reactive oxygen species in phytopathogenic fungi

8/12/2019 Reactive Oxygen Species in Phytopathogenic Fungi

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Reactive Oxygen Speciesin Phytopathogenic Fungi:Signaling, Development,and Disease

Jens Heller and Paul Tudzynski

Molecular Biology and Biotechnology of Fungi, Institute of Biology and Biotechnol

Plants, Westfalische Wilhelms-Universitat Munster, Germany;email: [email protected], [email protected]

Annu. Rev. Phytopathol. 2011. 49:36990

TheAnnual Review of Phytopathologyis online atphyto.annualreviews.org

This articles doi:

10.1146/annurev-phyto-072910-095355

Copyright c2011 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

AbstractReactive oxygen species (ROS) play a major role in pathogen-pinteractions: recognition of a pathogen by the plant rapidly trigger

oxidative burst, which is necessary for further defense reactions.

specific role of ROS in pathogen defense is still unclear. Studiethe pathogen so far have focused on the importance of the oxid

stress response (OSR) systems to overcome the oxidative burst its avoidance by effectors. This review focuses on the role of RO

fungal virulence and development. In the recent years, it has becobviousthat(a)fungalOSRsystemsmightnothavethepredictedcr

role in pathogenicity, (b) fungal pathogens, especially necrotrophs

actively contribute to the ROS level in planta and even take advanof the hosts response, (c) fungi possess superoxide-generating NADoxidases similar to mammalian Nox complexes that are importan

pathogenicity; however, recent data indicate that they are not dir

involved in pathogen-host communication but in fungal differentiprocesses that are necessary for virulence.

369

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ROS: reactive oxygenspecies

NADPH-dependantoxidase complex(Nox): superoxidegenerating enzymesystem

Oxidative burst:reactive oxygen speciesspike in response topathogen attack

INTRODUCTION

Single-celled organisms were the first to appear

on Earth under an atmosphere very low inoxygen. The introduction of oxygen into the

atmosphere provided an environment that

allowed the evolution of complex multicellularorganisms with high-energy demands. How-

ever, simultaneously it brought about a newsource of toxins. Although oxygen is by nature a

weak reactant, it has a tendency to readily formradicals, either by energy transfer reactions or

by electron transfer reactions forming incom-pletely reduced reactive oxygen species (ROS),

which are highly potent oxidants. Energytransfer leads to the formation of singlet oxy-

gen (1O2), whereas electron transfer results in

the sequential reduction to superoxide (O2),hydrogen peroxide (H2O2), and hydroxyl radi-

cal (OH

) (51). Within the cell, these and othershort-lived ROS can react nonspecifically 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 progressiveaging 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 havehistorically been observed as a harmful but

unavoidable outcome of an aerobic lifestyle.Under physiological steady state conditions,

these molecules are scavenged by differentantioxidative defense components. However, if

the equilibrium between production and scav-enging of ROS is perturbedfor example, by

adverse environmental factorsintracellularlevels of ROS can rise, resulting in changes

in the cell transcriptome. In addition, local

bursts of ROS have been shown to be involvedin various differentiation processes. This

indicates that in spite of their toxic effect, mostorganisms have adapted to the existence of

ROS and have evolved processes to use them assecond messengers to transduce extracellular

signals to the nucleus (81). However, ROS are

not only involved in intracellular signalin

they also play a role in cell-cell interactioand in the interaction of different organism

In plant-microbe and phagocyte-pathoginteractions, ROS are mainly involved

recognition or defense reactions.

In this review, we discuss the role of RO

in the interaction of phytopathogenic fungi aplants. In the past few years, many excellent acomprehensive reviews have been publishe

mainly describing the importance of ROS fdefense reactions of plants against pathoge

(76, 84, 98, 107, 108). Here, we focus on theffects of ROS in the fungal partner during t

interaction and in this context outline the importance of ROSfor signalingand developme

in filamentous fungi.

Reactive Oxygen SpeciesGenerating Systems

Mitochondria are the major source of intracelular ROS. However, there areseveral other e

zymatic and nonenzymatic systems involved cellular ROSproduction (36). Themost impo

tant enzymatic ROS generating system is thNADPH-dependent oxidase complex (No

The best-studied member of this group of ezymes is the mammalian gp91phox (also know

as Nox2) responsible for the neutrophil oxidtive burst defense response (57). Nox2 catalyz

the production of superoxide by a one-electro

reduction of oxygen using NADPH as electrdonor. Generation of ROS by Nox requires t

assembly of a multi-subunit complex. Thiscomposed of the regulatory cytosolic comp

nents p40phox, p47phox, p67phox, and the smGTPase Rac, which come together as a com

plex, and the integral membrane protein flavcytochromeb558, composed of thecatalytic su

unit gp91phox and the adaptor protein p22ph

which are localized in endosomal membran

Upon cell stimulation, the p47phox subunitphosporylated and the entire cytosolic compl

is recruited to the membrane, where it assciates with the two membrane-bound comp

nents to assemble the active oxidase compl

and produce O2. O2 can be converted to

370 Heller Tudzynski


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MAPK:mitogen-activatprotein kinase

number of other reactive oxidants that are used

by phagocytes to kill invading microorganisms.Along with gp91phox, 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 widerange of other organisms, where presence ofthis enzymehas been proposed to correlatewith

multicellular status (54).

