposed to be
controlled by localized bursts of ROS across
the plasma membrane in response to signaling
from the grass host (101).
Pathogenic Interaction: Dealing With
Plant Oxidative Burst
For pathogenic interactions between plants and
microbes, ROS are of considerable importance.
There are two major types of plant resistance
mechanisms against pathogen attack: the
general pathogen-associated molecular pattern
(PAMP)-triggered immunity (PTI) and the
more specic effector-triggered immunity
(ETI) stimulated by plant surveillance proteins
(R-proteins) recognizing specic effector
proteins of the pathogen (AVR protein) (48).
ROS production at the infection site is the
earliest response of PTI. Apart from primary
effects such as cell wall strengthening and
induction of antimicrobial activity, ROS
function as diffusible second messengers,
inducing several resistance responses including
synthesis of pathogenesis-related proteins and
phytoalexins, and programmed cell death in
neighboring cells (27, 117). The lifestyles
of phytopathogenic fungi can be generally
divided into two different groups. On the one
hand, there are biotrophic pathogens that
depend on living plant material and therefore
may not kill their host. On the other hand,
there are necrotrophic pathogens that subsist
from dead plant tissues. However, several
important plant pathogens like M. oryzae and
Colletotrichum species show an intermediate
infection strategy, termed hemibiotrophism,
characterized by an initial biotrophic phase
that changes into a necrotrophic one during
infection. The infection strategies of the partic-
ular groups differ from each other. Biotrophic,
and hemibiotrophic, fungi have developed
strategies to inhibit or overcome the plant PTI,
e.g., by effector-triggered susceptibility (ETS)
in which pathogen effectors counteract the
PTI. Necrotrophic pathogens seem to exploit
(at least in part) these defense mechanisms to
facilitate infection and colonization of the host
(34). Accordingly, the consequence of the plant
oxidative burst appears to differ between both
groups: Although this early response reaction
(if not blocked) can prevent the penetration
and spread of biotrophic fungi (67), it does
not inhibit the successive colonization of
necrotrophic fungi. However, this simple
black and white view does not really account
for the complex reality of ROS function in
such interactions (see below). As H uckelhoven
& Kogel (41) pointed out, different types of
ROS have different functions, and timing and
concentration are also important.
Biotrophs/hemibiotrophs. During infection
of maize by the basidiomycete Ustilago maydis,
plant cells stay alive and no apparent defense
responses are triggered (11). Nevertheless, this
biotrophic fungus needs to be able to respond
to an oxidative burst to be fully virulent. The
response is mediated by a system related to the
Yap1p regulator. U. maydis yap1 deletion mu-
tants show a higher sensitivity to H
2
O
2
than
wild-type (wt) cells and display reduced prolif-
eration in the infected tissue (72). H
2
O
2
accu-
mulates around the intracellular hyphae of the
yap1 mutant, but this response is not seen for wt
www.annualreviews.org ROS in Phytopathogenic Fungi 373
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PY49CH18-Tudzynski ARI 8 July 2011 9:34
Hemibiotrophic
fungi: pathogens with
an early biotrophic
growth phase that
switches into a
necrotrophic one
during infection
hyphae. Transcriptome analysis revealed that
a number of genes, including two peroxidase
genes, are downregulated in the mutant. More-
over, pharmacological inhibition of Noxs, the
main producers of defense-linked ROS in plant
tissue, largely restored virulence of yap1 dele-
tion strains (72). These ndings suggest that
virulence of U. maydis depends on its ability to
detoxify ROS. Nevertheless, it cannot be ex-
cluded that the Yap1p controls, in addition to
ROS scavenging enzymes, fungal effectors that
suppress the host oxidative burst.
The rust fungus Uromyces fabae converts
much of the carbohydrates it takes up during
the infection process of its host Vicia faba into
the C6-polyol mannitol. This compound is a
good quencher of ROS. Indeed, the scavenging
capacity of mannitol found in the apoplastic u-
ids of the host during infection is sufcient to
suppress the ROS built up during the defense
reaction to about one-half the level present in
the absence of mannitol. These results suggest
that quenching of ROS might be essential for
successful infection of this biotrophic fungus
(114).
For the biotrophic pathogen Claviceps
purpurea, which infects owers of grasses and
cereals, a classical oxidative burst-like plant de-
fense reaction has never been observed during
the successful colonization of rye ovarian tissue.
Only in the outer cell layer of the stigmatic
hairs is ROS formation detectable during pen-
etration. Once hyphae are inside the hair and
grow within the plant tissue toward the ovary
there is no change in ROS levels (90). Deletion
of genes encoding the single-secreted SODand
the major-secreted CAT(which is even present
in large amounts in the honeydew of infected
plants) did not signicantly reduce fungal vir-
ulence (28, 73). However, deletion of the gene
cptf1 encoding a homolog of the ssion yeast
transcription factor ATF1, which is a general
regulator of CAT activity in C. purpurea, led
to an oxidative burstlike reaction in the plant
tissue around the hyphae and in distal areas
(78). Preliminary macroarray data indicate that
Cptf1 controls a large set of genes, in addition
to those encoding ROS-scavenging enzymes
(E. Nathues & P. Tudzynski, unpublished
data). Therefore, like in U. maydis, it remains to
be elucidated whether the absence of an oxida-
tive burst in the pathogen-host interaction is
caused by active fungal suppression of the plant
defense reaction. Regardless, ROS and the
general redox homoeostasis appear to be essen-
tial also for this biotrophic interaction. This
conclusion is substantiated by the observation
that the histidine kinase CpHK2, a candidate
for a sensory component of ROS signaling, is
involved in virulence of this fungus (77).
Hemibiotrophic fungi provide an interest-
ing system to compare the necrotrophic and
the biotrophic lifestyle in one system. Dur-
ing the biotrophic early infection phase of the
hemibiotrophic wheat pathogen Septoria tritici,
noaccumulationof H
2
O
2
is detectable, whereas
in the necrotrophic phase, just before sporu-
lation, there is a massive production of ROS
(99). A recent study by Chi et al. (20) of the
hemibiotrophic fungus M. oryzae showed that
there are important fungal factors inuencing
the ROS status in the pathogen-plant interac-
tion beyond the usual candidates involved in
OSR and ROS generation. The gene des1 (de-
fense suppressor 1) was identied during a ran-
dommutagenesis screen using a T-DNA inser-
tional mutant library. It encodes a serine-rich
protein with unknown biochemical properties,
which is highly conserved within lamentous
ascomycetes. Mutants lacking Des1 show nor-
mal vegetative growth, formationof appressoria
and penetration of host tissue. However, they
are highly sensitive toROSinaxenic culture and
are impaired in secretion of ROS scavenging
enzymes (e.g., peroxidases). In planta mutant
growth is signicantly impaired and accompa-
nied by a strong oxidative burst and establish-
ment of general resistance reactions (in a nor-
mally susceptible rice cultivar). Addition of the
Nox inhibitor DPI reduced the plant defense
reaction and restored virulence of the fungus.
However, it cannot be excluded that DPI also
affects the fungal Noxs. Thus, Des1 represents
a new type of fungal pathogenicity factor in-
volved in modulating ROS-mediated plant de-
fense. Obviously, it has no signicant effect on
374 Heller

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Hypersensitive
reaction (HR):
mechanism used by
plants to prevent the
spread of infection by
microbial pathogens,
which includes the
rapid cell death in the
local region
surrounding an
infection
the intracellular ROS status. The authors spec-
ulate that Des1 might be involved in metal ion
homoeostasis.
These ndings lead to the assumption that
biotrophic and hemibiotrophic fungi depend
on the prevention of a strong oxidative burst
and the hypersensitive response of their host,
either by completely suppressing the PTI de-
fense reactions by suppressors or by scavenging
the ROS built by their hosts during the initial
phase of the infection process.
Necrotrophs. Most necrotrophic fungi be-
have completely differently. During successful
infection of the gray mold fungus B. cinerea, a
classical necrotrophic pathogen with high eco-
nomic importance, high levels of ROS can be
detected in all analyzed tissues indicating that
a strong oxidative burst takes place. This has
been demonstrated by classical staining tech-
niques using DAB (diamino benzaledehyde),
NBT (nitroblue tetrazolium chloride), and u-
orescent dyes (e.g., dichlorodihydrouorescein
diacetate, H
2
DCF-DA), by the more sensitive
electron microscopic cerium chloride staining
technique andby physicochemical methods (68,
92) (see Figures 1 and 2). The evidence for a
strong oxidative burst indicates that B. cinerea
needs an effective ROS scavenging system to
protect itself against the plant-derived ROS.
However, deletion of a gene coding for the
major secreted CAT had no effect on viru-
lence, although the mutant has reduced H
2
O
2
-
resistance in axenic culture (92). Although this
observation could be explained by the pres-
ence of multiple ROS detoxication systems
as revealed by the candidate proteins predicted
from the genome sequence, functional analysis
of bap1 (encoding a homolog of Yap1p) showed
that this major (and in axenic culture essential)
H
2
O
2
scavenging systemis neither required for
plant infection nor is it induced in planta (104).
This strongly suggests that B. cinerea does not
face oxidative stress in planta, at least not via
H
2
O
2
. It cannot be excluded, however, that al-
ternative OSRsystems provide protection from
plant-generated ROS, e.g., the ortholog of the
yeast Skn7 (see above). A secreted SOD has
been shown to be important for full virulence,
as deletion of the gene bcsod1 led to retarded
lesion development (87). However, this might
be due to its function in O
2

detoxication
or to its ability to produce H
2
O
2
. The latter
hypothesis is supported by data presented by
Tiedemann (106), demonstrating that the ag-
gressiveness of a given isolate of B. cinerea cor-
relates with the intensity of the oxidative burst
it induces. Accordingly, B. cinerea is almost
apathogenic on hypersensitive reaction (HR)-
decient Arabidopsis mutants. Moreover, pre-
treatment of Arabidopsis plants withHR-causing
bacteria enhances the spreading necrosis of
B. cinerea, whereas pretreatment with viru-
lent bacteria that cause no HR does not (34).
Thus, instead of suppressing the plants oxida-
tive burst, B. cinerea seems to exploit this de-
fense reaction and might even contribute to it.
Similar results have alsobeenobtainedfor other
necrotrophic fungi, e.g., in Leptosphaeria macu-
lans, a pathogen of Brassica napus (64, 65).
In Colletotrichum coccodes secretion of ammo-
nium ions increases the oxidative burst and en-
hances virulence on tomato (5; see Reference
84). However, Shetty et al. (99) pointed out
that the ability of fungi to grow within the host
tissue in spite of ROS accumulation does not
necessarily mean that they need the oxidative
burst: reduction of ROS formed in planta dur-
ing the necrotrophic growth of S. tritici by in-
ltrating CAT resulted in enhanced growth of
the pathogen. Obviously, the fungus can grow
in the presence of ROS but does not necessarily
need to produce this product to be virulent.