Reactive Oxygen Species Scavenging

Most likely all organisms have to cope withoxidative stress conditions during their life-

span. These conditions can be caused eitherby ROS produced as byproducts during normal

metabolic processes or by an environment withunfavorable 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 typicallyconsist 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, atripeptide -L-glutamyl-L-cysteinyl-glycine),

but also other compounds like phytochelatins,

ascorbic acid, polyamines, flavonoids, alkaloids,andcarotenoids (46). GSH is a ubiquitous thiol-

containing reductant that maintains the intra-cellular redox homeostasis by reducing cellu-

lar disulfide bonds and detoxifying damagingmolecules, such as xenobiotics and heavy met-

als. During the reaction, GSH is converted toits oxidized form,glutathione disulfide (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 NADPH as the electron donor(6).

Enzymatic ROS scavenging mechanismsinclude superoxide dismutase (SOD) and vari-

ous peroxidases, such as glutathione peroxidase

(GPX), peroxiredoxin, and catalase (CAT).

SODs dismutate O2 to H2O2 and act as thefirst line of defense. H2O2 can then be con-

verted to H2O by the action of GPX and CAT.ROS scavenging systems are crucial for sup-

pressing toxic ROS levels in a cell, and the OSRhas to be regulated very tightly. In yeast and

fission yeast, there are several signaling compo-nents that control the OSR at a transcriptionallevel, including a mitogen-activated protein ki-

nase (MAPK) cascade (Hog1/Sty1) and a redoxsensitive transcription factor (Yap1/Pap1) (39,

44). The MAPK pathway is activated not onlyin response to oxidative stress, but in response

to multiple stresses (18). After activation, theMAPK acts as a transcription factor itself or

activates the transcription machinery by phos-phorylating other transcription factors and pro-

teins, resulting in modulationof proteinactivityand altered gene expression (21).

The mode of action of Yap1 is based on

redox-sensitive nuclear export: H2O2mediatesthe oxidation of a peroxiredoxin that oxidizes

the highly conserved Cys-residues of thetranscription factor, causing nuclear retention.

Upon exposure to oxidative stress, Yap1becomes localized to the nucleus, where it

induces the expression of OSR genes (53). Infission 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, ina recent study, G onzalez-Parraga et al. (33)

proposed that in Candida albicans there areCap1 (=Yap1)-independent OSR mechanisms

that include the transcription factors Skn7and Msn2/4 (29). In addition, the thioredoxin

system is one of the major control systems forcellular redox homoeostasis. It is composed

of two enzymes, the thioredoxin (Trx) and its

thioredoxin reductase (TrxR). The reduceddithiol [Trx-(SH)2] is able to directly reducedisulfides of target proteins, which is required

for several intracellular processes like sulfur

assimilation, detoxification of ROS, protein

www.annualreviews.org ROS in Phytopathogenic Fungi 371


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RBOH: respiratoryburst oxidase homolog

repair, and redox regulation of enzymes and

transcription factors (7).Although the OSR has been studied ex-

tensively in prokaryotes and eukaryotes, theinformation available on filamentous fungi is

fragmentary. However, in Botrytis cinerea, As-

pergillus fumigatus, Aspergillus nidulans, and

Cochliobolus heterostrophusHog1 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 specific just for oxidative stress. Incontrast, Yap1 homologs seem to be the main

regulators of OSR genes in filamentous fungi(61, 62, 104). Furthermore, homologs of the re-

sponse regulator Skn7 are involved in the OSRas they confer H2O2 resistance and regulate

CAT gene expression inAspergillusspecies (55,111). However, the Skn7 homolog of the phy-

topathogenic fungusMagnaporthe oryzaeis nei-

ther involved in OSR nor is it important forpathogenicity (74). Other cellular components

controlling the OSR in filamentous fungi re-main to be identified.

REACTIVE OXYGEN SPECIES INEARLY PLANT MICROBEINTERACTIONS

In the early events of plant-microbe interac-

tions, the rapid and transient production of

ROS (oxidative or respiratory burst) by plantsis mainly caused by membrane-associated res-

piratory burst oxidase homologs (RBOHs),which are homologs of the mammalian gp91phox

(109). This enzyme complex is involved in bothsymbiotic and pathogenic interactions. There-

fore, this typically apoplastic ROS productioncould be considered as a specific signal during

the interaction process (63). Nevertheless, thecourse of ROS formation differs between sym-

biotic and pathogenic interactions. Avirulentpathogens induce a biphasic ROS accumulation

in plants with a transitory, low amplitude first

phase that usually takes place within minutesafter the first contact and a second one con-

sisting of a sustained phase that occurs hours

afterward and that is usually associated with t

establishment of defense reactions and the hpersensitive response (2, 35, 83). Just the fir

burst has been observed in virulent pathogeand in symbiotic interactions, suggesting the

is suppression of the second wave of ROS (97The mechanism that underlies this suppressi

is still unknown. Nevertheless, the second rsponse obviously plays an important role for texclusion of pathogens showing that ROS a

key factors in the establishment of plant defenresponses (63).

Mutualistic Interactions

The best-studied microbial symbiotic intera

tions of plants are those between plants of tfamily Leguminosae and bacteria (rhizob