Inthe maize pathogenCochliobolus heterostro-
phus, deletion of a gene encoding a secreted
CAT(Cat3) that confers H
2
O
2
resistance in ax-
enic culture had no effect on pathogenicity (86).
As in B. cinerea deletion of chap1 (encoding a
Yap1p homolog) in C. heterostrophus resulted in
increased sensitivity to oxidative stress caused
by hydrogen peroxide and menadione in axenic
culture, but it did not affect the virulence (62).
However, the expression of Chap1-controlled
genes in planta has not yet been analyzed.
These examples indicate that several
necrotrophic fungi, although inducing a strong
www.annualreviews.org ROS in Phytopathogenic Fungi 375
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PY49CH18-Tudzynski ARI 8 July 2011 9:34
50 m
10 m
10 m
50 m
15 m
5 m
a
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d
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PY49CH18-Tudzynski ARI 8 July 2011 9:34
oxidative burst, do not depend on an effective
ROS scavenging system. The reasons for this
are still unknown. A similar situation has been
shown for animal pathogens: ROS hyper-
sensitive mutants (obtained, for example, by
deletion of yap1 and sod genes) in A. fumigatus
were not reduced in virulence (58, 61). Inter-
estingly, in a phylogenetic study comparing
stress signaling pathways in fungi, Nikolaou
et al. (80) pointed out that plant pathogenic
fungi in general are more sensitive to oxidative
stress than animal pathogens, indicating that
there is no strong selection for oxidative
stress resistance in fungus-plant interactions.
Alternaria alternata seems to represent an
exception. In this necrotrophic pathogen,
the Yap1p homolog AaAP1/RLAP1, which
regulates OSR enzymes, plays an essential role
during fungal pathogenesis, as the deletion
mutant does not cause any visible necrotic
lesions on wounded or unwounded leaves (66,
115). However, it cannot be excluded that the
transcription factor controls other components
essential for infection. Microscopic analyses
revealed the presence of H
2
O
2
at cell walls of
appressoria and penetration pegs but not at
host cell walls underneath the appressoria of
A. alternata wt. Therefore, Jennings et al. (47)
proposed that most ROS in the interaction of
A. alternata and its host do not originate from
the plant but from the fungus. Thus, there
might be reasons for the pathogenicity defect
of AaAP1/RlAP1 deletion strains other than
enhanced sensitivity against the plant oxidative
burst.
Role of Fungal Reactive
Oxygen Species
The importance of ROS originating from the
pathogen rather than from the host for viru-
lence is described for other systems as well. The
recent discovery of functional members of the
Nox family within lamentous fungi has led
to increased speculation regarding the possi-
ble role of pathogen-derived ROS in virulence
(60). Filamentous fungi possess three distinct
subfamilies of Nox enzymes (4). Two of them
(NoxA and NoxB) are homologs of the mam-
maliancatalytic subunit gp91
phox
anda thirdone
(NoxC) contains putative calcium-binding EF-
hand motifs, a feature of human Nox5 and the
plant Nox homologs. Furthermore, fungi con-
taina homologof the regulatory subunit p67
phox
(NoxR) and of the small GTPase Rac (102).
Other components of the fungal Nox complex
are yet to be unequivocally identied.
Cytochemical analysis showed that during
the infection process of B. cinerea, O
2

ac-
cumulates in fungal hyphal tips and H
2
O
2
is
generated in and around the penetrated cell
wall as well as in the plant plasma membrane

Figure 1
In vivo localization of reactive oxygen species (ROS) produced by plants during the infection process of
Botrytis cinerea using the uorescent probe dichlorodihydrouorescein diacetate (H
2
DCF-DA). Bean leaves
were inoculated with droplets of a conidia suspension. All micrographs represent confocal laser scanning
microscopy images of different plant tissues underneath the site of infection. The dye was injected into the
leaf at the infection area prior to imaging. Left columns show brighteld images. After oxidation by ROS,
H
2
DCF-DA exhibits green uorescence (right middle). Chlorophyll autouorescence is shown in red (left
middle). The right column shows an overlay of the two middle columns. (a) In the negative control,
H
2
DCF-DA was injected into a noninfected leaf. Fluorescence of the dye is neither detectable in the
epidermis nor in the palisade parenchyma. (b) Three hours post-inoculation (hpi), ROS are detectable in
structures attached to the chloroplasts within the palisade parenchyma, showing the beginning oxidative
burst. These structures are probably peroxisomes. (c) Eighteen hpi, ROS are produced within the cell wall of
epidermal cells, probably by the action of RBOHs (top). Due to the strong ROS production in the palisade
parenchyma, ROS are even present in the intercellular space (bottom). (d ) Twenty-four hpi, the intensity of
ROS production increases in the epidermal cell wall. ROS are also detectable in nuclei of epidermal cells
(top). In the palisade parenchyma, there is a growing number of peroxisomes attached to the chloroplasts that
show strong uorescence.
www.annualreviews.org ROS in Phytopathogenic Fungi 377
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PY49CH18-Tudzynski ARI 8 July 2011 9:34
Botrytis cinerea
appressorium-like
structure
Botrytis cinerea
appressorium-like
structure
Mitochondrium Mitochondrium
O
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gt
1 m
aa b
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1 mm
Figure 2
In situ localization of the accumulation of superoxide and hydrogen peroxide in
bean and tomato leaves infected with Botrytis cinerea. (a) Localization of H
2
O
2
by the cerium chloride technique at the interface of a B. cinerea appressorium-
like structure and the outer epidermal wall of a tomato leaf at 16 hours post-
inoculation (hpi). Reactive oxygen species (ROS) of fungal origin produced by
mitochondria are located in the direct contact zone of the fungus and its host.
In the host leaf, a halo (white arrowheads) is present in the outer cell wall layer,
indicating the existence of an ROS-depleted zone. If this is due to the activity
of scavenging enzymes originating from the host or the pathogen remains to be
elucidated (picture by K.B. Tenberge). (b) Localization of superoxide by
nitroblue tetrazolium chloride (NBT) staining of bean leaves at 6 hpi. Surface
view of a germinating conidiospore (co). O
2

accumulation of fungal origin


can be detected in a hyphal tip swelling at the end of the germ tube (gt).
(picture by K.B. Tenberge and B. Hoppe) (c) Localization of superoxide by
NBT staining of a tomato leaf at 72 hpi (picture by K.B. Tenberge and
B. Hoppe). (d ) Localization of H
2
O
2
by diamino benzaldehyde staining of a
bean leaf at 48 hpi (picture by N. Temme). Most O
2

and H
2
O
2
can be
detected at the edge of the lesion, where the direct interaction of the fungus
and its host takes place. The origin of these ROS is not known.
(105) (Figure 2). These ROS are thought to
arise from the fungus and indeed B. cinerea pos-
sesses two genes encoding catalytic subunits of
NADPH oxidases. Both proteins have a great
impact on pathogenicity: Whereas bcnoxA mu-
tants are still able to penetrate host tissue in the
same way as the wt but colonization is slower,
bcnoxB mutants show a retarded formation of
primary lesions, probably due to an impaired
function of appressoria. Deletion of bcnoxR,
encoding the putative regulatory subunit
for both, NoxA and NoxB, yields the same
phenotype as the double mutant: Both strains
are almost apathogenic as they have a penetra-
tiondefect. The bcnoxRdeletionmutant canstill
form appressoria, but it is unable to use them
for penetration (Figure 3). Instead, they give
rise to new hyphal outgrowth and new rounds
of appressoria development and penetration
initiation (95). However, neither mutant is
signicantly impaired in intra- or extracellular
ROS production, suggesting that an alterna-
tive, as-yet-unknown source of ROS is involved
in ROS accumulation during plant infection.
M. oryzae seems to undergo a local oxida-
tive burst itself during plant infection, which
is associated with development of appressoria.
Scavenging of oxygen radicals as well as DPI-
mediated inhibition of Nox enzymes led to sig-
nicant delay of appressoria development and
altered morphology (25). Deletion of both nox1
and nox2 caused apathogenicity. Although in
this case the double mutant showed a faint re-
duction of ROS in the appressoria, surprisingly
the ROS level in hyphae of these mutants was
elevated, supporting the idea that the Nox com-
plex is not the only and not even a major pro-
ducer of ROS in fungi. Because other ROS gen-
erating systems must exist, it has beensuggested
that enzymes normally involved in oxidative
degradation of substrates, such as glucose ox-
idase, might be good candidates (15, 36).
The complex situation of ROS generation
and homoeostasis is illustrated in a study pub-
lished by Kim et al. (50). They showed that a
novel transmembrane protein, TmpL, has ma-
jor impact on redox homeostasis and virulence
in both a plant pathogen (Alternaria brassicicola)
and an animal pathogen (A. fumigatus). Dele-
tion of tmpl led to a massive increase in intrahy-
phal ROS levels, which was most probably not
caused by increased Nox activity. This suggests
once again that there are alternative fungal
ROS production systems. Overexpression
of the Yap1p homolog in the mutant sup-
pressed overproduction of intracellular ROS
and partly restored virulence. Interestingly,
overexpression of the Yap1p homolog in the wt
as well as treatment of wt spores with the Nox
inhibitor DPI or antioxidants led to reduced
378 Heller

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PY49CH18-Tudzynski ARI 8 July 2011 9:34
100 m
10 m
20 m
100 m
10 m
20 m
a b
c d
e f
Figure 3
Conidial germination and penetration of the plant surface by Botrytis cinerea. Detached bean leaves were
sprayed with spore suspensions and incubated for 22 hours under humid conditions. Scanning electron
microscopy (SEM) images with distinct resolutions of the wild-type (wt) strain B05.10 (a, c, e) and the
deletion strain noxR (b, d, f ) are shown. (a) The wt forms very short germ tubes before penetrating the host
surface. (b) The noxR strain forms elongated germ tubes that do not penetrate the host surface
immediately. The mutant grows on top of the host surface instead. (c) At the end of the germ tubes the wt
differentiates appressoria-like structures (white arrowhead ) and penetrates the host surface. (d ) The deletion
mutant produces appressoria-like structures as well (white arrowhead ), but it does not penetrate the host
surface. Instead, another hypha originates from the appressoria-like structure that elongates on top of the
host surface. (e) After detaching the appressoria-like structure the penetration site can be detected in the wt
(insert, white arrow head ). ( f ) No penetration site can be found in the noxR strain, showing that the Nox
complex is essential for penetration via appressoria-like structures of B. cinerea (pictures by M. Becker).
www.annualreviews.org ROS in Phytopathogenic Fungi 379
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PY49CH18-Tudzynski ARI 8 July 2011 9:34
virulence. The authors explain the pleiotrophic
phenotype observed by these treatments with
disturbance of oxidative stress homoeostasis,
substantiating the idea that intra- and extracel-
lular ROS concentrations are crucial for proper
development and virulence. Hence, it is not
surprising that in biotrophic pathogens and in
endophytes ROS generating systems also have
an inuence on the fungus host interaction.