(75). Although rhizobia colonize roots inway that resembles colonization by pathogen

microorganisms, almost no host plant defen

reactions are triggered during successful symbioses. In these symbiotic interactions, there

an initial response by the host to the bacterbut they then appear to suppress the pla

defense responses (9, 79, 112). Accordingthe involvement of ROS in this interacti

has been shown: Alfalfa responds to infectiowith Sinorhizobium meliloti by production

superoxide and hydrogen peroxide (89). Tsymbionts use protecting mechanisms li

ROS-scavenging enzymes to counteract t

plant defense response and particularly sutained ROS accumulation (45). However, sin

both application of extracellular ROS and inhbition of plant NADPH oxidases by diphen

lene iodonium (DPI) treatment suppressthe root hair curling and infection thre

formation, ROS are suggested to regularather than directly form, part of the defen

system in rhizobia-legume symbioses (82).Mutualistic fungus-plant interactions ha

been analyzed in detail using functional gene

and genomics approaches. There is increasinevidence that during the process of plant ro

colonization by mycorrhizal fungi, ROS plan important role; e.g., volatile compoun

secreted by the truffle Tuber melanosporu



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Arbuscularmycorrhiza: mwidespread formmycorrhiza thatinclude formatiointricately branc

haustoria in corcells of plant roarbuscules

PAMP: pathogassociated molepattern

PTI: PAMP-triggered immu

Biotrophic funpathogens feedidepending on licells

Necrotrophic fpathogens able their host cells afeed on dead platissue

Appressoria:unicellular fungpenetration orgwhich high osmpressure is gene

induce an oxidative burst inArabidopsis thaliana(100). In arbuscular mycorrhizal interactions,the most widespread terrestrial symbiosis (14)

accumulation of ROS, catalase, peroxidase, andSOD transcripts has been observed (26, 32, 56).

A proteomics study of the interaction of two

Glomus species and Medicago truncatula iden-

tified several fungal proteins involved in redoxhomoeostasis, including the Cu/Zn-SOD,thioredoxin peroxidase, thioredoxin reductase,

and other enzymes (85). In the ericoid mycor-rhizal fungusOidiodendron maius, disruption of

a sodgene caused increased sensitivity againstexternal ROS and a reduction in mycorrhiza-

tion (1). These data strongly suggest that ROSand 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 endophyticpartnerships; e.g., for the interaction ofEpichloe

festucae and perennial ryegrass growth of the

hyphal tip, and branching is proposed to becontrolled by localized bursts of ROS across

the plasma membrane in response to signalingfrom the grass host (101).

Pathogenic Interaction: Dealing WithPlant Oxidative Burst

For pathogenic interactions between plants andmicrobes, ROS are of considerable importance.

There are two major types of plant resistance

mechanisms against pathogen attack: thegeneral pathogen-associated molecular pattern

(PAMP)-triggered immunity (PTI) and themore specific effector-triggered immunity

(ETI) stimulated by plant surveillance proteins(R-proteins) recognizing specific effector

proteins of the pathogen (AVR protein) (48).ROS production at the infection site is the

earliest response of PTI. Apart from primaryeffects such as cell wall strengthening and

induction of antimicrobial activity, ROSfunction as diffusible second messengers,

inducing several resistance responses including

synthesis of pathogenesis-related proteins andphytoalexins, and programmed cell death in

neighboring cells (27, 117). The lifestyles

of phytopathogenic fungi can be generally

divided into two different groups. On the onehand, there are biotrophic pathogens that

depend on living plant material and thereforemay 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 andColletotrichum species show an intermediateinfection strategy, termed hemibiotrophism,

characterized by an initial biotrophic phasethat changes into a necrotrophic one during

infection. The infection strategies of the partic-ular groups differ from each other. Biotrophic,

and hemibiotrophic, fungi have developedstrategies 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 tofacilitate infection and colonization of the host

(34). Accordingly, the consequence of the plantoxidative 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 doesnot inhibit the successive colonization of

necrotrophic fungi. However, this simpleblack and white view does not really account

for the complex reality of ROS function insuch interactions (see below). As Huckelhoven

& Kogel (41) pointed out, different types of

ROS have different functions, and timing andconcentration are also important.

Biotrophs/hemibiotrophs. During infection

of maize by the basidiomyceteUstilago maydis,plant cells stay alive and no apparent defense

responses are triggered (11). Nevertheless, thisbiotrophic fungus needs to be able to respond

to an oxidative burst to be fully virulent. Theresponse is mediated by a system related to the

Yap1p regulator.U. maydis yap1 deletion mu-

tants show a higher sensitivity to H2O2 than

wild-type (wt) cells and display reduced prolif-eration in the infected tissue (72). H2O2accu-

mulates around the intracellular hyphae of the

yap1 mutant, but this response is not seen for wt



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Hemibiotrophicfungi: pathogens withan early biotrophicgrowth phase thatswitches into anecrotrophic one

during infection

hyphae. Transcriptome analysis revealed that

a number of genes, including two peroxidasegenes, are downregulated in the mutant. More-

over, pharmacological inhibition of Noxs, themain producers of defense-linked ROS in plant

tissue, largely restored virulence ofyap1dele-tion strains (72). These findings suggest that

virulence ofU. maydisdepends on its ability todetoxify ROS. Nevertheless, it cannot be ex-cluded that the Yap1p controls, in addition to

ROS scavenging enzymes, fungal effectors thatsuppress the host oxidative burst.

The rust fungus Uromyces fabae convertsmuch of the carbohydrates it takes up during

the infection process of its hostVicia fabaintothe C6-polyol mannitol. This compound is a

good quencher of ROS. Indeed, the scavengingcapacity of mannitol found in the apoplasticflu-

ids of the host during infection is sufficient tosuppress the ROS built up during the defense

reaction to about one-half the level present in

the absence of mannitol. These results suggestthat quenching of ROS might be essential for

successful infection of this biotrophic fungus(114).