The genome of the biotroph C. purpurea
contains two nox genes, and hence the fun-
gus probably possesses two different Nox com-
plexes. In contrast to B. cinerea and M. oryzae,
neither of these enzymes is required for pen-
etration, which in C. purpurea is not achieved
via specialized infection structures. However,
Cpnox1 is required for normal pathogenic de-
velopment, as the deletion mutant is impaired
in colonization of host tissue and cannot gain
access to the plants phloem exudate and hence
cannot produce macroscopic signs of infection
like honeydewandsclerotia (31). Amutant lack-
ing the catalytic subunit of the second Nox
complex, CpNox2, has a quite striking pheno-
type: It colonizes the host tissue very efciently
and produces vast amounts of honeydew, much
more and for a longer period than the wt; so
it could be considered to be even more viru-
lent. However, it never develops sclerotia, so it
is restricted to the sporulating stage, and would
therefore not survive in nature (D. Buttermann
& P. Tudzynski, unpublished data). Obviously,
in this fungus both Nox complexes have func-
tions in later stages of infection. One could even
argue that in C. purpurea at least the Cpnox2
complex is neededfor a balancedinteractionbe-
cause the mutant appears to be more virulent.
This would support the idea that C. purpurea
has established some kind of mutualismwith its
host, e.g., contributing some advantage for the
plant through its secondary metabolites, ergot
alkaloids, which are toxic to animals (see discus-
sion in Reference 37). Still, these data substan-
tiate the observation that endogenous ROS are
also important for this biotrophic fungus.
In the closely related endophyte E. festucae,
endogenous ROS production by a Nox com-
plex is necessary to maintain a mutualistic,
balanced interaction as well. Here, inactivation
of the noxA gene led to a switch from a mu-
tualistic to an antagonistic interaction with its
host (103). Plants infected with the E. festucae
noxA mutant showed disease symptoms and the
fungal biomass dramatically increased. ROS
accumulation was observed in the extracellular
matrix of the endophyte and at the interface
between extracellular matrix and host cell
walls of meristematic tissue in the wt but not
in noxA mutants. Deletion of the second nox
gene in this fungus, noxB, had no effect on the
plant-interaction phenotype (103).
Taken together, these data show that in ad-
dition to the requirement of ROS for an ox-
idative attack in necrotrophs, fungal-derived
ROS have a far more basic role in many host-
pathogen interactions of a completely different
nature.
REACTIVE OXYGEN
SPECIES SIGNALING
AND DEVELOPMENT
The Janus-faced nature of ROS has long been
known in animal and plant systems (10, 52):
They are toxic to cells because of the damage to
macromolecules and membranes, but they also
represent important signaling molecules acting
as secondary messengers and are involved in
differentiation processes. Hansberg & Aguirre
(38) have proposed that ROS are critical
for fungal growth and differentiation: They
suggested that differentiation is a response
to oxidative stress. Under normal growth
conditions, ROS levels are low, and generation
and scavenging are balanced. Differentiation
is dened by a (localized) increase in the ROS
level, which is transient, as it also induces
the upregulation of ROS scavenging systems.
This theory is supported by solid experimental
evidence, e.g., the upregulation of genes en-
coding specic antioxidant enzymes connected
to developmental processes, the appearance of
ROS spikes in conidiogenesis or during fruiting
body development, and by the observation
that addition of antioxidants inhibits differ-
entiation processes (see detailed discussion in
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PY49CH18-Tudzynski ARI 8 July 2011 9:34
Reference 4). Many examples exist for mutants
impaired in ROS scavenging, generation, or
signaling that have defects in differentiation
processes, e.g., the above mentioned tmpl mu-
tants of A. brassicicola and A. fumigatus, which
are impaired in conidiogenesis (50). Recently,
it has been shown by Hutchison et al. (42) that
in N. crassa ROS are involved in programmed
cell death (PCD) associated with heterokaryon
incompatibility. The authors speculate that
ROS might be a downstream effectors of PCD.
In mammalian systems, precisely localized
and timed ROS production by a set of NADPH
oxidases correlated with differentiation pro-
cesses (3, 13). Lara-Ortz et al. (60) were the
rst to establish a link between Nox activ-
ity and differentiation in fungi: They showed
that the single NoxA in A. nidulans is neces-
sary for fruiting body development. Later stud-
ies showed that the corresponding enzymes
(here termed Nox1) in the closely related as-
comycetes Podospora anserina and Neurospora
crassa are necessary for fruiting body develop-
ment; both fungi also possess a member of the
class NoxB (Nox2), which is important for as-
cospore germination (17, 69, 70). In A. nidu-
lans, apical dominance is regulated by ROS,
probably produced by the Nox complex (96).
In B. cinerea both NoxA and NoxB are nec-
essary for the formation of sclerotia, which
are the basis for the development of sexual
fruiting bodies (95). Brun et al. (15) recently
showed that the Nox complexes in P. anserina
are involved in regulation of cellulose degra-
dation. This saprophytic fungus differentiates
specic structures designed to colonize the cel-
lulose substratesimilar to appressoria in plant
pathogens. Differentiation of these structures
depends onNox activity. These results establish
a bridge to the above mentioned virulence de-
fects linked to nox mutants in plant pathogens:
Most likely, the primary role of Noxs in phy-
topathogens (B. cinerea, C. purpurea, M. oryzae)
is the generationof spatio-temporal ROSspikes
necessary for differentiation, e.g., of penetra-
tion hyphae and sclerotia primordials, and not
the communication with or attack of the host
(Figure 4).
NITRIC OXIDE (NO) SIGNALING
Nitric oxide (NO) and NO-derived reactive nitrogen species
(RNS) play an essential role in plant development and defense
reactions in cooperation with reactive oxygen species (ROS) (22,
40). However, a direct impact on pathogens has never been
shown. The role of NO as internal signal component in fungi
is also unclear. It has been postulated that because of the ability
of NOto pass the plasma membrane by free diffusion, it may have
a central role in nitrogen signaling. In Aspergillus nidulans genes
encoding NO-detoxifying enzymes, avohemoglobins (FHGs)
are coregulated with nitrate assimilatory genes (91). In Botrytis
cinerea, the singular Bcfhg enzyme is important for response to
nitrosative stress but has no impact on virulence (110), although
NO plays a crucial role in resistance of Nicotiana benthamiana
against B. cinerea (8). In Aspergillus oryzae, FHGs promote ox-
idative damage by hydrogen peroxide, corroborating a close link
between ROS and RNS metabolism and signaling also in fungi
(116).
In spite of the intensive research on fungal
Noxs during the past years, our knowledge of
their precise function is still limited. Because
the catalytic subunits are transmembrane pro-
teins, the complexes must be localized at some,
still undened, membrane. If they are localized
at the plasmalemma, then the O
2

would be
generated outside the hyphae, which raises the
question as to how this would affect the intra-
cellular redox status. Or are the ROS generated
by a Nox signal that is immediately taken up
by sensors and translated to intracellular sig-
nals (Figure 4)? A convincing answer to this
question is hampered by the fact that local-
ization experiments using functional Nox:GFP
fusion proteins are still to be carried out. An-
other open question is connected with the spe-
cic role of the different Nox complexes: Apart
from M. oryzae, in all investigated systems pos-
sessing twogp91
phox
homologs bothhave differ-
ent functions, although both are most probably
activated by the same regulator NoxR. How
are they recruited to their specic site of ac-
tion? Semighini & Harris (96) speculate that in
A. nidulans localization of the NoxA complex is
mediated by the scaffold protein Bem1 together
www.annualreviews.org ROS in Phytopathogenic Fungi 381
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Nucleus
RBOH
ROS
RBOH
ROS
RBOH
ROS
ROS
ROS
H
2
O
2
Ap1
ox
Sak
TF
Mitochondria
Target
proteins
Respiration
ROS
?
ROS
Signal
Other ROS
producer
NoxR
NoxA/
NoxB
Rac
?
Mitochondria
Chloroplast
Chloroplast
SOD/
CAT/PX
Vesicle
?
Vacuole
Development
ROS
ROS
ROS
ROS ROS
ROS
?
Ap1
red
Signal
Attack?
Attack?
Nucleus
(e.g., GSH)
(e.g., GSSG)
Development
PCD
Nucleus
Figure 4
Reactive oxygen species (ROS) in the interaction of a necrotrophic fungal pathogen and its host plant. The
schematic summarizes data and hypotheses based, e.g., on the Botrytis cinerea patho system, without claim of
exhaustion. Upon recognition of the pathogen, the plant cell produces ROS by respiratory burst oxidase
homologs (RBOHs) in the plasma membrane and from several internal sources. These plant-derived ROS
serve as signal to warn neighboring cells and affect the pathogen. The fungal hypha produces ROS by Nox
complexes, localized at the plasma membrane or in yet unknown vesicles, which mainly serve as signals.
Other internal ROS sources contribute to the ROS level in the interaction zone, probably stimulating/
increasing the oxidative burst. Scavenging systems from both partners contribute to the intra- and
extracellular redox homoeostasis. SOD, superoxide dismutase; CAT, catalase; PX, peroxidase; Ap1, Ap1-like
transcription factor; Sak, stress activated MAPK; GSH/GSSG, glutathione system; TF, transcription factors;
PCD, programmed cell death.
withthe small GTPase Cdc42 and NoxR. Bem1
could function as a Nox complex organizer like
p40
phox
in mammals. At least in B. cinerea such
a central function of Bem1 is unlikely: Dele-
tion of the singular bem1 homolog had no ef-
fect on virulence in this fungus (S. Giesbert &
P. Tudzynski, unpublished data), in contrast to
the nox knockouts (see above). However, it can-
not be ruled out that there is some redundancy
in the system that we still do not understand
well.
In P. anserina, a connection between the
Nox complex and a transmembrane protein,
the tetraspanin Pls1, could be established, as
deletion of both nox2 and pls1 resulted in a sim-
ilar spore germination defect (59). It turned out
that mutants in genes encoding Pls1 homologs
in B. cinerea and M. oryzae also have a very
similar phenotype as the corresponding noxB
mutants, and a phylogenetic analysis showed a
close correlation between the presence of NoxB
and Pls1 encoding genes (16; see discussion in
Reference 69). Thus, Pls1 could be a good can-
didate for a NoxB-interacting protein involved
in its recruitment to the site of action, e.g., the
appressoria. However, noproteininteractionor
382 Heller

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PY49CH18-Tudzynski ARI 8 July 2011 9:34
colocalization studies for these two proteins are
yet available. The exact composition and regu-
lation of the Nox complex(es) remains an open
question. Although there is strong genetic evi-
dence that NoxRis necessary for NoxA/Bfunc-
tion (see above), a direct physical interaction
has not yet been proven. So far only in Epichloe
direct binding of Rac to NoxR was demon-
strated (102). In C. purpurea, it was shown that
Rac and an important partner of Rac, the P21
kinase Cla4, are involved in regulation of the
nox1 gene (88). The report by Chen et al. (19)
on a direct interaction of Rac with Nox1/2 in
M. oryzae must be interpreted cautiously be-
cause the yeast-two-hybrid experiments were
performed with the full coding regions of these
transmembrane proteins, so the interaction in
the yeast nucleus could be articial.