For the biotrophic pathogen Claviceps

purpurea, which infects flowers 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 andgrow within the plant tissue toward the ovary

there is no change in ROS levels (90). Deletionof genes encoding the single-secreted SOD and

the major-secreted CAT (which is even presentin large amounts in the honeydew of infected

plants) did not significantly reduce fungal vir-ulence (28, 73). However, deletion of the gene

cptf1 encoding a homolog of the fission yeasttranscription factor ATF1, which is a general

regulator of CAT activity in C. purpurea, led

to an oxidative burstlike reaction in the planttissue around the hyphae and in distal areas

(78). Preliminary macroarray data indicate thatCptf1 controls a large set of genes, in addition

to those encoding ROS-scavenging enzymes

(E. Nathues & P. Tudzynski, unpublish

data). Therefore, like in U. maydis, it remainsbe elucidated whether the absence of an oxid

tive burst in the pathogen-host interactioncaused by active fungal suppression of the pla

defense reaction. Regardless, ROS and t

general redox homoeostasis appear to be esse

tial also for this biotrophic interaction. Thconclusion is substantiated by the observatithat the histidine kinase CpHK2, a candida

for a sensory component of ROS signaling,involved in virulence of this fungus (77).

Hemibiotrophic fungi provide an interesing system to compare the necrotrophic an

the biotrophic lifestyle in one system. Duing the biotrophic early infection phase of t

hemibiotrophic wheat pathogenSeptoria triti

no accumulation of H2O2is detectable, where

in the necrotrophic phase, just before sporlation, there is a massive production of RO

(99). A recent study by Chi et al. (20) of th

hemibiotrophic fungusM. oryzae showed ththere are important fungal factors influencin

the ROS status in the pathogen-plant interation beyond the usual candidates involved

OSR and ROS generation. The genedes1(dfense suppressor 1) was identified during a ra

dom mutagenesis screen using a T-DNA insetional mutant library. It encodes a serine-ri

protein with unknown biochemical propertiwhich is highly conserved within filamento

ascomycetes. Mutants lacking Des1 show no

mal vegetative growth, formation of appressoand penetration of host tissue. However, th

arehighlysensitivetoROSinaxeniccultureanare impaired in secretion of ROS scavengin

enzymes (e.g., peroxidases). In planta mutagrowth is significantly impaired and accomp

nied by a strong oxidative burst and establisment of general resistance reactions (in a no

mally susceptible rice cultivar). Addition of thNox inhibitor DPI reduced the plant defen

reaction and restored virulence of the fungu

However, it cannot be excluded that DPI alaffects the fungal Noxs. Thus, Des1 represen

a new type of fungal pathogenicity factor ivolved in modulating ROS-mediated plant d

fense. Obviously, it has no significant effect o



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Hypersensitivereaction (HR):mechanism usedplants to prevenspread of infectimicrobial patho

which includes trapid cell death local regionsurrounding aninfection

the intracellular ROS status. The authors spec-

ulate that Des1 might be involved in metal ionhomoeostasis.

These findings lead to the assumption thatbiotrophic and hemibiotrophic fungi depend

on the prevention of a strong oxidative burstand the hypersensitive response of their host,

either by completely suppressing the PTI de-fense reactions by suppressors or by scavengingthe 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 fungusB. cinerea, aclassical necrotrophic pathogen with high eco-

nomic importance, high levels of ROS can bedetected in all analyzed tissues indicating that

a strong oxidative burst takes place. This hasbeen demonstrated by classical staining tech-

niques using DAB (diamino benzaledehyde),

NBT (nitroblue tetrazolium chloride), and flu-orescent dyes (e.g., dichlorodihydrofluorescein

diacetate, H2DCF-DA), by the more sensitiveelectron microscopic cerium chloride staining

technique and by physicochemicalmethods (68,92) (seeFigures 1 and 2). The evidence for a

strong oxidative burst indicates thatB. cinereaneeds 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 H2O2-resistance in axenic culture (92). Although this

observation could be explained by the pres-ence of multiple ROS detoxification systems

as revealed by the candidate proteins predictedfrom the genome sequence, functional analysis

ofbap1(encoding a homolog of Yap1p) showedthat this major (and in axenic culture essential)

H2O2scavenging system is neither required forplant infection nor is it induced in planta (104).

This strongly suggests thatB. cinereadoes notface oxidative stress in planta, at least not via

H2O2. It cannot be excluded, however, that al-ternative OSR systems 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 retardedlesion development (87). However, this might

be due to its function in O2 detoxificationor to its ability to produce H2O2. The latter

hypothesis is supported by data presented byTiedemann (106), demonstrating that the ag-

gressiveness of a given isolate ofB. cinereacor-relates with the intensity of the oxidative burstit induces. Accordingly, B. cinerea is almost

apathogenic on hypersensitive reaction (HR)-deficientArabidopsismutants. Moreover, pre-

treatment ofArabidopsisplantswith HR-causingbacteria 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 resultshave also been obtained forother

necrotrophic fungi, e.g., inLeptosphaeria macu-

lans, a pathogen ofBrassica napus(64, 65).InColletotrichum coccodessecretion of ammo-

nium ions increases the oxidative burst and en-hances virulence on tomato (5; see Reference

84). However, Shetty et al. (99) pointed outthat the ability of fungi to grow within the host

tissue in spite of ROS accumulation does notnecessarily mean that they need the oxidative

burst: reduction of ROS formed in planta dur-ing the necrotrophic growth ofS. triticiby in-

filtrating CAT resulted in enhanced growth of

the pathogen. Obviously, the fungus can growin the presence of ROS but does not necessarily

need to produce this product to be virulent.In themaize pathogen Cochliobolus heterostro-

phus, deletion of a gene encoding a secretedCAT (Cat3) that confers H2O2resistance in ax-

enic culture hadno effect on pathogenicity (86).As in B. cinerea deletion of chap1 (encoding a

Yap1p homolog) inC. heterostrophusresulted inincreased sensitivity to oxidative stress caused

by hydrogen peroxide and menadione in axenicculture, 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



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10 m

10 m

50 m

15 m

5 m

a

b

c

d



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oxidative burst, do not depend on an effective

ROS scavenging system. The reasons for thisare still unknown. A similar situation has been

shown for animal pathogens: ROS hyper-sensitive mutants (obtained, for example, by

deletion ofyap1and sodgenes) inA. fumigatuswere not reduced in virulence (58, 61). Inter-

estingly, in a phylogenetic study comparingstress signaling pathways in fungi, Nikolaouet al. (80) pointed out that plant pathogenic

fungi in general are more sensitive to oxidativestress than animal pathogens, indicating that

there is no strong selection for oxidativestress resistance in fungus-plant interactions.