As with mammalian systems, MAPKs obvi-
ously are involved in nox regulation: In A. nidu-
lans, the P38 homolog SakA has a repressing
function on noxA (60); there is some evidence
that in Epichloe SakA could inuence Nox ac-
tivity post-translationally (24). In contrast, in
B. cinerea the stress-activated kinase pathway
does not seemto have any inuence on the Nox
complex (at least not at transcriptional level).
Here, the MAPK Bmp3 (homolog of the yeast
Slt2 involved inthe cell integrity pathway) is the
major transcriptional regulator of the nox genes
(95). Interestingly, the regulation of the two
nox genes is inverse: In bmp3 deletion mutants
expression of noxA is downregulated, whereas
noxB is strongly upregulated, suggesting that
there is a link between MAPK signaling and
the Nox complexes. However, the exact nature
of this link remains to be elucidated.
In contrast to our rapidly growing under-
standing of the obvious role of ROS in develop-
mental processes and signaling (see discussions
in 4; 93), the exact mechanisms of these effects
are still unknown. How are ROS sensed extra-
and intracellularly? The transmission of infor-
mation to the nucleus could be mediated by
three principal modes of action. ROS could ac-
tivate special sensors that induce signaling cas-
cades, which ultimately regulate gene expres-
sion. Components of signaling pathways could
be directly oxidized by ROS; and ROS might
change gene expression by modifying the activ-
ity of transcription factors (Figure 4). A major
mode of action is probably their drastic effect
on protein structure via modication of thiol
(SH) groups, which is important for regula-
tion of proteins like thioredoxins, peroxiredox-
ins, etc. A classical example of the direct sens-
ing of redox status is the activation of Yap1p
via the thiolperoxidase Gpx3 (30). Compara-
ble effects might be important for many signal-
ing components. However, the exact control
mechanism of time and place of such regula-
tory effects remain to be resolved. A detailed
analysis of these processes would require high-
resolution ROS/redox status detection systems.
However, preliminary results using a redox-
sensitive reporter gene system, roGFP2 (71),
to measure the redox status of the GSH pool in
living hyphae of B. cinerea seem promising ( J.
Heller, A. Meyer, P. Tudzynski, unpublished
data).
SUMMARY POINTS
1. ROSare ubiquitous inliving cells; they have highdamaging potential but are alsoessential
for signaling and development.
2. Fungi have, like other eukaryotic cells, developed efcient ROS scavenging systems that
are under complex regulatory control; not much is known so far about ROS sensing
systems.
3. ROS play an important role in all fungus-plant interactions, mostly as signaling
components.
www.annualreviews.org ROS in Phytopathogenic Fungi 383
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PY49CH18-Tudzynski ARI 8 July 2011 9:34
4. The major resistance response by the plant, the PAMP-triggered immunity (PTI), in-
cludes a massive ROS production, the oxidative burst.
5. In mutualistic, endophytic, and biotrophic interactions, avoidance or suppression of
the ROS-mediated plant response is necessary to establish an interaction, whereas
necrotrophic fungi seem to stimulate or even to need the oxidative burst response of
the plant; they can produce ROS and contribute to the oxidative status in planta.
6. ROS are crucial in many fungal developmental processes.
7. Fungi posses NADPH oxidase (Nox) complexes, comparable to those of mammalian
cells. They are involved in sexual differentiation and virulence, but they do not seem to
contribute signicantly to (detectable) overall intra- and extracellular ROS levels. Most
likely, their major role is the generation of exactly localized and timed ROS peaks.
8. The spatio-temporal control of Nox activity is complex and not well understood; MAPK
cascades and small GTPases are directly involved in these processes.
FUTURE ISSUES
1. Why do fungi like B. cinerea not react with typical H
2
O
2
-triggered OSR in planta, in
spite of the oxidative burst? Why are ROS scavenging systems not relevant for virulence
in most cases?
2. What are the sources of intracellular and extracellular ROS production in fungi? What
is their relevance in interactions?
3. Can high resolution reporter systems be developed that allow unequivocal detection of
ROS peaks in subcellular structures?
4. What is the exact composition of Nox complexes in fungi?
5. How are Nox complexes recruited to their site of action?
6. Which spatio-temporal expression pattern do Nox complexes show, which regulatory
circuits are essential?
7. What is the exact role of ROS in developmental processes; what are their targets?
8. Although most ROS species are interconvertible, are there specic functions/roles in
developmental/interaction processes?
DISCLOSURE STATEMENT
The authors are not aware of any afliations, memberships, funding, or nancial holdings that
might be perceived as affecting the objectivity of this review.
ACKNOWLEDGMENTS
We have to apologize to all colleagues whose work we could not (or not adequately) cite due to
restrictions of space. We thank Barry Scott and Jesus Aguirre for critically reading the manuscript,
Sabine Giesbert and Nora Temme for discussions, and Klaus B.Tenberge, Matthias Becker, Nora
Temme for providing material for gures.
384 Heller

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PY49-FrontMatter ARI 8 July 2011 9:55
Annual Review of
Phytopathology
Volume 49, 2011
Contents
Not As They Seem
George Bruening p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1
Norman Borlaug: The Man I Worked With and Knew
Sanjaya Rajaram p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 17
Chris Lamb: A Visionary Leader in Plant Science
Richard A. Dixon p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 31
A Coevolutionary Framework for Managing Disease-Suppressive Soils
Linda L. Kinkel, Matthew G. Bakker, and Daniel C. Schlatter p p p p p p p p p p p p p p p p p p p p p p p p p p p 47
A Successful Bacterial Coup d

Etat: How Rhodococcus fascians Redirects


Plant Development
Elisabeth Stes, Olivier M. Vandeputte, Mondher El Jaziri, Marcelle Holsters,
and Danny Vereecke p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 69
Application of High-Throughput DNA Sequencing in Phytopathology
David J. Studholme, Rachel H. Glover, and Neil Boonham p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 87
Aspergillus avus
Saori Amaike and Nancy P. Keller p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 107
Cuticle Surface Coat of Plant-Parasitic Nematodes
Keith G. Davies and Rosane H.C. Curtis p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 135
Detection of Diseased Plants by Analysis of Volatile Organic
Compound Emission
R.M.C. Jansen, J. Wildt, I.F. Kappers, H.J. Bouwmeester, J.W. Hofstee,
and E.J. van Henten p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 157
Diverse Targets of Phytoplasma Effectors: From Plant Development
to Defense Against Insects
Akiko Sugio, Allyson M. MacLean, Heather N. Kingdom, Victoria M. Grieve,
R. Manimekalai, and Saskia A. Hogenhout p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 175
Diversity of Puccinia striiformis on Cereals and Grasses
Mogens S. Hovmller, Chris K. Srensen, Stephanie Walter,
and Annemarie F. Justesen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 197
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PY49-FrontMatter ARI 8 July 2011 9:55
Emerging Virus Diseases Transmitted by Whiteies
Jes us Navas-Castillo, Elvira Fiallo-Oliv e, and Sonia S anchez-Campos p p p p p p p p p p p p p p p p p 219
Evolution and Population Genetics of Exotic and Re-Emerging
Pathogens: Novel Tools and Approaches
Niklaus J. Gr unwald and Erica M. Goss p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 249
Evolution of Plant Pathogenesis in Pseudomonas syringae:
A Genomics Perspective
Heath E. OBrien, Shalabh Thakur, and David S. Guttman p p p p p p p p p p p p p p p p p p p p p p p p p p p 269
Hidden Fungi, Emergent Properties: Endophytes and Microbiomes
Andrea Porras-Alfaro and Paul Bayman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 291
Hormone Crosstalk in Plant Disease and Defense: More Than Just
JASMONATE-SALICYLATE Antagonism
Alexandre Robert-Seilaniantz, Murray Grant, and Jonathan D.G. Jones p p p p p p p p p p p p p 317
Plant-Parasite Coevolution: Bridging the Gap between Genetics
and Ecology
James K.M. Brown and Aur elien Tellier p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 345
Reactive Oxygen Species in Phytopathogenic Fungi: Signaling,
Development, and Disease
Jens Heller and Paul Tudzynski p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 369
Revision of the Nomenclature of the Differential Host-Pathogen
Interactions of Venturia inaequalis and Malus
Vincent G.M. Bus, Erik H.A. Rikkerink, Val erie Cafer, Charles-Eric Durel,
and Kim M. Plummer p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 391
RNA-RNA Recombination in Plant Virus Replication and Evolution
Joanna Sztuba-Soli nska, Anna Urbanowicz, Marek Figlerowicz,
and Jozef J. Bujarski p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 415
The Clavibacter michiganensis Subspecies: Molecular Investigation
of Gram-Positive Bacterial Plant Pathogens
Rudolf Eichenlaub and Karl-Heinz Gartemann p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 445
The Emergence of Ug99 Races of the Stem Rust Fungus is a Threat
to World Wheat Production
Ravi P. Singh, David P. Hodson, Julio Huerta-Espino, Yue Jin, Sridhar Bhavani,
Peter Njau, Sybil Herrera-Foessel, Pawan K. Singh, Sukhwinder Singh,
and Velu Govindan p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 465
The Pathogen-Actin Connection: A Platform for Defense
Signaling in Plants
Brad Day, Jessica L. Henty, Katie J. Porter, and Christopher J. Staiger p p p p p p p p p p p p p p p 483
vi Contents
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PY49-FrontMatter ARI 8 July 2011 9:55
Understanding and Exploiting Late Blight Resistance in the Age
of Effectors
Vivianne G.A.A. Vleeshouwers, Sylvain Raffaele, Jack H. Vossen, Nicolas Champouret,
Ricardo Oliva, Maria E. Segretin, Hendrik Rietman, Liliana M. Cano,
Anoma Lokossou, Geert Kessel, Mathieu A. Pel, and Sophien Kamoun p p p p p p p p p p p p p p p 507
Water Relations in the Interaction of Foliar Bacterial Pathogens
with Plants
Gwyn A. Beattie p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 533
What Can Plant Autophagy Do for an Innate Immune Response?
Andrew P. Hayward and S.P. Dinesh-Kumar p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 557
Errata
An online log of corrections to Annual Review of Phytopathology articles may be found at
http://phyto.annualreviews.org/
Contents vii
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