Alternaria alternata seems to represent anexception. In this necrotrophic pathogen,

the Yap1p homolog AaAP1/RLAP1, whichregulates OSR enzymes, plays an essential role

during fungal pathogenesis, as the deletionmutant does not cause any visible necrotic

lesions on wounded or unwounded leaves (66,

115). However, it cannot be excluded that thetranscription factor controls other components

essential for infection. Microscopic analysesrevealed the presence of H2O2 at cell walls of

appressoria and penetration pegs but not athost cell walls underneath the appressoria of

A. alternatawt. Therefore, Jennings et al. (47)proposed that most ROS in the interaction of

A. alternataand its host do not originate fromthe plant but from the fungus. Thus, there

might be reasons for the pathogenicity defect

of AaAP1/RlAP1 deletion strains other thanenhanced sensitivity against the plant oxidative

burst.

Role of Fungal ReactiveOxygen Species

The importance of ROS originating from thepathogen rather than from the host for viru-

lence is described for other systems as well. Therecent discovery of functional members of the

Nox family within filamentous fungi has ledto 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-

malian catalytic subunit gp91phox andathirdone(NoxC) contains putative calcium-binding EF-

hand motifs, a feature of human Nox5 and the

plant Nox homologs. Furthermore, fungi con-tainahomologoftheregulatorysubunitp67phox

(NoxR) and of the small GTPase Rac (102).Other components of the fungal Nox complex

are yet to be unequivocally identified.Cytochemical analysis showed that during

the infection process of B. cinerea, O2 ac-cumulates in fungal hyphal tips and H2O2 is

generated in and around the penetrated cellwall 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 ofBotrytis cinereausing the fluorescent probe dichlorodihydrofluorescein diacetate (H2DCF-DA). Bean leaveswere inoculated with droplets of a conidia suspension. All micrographs represent confocal laser scanningmicroscopy images of different plant tissues underneath the site of infection. The dye was injected into theleaf at the infection area prior to imaging. Left columns show brightfield images. After oxidation by ROS,H2DCF-DA exhibits green fluorescence (right middle). Chlorophyll autofluorescence is shown in red (leftmiddle). The right column shows an overlay of the two middle columns. ( a) In the negative control,

H2DCF-DA was injected into a noninfected leaf. Fluorescence of the dye is neither detectable in theepidermis nor in the palisade parenchyma. (b) Three hours post-inoculation (hpi), ROS are detectable instructures attached to the chloroplasts within the palisade parenchyma, showing the beginning oxidativeburst. These structures are probably peroxisomes. (c) Eighteen hpi, ROS are produced within the cell wall ofepidermal cells, probably by the action of RBOHs (top). Due to the strong ROS production in the palisadeparenchyma, ROS are even present in the intercellular space (bottom). (d) Twenty-four hpi, the intensity ofROS 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 thatshow strong fluorescence.



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Botrytis cinereaotrytis cinereaappressorium-likeppressorium like

structuretructureBotrytis cinerea

appressorium-likestructure

MitochondriumitochondriumMitochondrium Outer

epide

rmal

wall

5 mm

5 m

co

gt

1 m

aa b

c

d

1 mm

Figure 2

In situ localization of the accumulation of superoxide and hydrogen peroxide inbean and tomato leaves infected withBotrytis cinerea. (a) Localization of H2O2by the cerium chloride technique at the interface of a B. cinereaappressorium-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 bymitochondria 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 activityof scavenging enzymes originating from the host or the pathogen remains to beelucidated (picture by K.B. Tenberge). (b) Localization of superoxide bynitroblue tetrazolium chloride (NBT) staining of bean leaves at 6 hpi. Surfaceview of a germinating conidiospore (co). O2 accumulation of fungal origincan 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 byNBT staining of a tomato leaf at 72 hpi (picture by K.B. Tenberge andB. Hoppe). (d) Localization of H2O2by diamino benzaldehyde staining of abean leaf at 48 hpi (picture by N. Temme). Most O2 and H2O2can bedetected at the edge of the lesion, where the direct interaction of the fungusand its host takes place. The origin of these ROS is not known.

(105) (Figure 2). These ROS are thought toarise from the fungus and indeedB. cinereapos-

sesses two genes encoding catalytic subunits ofNADPH oxidases. Both proteins have a great

impact on pathogenicity: WhereasbcnoxAmu-tants are still able to penetrate host tissue in the

same way as the wt but colonization is slower,

bcnoxBmutants 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 strai

are almost apathogenic as they have a penetrtion defect. ThebcnoxR deletion mutantcan st

form appressoria, but it is unable to use thefor penetration (Figure 3). Instead, they gi

rise to new hyphal outgrowth and new rounof appressoria development and penetrati

initiation (95). However, neither mutant significantly impaired in intra- or extracellulROS production, suggesting that an altern

tive, as-yet-unknown source of ROS is involvin ROS accumulation during plant infection

M. oryzae seems to undergo a local oxidtive burst itself during plant infection, whi

is associated with development of appressorScavenging of oxygen radicals as well as DP

mediated inhibition of Nox enzymes led to sinificant delay of appressoria development a

altered morphology (25). Deletion of both noand nox2 caused apathogenicity. Although

this case the double mutant showed a faint r

duction of ROS in the appressoria, surprisingthe ROS level in hyphae of these mutants w

elevated, supporting the ideathat the Nox complex is not the only and not even a major pr

ducer of ROS in fungi. Because other ROS geerating systems must exist, it hasbeen suggest

that enzymes normally involved in oxidatidegradation of substrates, such as glucose o

idase, might be good candidates (15, 36).The complex situation of ROS generati

and homoeostasis is illustrated in a study pu

lished by Kim et al. (50). They showed thanovel transmembrane protein, TmpL, has m

jor impact on redox homeostasis and virulenin both a plant pathogen (Alternaria brassicicoand an animal pathogen (A. fumigatus). Deltion oftmplled to a massive increase in intrah

phal ROS levels, which was most probably ncaused by increased Nox activity. This sugges

once again that there are alternative fungROS production systems. Overexpressi

of the Yap1p homolog in the mutant su

pressed overproduction of intracellular ROand partly restored virulence. Interesting

overexpression of the Yap1p homolog in the was well as treatment of wt spores with the N

inhibitor DPI or antioxidants led to reduc



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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 byBotrytis cinerea. Detached bean leaves weresprayed with spore suspensions and incubated for 22 hours under humid conditions. Scanning electronmicroscopy (SEM) images with distinct resolutions of the wild-type (wt) strain B05.10 ( a,c,e) and thedeletion strainnoxR (b,d,f) are shown. (a) The wt forms very short germ tubes before penetrating the hostsurface. (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 wtdifferentiates appressoria-like structures (white arrowhead) and penetrates the host surface. (d) The deletionmutant produces appressoria-like structures as well (white arrowhead), but it does not penetrate the hostsurface. Instead, another hypha originates from the appressoria-like structure that elongates on top of thehost 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 Noxcomplex is essential for penetration via appressoria-like structures ofB. cinerea(pictures by M. Becker).



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virulence. The authors explain the pleiotrophic

phenotype observed by these treatments withdisturbance of oxidative stress homoeostasis,

substantiating the idea that intra- and extracel-lular ROS concentrations are crucial for proper

development and virulence. Hence, it is notsurprising that in biotrophic pathogens and in

endophytes ROS generating systems also havean influence 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 toB. cinereaand M. oryzae,neither of these enzymes is required for pen-

etration, which in C. purpurea is not achievedvia 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 gainaccess to the plants phloem exudate and hence

cannot produce macroscopic signs of infection

likehoneydewandsclerotia(31).Amutantlack-ing the catalytic subunit of the second Nox

complex, CpNox2, has a quite striking pheno-type: It colonizes the host tissue very efficiently

and produces vast amounts of honeydew, muchmore 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 wouldtherefore 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 Cpnox2complexisneededforabalancedinteractionbe-

cause the mutant appears to be more virulent.This would support the idea thatC. purpureahas established some kind of mutualism with itshost, e.g., contributing some advantage for the

plant through its secondary metabolites, ergotalkaloids, which are toxic to animals (see discus-

sion in Reference 37). Still, these data substan-

tiate the observation that endogenous ROS arealso 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, inactivati

of the noxA gene led to a switch from a mtualistic to an antagonistic interaction with

host (103). Plants infected with theE. festuc

noxAmutant showed disease symptoms and t

fungal biomass dramatically increased. RO

accumulation was observed in the extracellul

matrix of the endophyte and at the interfabetween extracellular matrix and host cwalls of meristematic tissue in the wt but n

in noxA mutants. Deletion of the second ngene in this fungus,noxB, had no effect on t

plant-interaction phenotype (103).Taken together, these data show that in a

dition to the requirement of ROS for an oidative attack in necrotrophs, fungal-deriv

ROS have a far more basic role in many hos

pathogen interactions of a completely differenature.

REACTIVE OXYGENSPECIES SIGNALING

AND DEVELOPMENT

The Janus-faced nature of ROS has long be

known in animal and plant systems (10, 52They are toxic to cells because of the damage

macromolecules and membranes, but they alrepresent important signaling molecules acti

as secondary messengers and are involved differentiation processes. Hansberg & Aguir

(38) have proposed that ROS are critic

for fungal growth and differentiation: Thsuggested that differentiation is a respon

to oxidative stress. Under normal growconditions, ROS levels are low, and generatio

and scavenging are balanced. Differentiatiis defined by a (localized) increase in the RO

level, which is transient, as it also inducthe upregulation of ROS scavenging system

This theory is supported by solid experimenevidence, e.g., the upregulation of genes e

coding specific antioxidant enzymes connectto developmental processes, the appearance

ROS spikes in conidiogenesis or during fruiti

body development, and by the observatithat addition of antioxidants inhibits diffe

entiation processes (see detailed discussion



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Reference 4). Many examples exist for mutants

impaired in ROS scavenging, generation, orsignaling that have defects in differentiation

processes, e.g., the above mentioned tmplmu-tants ofA. brassicicolaand A. fumigatus, which

are impaired in conidiogenesis (50). Recently,it has been shown by Hutchison et al. (42) that

inN. crassaROS are involved in programmedcell death (PCD) associated with heterokaryonincompatibility. 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 NADPHoxidases correlated with differentiation pro-

cesses (3, 13). Lara-Ort z et al. (60) were thefirst to establish a link between Nox activ-

ity and differentiation in fungi: They showedthat 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 Neurosporacrassaare necessary for fruiting body develop-

ment; both fungi also possess a member of theclass 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, whichare the basis for the development of sexual

fruiting bodies (95). Brun et al. (15) recently

showed that the Nox complexes inP. anserinaare involved in regulation of cellulose degra-

dation. This saprophytic fungus differentiatesspecific structures designed to colonize the cel-

lulose substratesimilar to appressoria in plantpathogens. Differentiation of these structures

depends on Noxactivity. These results establisha bridge to the above mentioned virulence de-

fects linked tonoxmutants in plant pathogens:Most likely, the primary role of Noxs in phy-

topathogens (B. cinerea,C. purpurea,M. oryzae)

is thegeneration of spatio-temporal ROSspikesnecessary for differentiation, e.g., of penetra-

tion hyphae and sclerotia primordials, and notthe communication with or attack of the host

(Figure 4).

NITRIC OXIDE (NO) SIGNALING

Nitric oxide (NO) and NO-derived reactive nitrogen spec(RNS) play an essential role in plant development and defe

reactions in cooperation with reactive oxygen species (ROS) (40). However, a direct impact on pathogens has never be

shown. The role of NO as internal signal component in fuis also unclear. It has been postulated that because of the abi

of NO to pass the plasma membrane by free diffusion, it may ha central role in nitrogen signaling. InAspergillus nidulansge

encoding NO-detoxifying enzymes, flavohemoglobins (FHare coregulated with nitrate assimilatory genes (91). In Botrcinerea, the singular Bcfhg enzyme is important for response

nitrosative stress but has no impact on virulence (110), althouNO plays a crucial role in resistance of Nicotiana benthami

againstB. cinerea (8). In Aspergillus oryzae, FHGs promote idative damage by hydrogen peroxide, corroborating a close l

between ROS and RNS metabolism and signaling also in fu(116).

In spite of the intensive research on fungalNoxs during the past years, our knowledge of

their precise function is still limited. Becausethe catalytic subunits are transmembrane pro-

teins, the complexes must be localized at some,still undefined, membrane. If they are localized

at the plasmalemma, then the O2 would begenerated 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 upby 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-

cific role of the different Nox complexes: ApartfromM. oryzae, in all investigated systems pos-

sessingtwogp91phox homologs both have differ-ent functions, although both are most probably

activated by the same regulator NoxR. Howare they recruited to their specific site of ac-

tion? Semighini & Harris (96) speculate that in

A. nidulanslocalization of the NoxA complex ismediated by the scaffold protein Bem1 together



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Nucleus

RBOH

ROS

RBOH

ROS

RBOH

ROS

ROS

ROS

H2O2

Ap1ox Sak

TF

Mitochondria

Targetproteins

Respiration

ROS

?

ROS

Signal

Other ROSproducer

NoxR

NoxA/NoxB

Rac

?

MitochondriaChloroplast

Chloroplast

SOD/CAT/PX

Vesicle

?

Vacuole

Development

ROSROS

ROS

ROS ROS

ROS

?

Ap1red

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. Theschematic summarizes data and hypotheses based, e.g., on the Botrytis cinereapatho system, without claim exhaustion. Upon recognition of the pathogen, the plant cell produces ROS by respiratory burst oxidasehomologs (RBOHs) in the plasma membrane and from several internal sources. These plant-derived ROSserve as signal to warn neighboring cells and affect the pathogen. The fungal hypha produces ROS by Noxcomplexes, 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- andextracellular redox homoeostasis. SOD, superoxide dismutase; CAT, catalase; PX, peroxidase; Ap1, Ap1-litranscription factor; Sak, stress activated MAPK; GSH/GSSG, glutathione system; TF, transcription factoPCD, programmed cell death.

with the small GTPase Cdc42 and NoxR. Bem1could function as a Nox complex organizer like

p40phox in mammals. At least inB. cinereasuch

a central function of Bem1 is unlikely: Dele-tion of the singular bem1homolog had no ef-

fect on virulence in this fungus (S. Giesbert &

P. Tudzynski, unpublished data), in contrast tothe nox knockouts (see above). However, it can-not be ruled out that there is some redundancy

in the system that we still do not understandwell.

In P. anserina, a connection between theNox complex and a transmembrane protein,

the tetraspanin Pls1, could be established, deletion of bothnox2andpls1resulted in a sim

ilar spore germination defect (59). It turned o

that mutants in genes encoding Pls1 homoloin B. cinerea and M. oryzae also have a ve

similar phenotype as the corresponding nox

mutants, and a phylogenetic analysis showedclose correlation betweenthe presence of Noand Pls1 encoding genes (16; see discussion

Reference 69). Thus, Pls1 could be a good cadidate for a NoxB-interacting protein involv

in its recruitment to the site of action, e.g., thappressoria. However, no proteininteraction



15/25

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 NoxR is necessary for NoxA/B func-

tion (see above), a direct physical interactionhas not yet been proven. So far only in Epichloe

direct binding of Rac to NoxR was demon-strated (102). InC. purpurea, it was shown thatRac and an important partner of Rac, the P21

kinase Cla4, are involved in regulation of thenox1gene (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 wereperformed with the full coding regions of these

transmembrane proteins, so the interaction inthe yeast nucleus could be artificial.

As with mammalian systems, MAPKs obvi-ously are involved innoxregulation: InA. nidu-lans, the P38 homolog SakA has a repressing

function onnoxA(60); there is some evidencethat in Epichloe SakA could influence Nox ac-

tivity post-translationally (24). In contrast, in

B. cinerea the stress-activated kinase pathway

does not seem to have any influence on the Noxcomplex (at least not at transcriptional level).

Here, the MAPK Bmp3 (homolog of the yeastSlt2 involved in thecellintegrity pathway) is the

major transcriptional regulator of the nox genes(95). Interestingly, the regulation of the two

noxgenes is inverse: In bmp3deletion mutants

expression ofnoxA is downregulated, whereasnoxB 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 bythree 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 couldbe directly oxidized by ROS; and ROS might

change gene expression by modifying the activ-

ity of transcription factors (Figure 4). A majormode of action is probably their drastic effect

on protein structure via modification 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 Yap1pvia 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 detailedanalysis 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 ofB. cinerea seem promising ( J.Heller, A. Meyer, P. Tudzynski, unpublished

data).

SUMMARY POINTS

1. ROSare ubiquitous in living cells; they have high damaging potentialbutarealso essentialfor signaling and development.

2. Fungi have, like other eukaryotic cells, developed efficient ROS scavenging systems thatare 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.



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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 ofthe 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 mammaliancells. They are involved in sexual differentiation and virulence, but they do not seem to

contribute significantly to (detectable) overall intra- and extracellular ROS levels. Mostlikely, 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; MAPKcascades and small GTPases are directly involved in these processes.

FUTURE ISSUES

1. Why do fungi like B. cinerea not react with typical H2O2-triggered OSR in planta, inspite 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 regulatorycircuits 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 specific functions/roles in

developmental/interaction processes?

DISCLOSURE STATEMENT

The authors are not aware of any affiliations, memberships, funding, or financial holdings thmight 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 restrictions of space. We thank Barry Scott and Jesus Aguirre for critically reading the manuscri

Sabine Giesbert and Nora Temme for discussions, and Klaus B.Tenberge, Matthias Becker, No

Temme for providing material for figures.



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Annual Review

Phytopatholog

Volume 49, 201Contents

Not As They Seem

George Bruening 1

Norman Borlaug: The Man I Worked With and Knew

Sanjaya Rajaram 17

Chris Lamb: A Visionary Leader in Plant Science

Richard A. Dixon 31

A Coevolutionary Framework for Managing Disease-Suppressive Soils

Linda L. Kinkel, Matthew G. Bakker, and Daniel C. Schlatter 47

A Successful Bacterial Coup d Etat: HowRhodococcus fasciansRedirects

Plant Development

Elisabeth Stes, Olivier M. Vandeputte, Mondher El Jaziri, Marcelle Holsters,

and Danny Vereecke 69

Application of High-Throughput DNA Sequencing in Phytopathology

David J. Studholme, Rachel H. Glover, and Neil Boonham 87

Aspergillus flavusSaori Amaike and Nancy P. Keller 107

Cuticle Surface Coat of Plant-Parasitic Nematodes

Keith G. Davies and Rosane H.C. Curtis 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 157

Diverse Targets of Phytoplasma Effectors: From Plant Development

to Defense Against InsectsAkiko Sugio, Allyson M. MacLean, Heather N. Kingdom, Victoria M. Grieve,

R. Manimekalai, and Saskia A. Hogenhout 175

Diversity ofPuccinia striiformison Cereals and Grasses

Mogens S. Hovmller, Chris K. Srensen, Stephanie Walter,

and Annemarie F. Justesen 197

v


24/25

Emerging Virus Diseases Transmitted by Whiteflies

Jes us Navas-Castillo, Elvira Fiallo-Olive, and Sonia S anchez-Campos 2

Evolution and Population Genetics of Exotic and Re-Emerging

Pathogens: Novel Tools and Approaches

Niklaus J. Gr unwald and Erica M. Goss 24

Evolution of Plant Pathogenesis inPseudomonas syringae:

A Genomics PerspectiveHeath E. OBrien, Shalabh Thakur, and David S. Guttman 2

Hidden Fungi, Emergent Properties: Endophytes and Microbiomes

Andrea Porras-Alfaro and Paul Bayman 2

Hormone Crosstalk in Plant Disease and Defense: More Than Just

JASMONATE-SALICYLATE Antagonism

Alexandre Robert-Seilaniantz, Murray Grant, and Jonathan D.G. Jones 3

Plant-Parasite Coevolution: Bridging the Gap between Genetics

and Ecology

James K.M. Brown and Aurelien Tellier

34

Reactive Oxygen Species in Phytopathogenic Fungi: Signaling,

Development, and Disease

Jens Heller and Paul Tudzynski 3

Revision of the Nomenclature of the Differential Host-Pathogen

Interactions ofVenturia inaequalisand Malus

Vincent G.M. Bus, Erik H.A. Rikkerink, Valerie Caffier, Charles-Eric Durel,

and Kim M. Plummer 3

RNA-RNA Recombination in Plant Virus Replication and Evolution

Joanna Sztuba-Solinska, Anna Urbanowicz, Marek Figlerowicz,

and Jozef J. Bujarski 4

TheClavibacter michiganensisSubspecies: Molecular Investigation

of Gram-Positive Bacterial Plant Pathogens

Rudolf Eichenlaub and Karl-Heinz Gartemann 44

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

4

The Pathogen-Actin Connection: A Platform for Defense

Signaling in Plants

Brad Day, Jessica L. Henty, Katie J. Porter, and Christopher J. Staiger 4

vi C on te nt s


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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 507

Water Relations in the Interaction of Foliar Bacterial Pathogens

with PlantsGwyn A. Beattie 533

What Can Plant Autophagy Do for an Innate Immune Response?

Andrew P. Hayward and S.P. Dinesh-Kumar 557

Errata

An online log of corrections toAnnual Review of Phytopathologyarticles may be found at

http://phyto.annualreviews.org/

reactive oxygen species in phytopathogenic fungi

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